CA1188245A - Chemokinesins and chemotaxins of leukocytes and inflamed tissues and process of their preparation - Google Patents
Chemokinesins and chemotaxins of leukocytes and inflamed tissues and process of their preparationInfo
- Publication number
- CA1188245A CA1188245A CA000398628A CA398628A CA1188245A CA 1188245 A CA1188245 A CA 1188245A CA 000398628 A CA000398628 A CA 000398628A CA 398628 A CA398628 A CA 398628A CA 1188245 A CA1188245 A CA 1188245A
- Authority
- CA
- Canada
- Prior art keywords
- leukocytes
- process according
- proteins
- ammonium sulfate
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
Abstract:
New chemokinesins and chemotaxins of leukocytes and inflamed tissue are described having the following properties: a) biological activities in vivo and in vitro:
selective reversible influence on the motility of leuko-cytes (chemokinesis) or selective chemical attraction of leukocytes (chemotaxis) in vitro; b) physico-chemical properties: soluble in aqueous media including in 15%
ethanol at a pH value of at least 4.0 to 10; insoluble in ammonium sulfate solution at 90% saturation (3.6 mol/1);
electrophoretic migration in acrylamide matrices at a pH
of 7.40 is anodic; adsorb reversibly in structure and biological activity on anion and cation exchangers, calcium phosphate gel and hydroxyapatite and can be subjected in native form to volume partition chromatography, character-ized by homogenizing leukocytes or inflamed tissue or culturing leukocytes and isolating the resultant chemo-kinesins and chemotaxins from the homogenates or the supernatant culture solution. The compounds selectively influence the motility of leukocytes or selectively attract leukocytes.
New chemokinesins and chemotaxins of leukocytes and inflamed tissue are described having the following properties: a) biological activities in vivo and in vitro:
selective reversible influence on the motility of leuko-cytes (chemokinesis) or selective chemical attraction of leukocytes (chemotaxis) in vitro; b) physico-chemical properties: soluble in aqueous media including in 15%
ethanol at a pH value of at least 4.0 to 10; insoluble in ammonium sulfate solution at 90% saturation (3.6 mol/1);
electrophoretic migration in acrylamide matrices at a pH
of 7.40 is anodic; adsorb reversibly in structure and biological activity on anion and cation exchangers, calcium phosphate gel and hydroxyapatite and can be subjected in native form to volume partition chromatography, character-ized by homogenizing leukocytes or inflamed tissue or culturing leukocytes and isolating the resultant chemo-kinesins and chemotaxins from the homogenates or the supernatant culture solution. The compounds selectively influence the motility of leukocytes or selectively attract leukocytes.
Description
BACKGROUND OF THE INVENTION
-Chemokinesins and chemotaxins of leukocytes are endogenous chemical self-compone~ts which activate and regulate the processes of emigration of leukocytes from the blood stream and their accumulation in tissue in inflammatory processes.
Local injuries in tissue cause an inflammatory reaction.
The inflammatory reaction is defined as process which begins with a sublethal tissue injury and ends with com-plete destruction or complete healing of the tissue.
The inflammatory reaction comprises numerous biological signal processes. Cells and mediators of inflammation belong to the structural equivalents of this cybernetic loop of the information network of the inflammatory pro-cess. Like the classical hormones of endocrine glands, the inflammatory mediators are also substances which are present in only minute concentrations as traces in tis-sues and in blood. For example, it can be sho~n that a dividing cell can maintain Qnly up to-5,900 of such media-tor molecules in a steady state equilibrium in its sur-rounding medium.
Many ~ifferent cell types participate in the biological information network. In particular, they include the dif ferent tpyes of leukocytes which accumulate at the reac-tion site of inflammation in the tissue. These leukocyte types can be subdivided into the following groups: Neutro-phils, eosinophils, basophils, monocytes (macrophages), lymphocytes and the different types of juvenile forms of these cells.
The complex biosynthesis from a common precursor form of these cells takes place in the bone marrow. After cell
-Chemokinesins and chemotaxins of leukocytes are endogenous chemical self-compone~ts which activate and regulate the processes of emigration of leukocytes from the blood stream and their accumulation in tissue in inflammatory processes.
Local injuries in tissue cause an inflammatory reaction.
The inflammatory reaction is defined as process which begins with a sublethal tissue injury and ends with com-plete destruction or complete healing of the tissue.
The inflammatory reaction comprises numerous biological signal processes. Cells and mediators of inflammation belong to the structural equivalents of this cybernetic loop of the information network of the inflammatory pro-cess. Like the classical hormones of endocrine glands, the inflammatory mediators are also substances which are present in only minute concentrations as traces in tis-sues and in blood. For example, it can be sho~n that a dividing cell can maintain Qnly up to-5,900 of such media-tor molecules in a steady state equilibrium in its sur-rounding medium.
Many ~ifferent cell types participate in the biological information network. In particular, they include the dif ferent tpyes of leukocytes which accumulate at the reac-tion site of inflammation in the tissue. These leukocyte types can be subdivided into the following groups: Neutro-phils, eosinophils, basophils, monocytes (macrophages), lymphocytes and the different types of juvenile forms of these cells.
The complex biosynthesis from a common precursor form of these cells takes place in the bone marrow. After cell
-2~ 8~'~5 formation, they are stored in the bone marrow as juvenile or mature cell forms. From their primary (so-called poie-tic) storage pools in the bone marrow they are mobilized and recruited as blood leukocytes into the blood stream, when necessary. The emigration of leukocytes ~rom the blood stream and their accumulation at the reaction site of tissue injury is not a passive process. It is an active process and occurs in well-defined emigration sequences of specific leukocyte populations. Therefore, time-depen-dently, either different homogenous or mixed populations of leukocytes may accumulate at the reaction site of inflammation.
Inflammatory mediators are soluble chemical substances which participate in the activation and regulation of the body's defence-system as carriers of specific information.
They are formed at the reaction site of inflammation by humoral or cellular mechanisms. The transmission of their specific information occurs systemically or locally to neigh~our or remote target cells. The mode of action of inflammatory mediators is often similar to that of the hormones of known endocrine glands.
The possible existence of inflammatory mediators with defined structure was first shown by Sir Thomas Lewis (1927) "The Vessels of the Human Skin and their Responses", Shaw, London. He showed that the so-called "triple res-ponse" can be mimicked by histamine, a substance of known structure. Today, many different kinds of inflammatory mediators are known. Inflammatory mediators can be simple or complex organic molecules, such as his-tamine, seroto-nin, prostaglandins, prostacyclins, thromboxans, leuko-triens, etc.
Chemotaxis is a reaction, by which the direction of loco-motion of cells or organisms is determined by chemical substances in their environment. This definition shows that chemotaxis is not a specific ability of leukocytes alone, but is a basic property of all living organisms.
Apart from directional locomotion to chemical stimuli (chemotaxis), cells can also be influenced chemically in their motility (chemokinesis). This may result in an in-hibition or a promotion of their random locomot~ion. ~ccord-ingly, chemokinesis is a reac-tion by which the motility of cells or organisms migrating randomly is determined by chemical substances in their environment; see W. Rothert, Flora, vol. 88, 1901, p. 371-421.
Positive and negative changes in the migration trace of a cell may result from alterations in cellular speed, pro-bability and frequency of migration or tumbling and direc-tion of locomotion. These changes in the random migration trace are called positive or negative chemokinesis. If all these changes cancel out each other, the substance inves-tigated has an indifferent chemokinetic activity.
Chemotaxis and chemokinesis of leukocytes (leukotaxis and leukokinesis~ can be objectively measured and differen-tiated only in well definedin vitro test systems. In vivo, such a measurement or differentiation of chemotaxis and chemokinesis is possible only with laborious and ingeneous device systems; see I.K. suckley,Exp. Mol Pathology, vol. II. p. 402 to 417.
Therefore, for a long time, a main research problem was the development of reproducible in vitro assay systems for chemokinesis and chemotaxis of leukocytes. The assess-ment in vitro occurs either by direct microscopic obser-vation ofsingle migration steps of single cells concomi-tantly to a measurement of locomotion speed of cells migrating in a concentration equilibrium or along a con-centration gradient of the substance to be investigated.
The results obtained have then to be examined as to whether or not they conform to or deviate from basic relations of random walk theory; see ~.C~ Peterson, s P.B. Noble, Biophys. J. vol. 12, (1972), p. 1048 to 1055.
When they conform to basic relations of random walk theory, the type of migration is called chemokinesis; If they deviate from the relations, the ~ype of migration of cells is called chemotaxis. Another test system for chemokinesis and chemotaxis has been developed by S.V.
Boyden in the form of a filter assay system consisting of a two compartment chamber. Today, many modifications of ~s assay system exist. In principle, the migration of many cells through the pores of a filter is measured;
see J.EI. Wissler et al.,Eur.~J. Immunol. vol. II, p. 90-96.
An alternative test system f or the measurement of negative chemokinetic activity of substances uses the inhibition of cell emigration from glass capillaries; see ~. R. ~ich and M.R. Lewis, Bull. John Hopkins Hosp. 7 vol. 50 (1932), p. 115 to 131. This inhibition is reversible, if the cells after the assay can be shown to be functionally viable~
This functional viability has to be demonstrated in a further assay system, i.e. whether or not they are still chemotactically responsive and motile. If the substances investigated are cytotoxic and therefore, the inhibi-tion o migration is positive, then the result of this second assay system is negative: The observed migration inhibition was not caused by reversible chemokinetic activity of the substance under investigation but the motility of the cells was inhibited by irreversible cyto-toxic actions and loss in the function of the cells.
V. ~lenkin, Biochemical Mechanisms in Inflammation, Char-les C. Thomas, ~pringfield, Illinois, 1956 has shown that soluble mediators are contributory factors in mecha-nisms which induce the emigration of blood leukocytes from blood vessels and their accumulation in tissues.
He isolated a crystallizable preparation of substances from inflammatory exudates whose nature has not been ~8~S
characterized in detail. However, with this preparation, he could induce an accumulation of leukocytes in tissues.
It has been assumed, however, that contamination with bacterial endoto~ins and other exogenous substances in the preparations obtained have contributed to the diffe-rent types of activities displayed. Such exogenous sub-stances, like endotoxins have a strong indirect biological action on blood plasma or blood cells. It is known that on the one hand, endotoxins may activate blood plasma pro-tein systems, such as kinine and complement protein systems. On the other hand, they have a mitogenic effect on mononuclear leukocytes (B-cell mitogens); see Ander-son et al. J. Exp. Med. 137 (1973), p. 943 to 953.
As a result of these findings, humoral serum protein pre-parations with chemotactic and/or chemokinetic activity on leukocytes have been prepared. However, these prepara-tions have been neither molecularly homogenous nor have they been biologically specific in their action. Nor have they been characterized in detail; see P.C. Wilkinson (ed.) Chemotaxis and Inflammation, Ch. Livingstone, ~din-burgh (1974).
Thus, some of these protein preparations also induce a leukocytosis reaction in vivo; see B. Damerau et al., Naunyn-Schmiedberg's Arch. Pharmacol. 302 (1978), p. 45 to 50. Detailed investigations of the mechanisms of for-mation of humoral chemotaxins for leukocytes derived from serum-proteins have shown their relationship with ana-phyJa:toxin activity which was detected by Friedberger in 1910; see J.A. Jensen, in ~ngram, D.G., (ed.): Biological Activities of Complement, Xarger, Basle, (1972), p. 132 to 157.
More recently, using modern chromatographical preparation techniques, such biological active humoral trace proteins could be isolated and characterized in molcularly homo-genous, crystalline and chemotactically acting form after .
~8~
about 5,000 to 20,000 fold purification; see J. H. Wissler, Eur. J. Immunol., vol. II, (1972), p. 73-96. These pro-tein preparations have neither a~leuko]cinetic activi~y nor can they mobilize and recruit leukoc~ytes from the bone marrow into blood circulation.
It is these molecular-biological properties, i e. the dis-tinct cell and action speci~icity, in which the natural humoral leukotaxin protein preparations prepared and highly purified from contact-activated serum basically differ from less purified natural and, especially, from synthetic low-mole~ular peptide leukotaxins (formyl-methio~
nyl deri~atives etc.). Taxis, kinesis, adhesion, aggre-gation and, in addition, phagocytosis of leukocytes are cbncomitantly and indiscriminately induced by such pre-parations and by the synthetic peptide.
Consequently, it has been postulated that a common recep-tor in the cell membrane of leukocytes exists whieh indis-criminately transmits information for taxis, kinesis, adhesion, aggregation and phagocytosis to the cell.
.
However, further investigation with highly purified, spe-cifically acting natural mediators show that these postu-~ates are not in conformity with reality. This can be dlrectly demonstrated by comparing the activity of the low-molecular synthetic peptide with the highly purified humoral, natural cell and reaction-specific leukotaxin preparatinS. While the synthetic peptides indiscriminate-ly activate cells to chemotaxis, chemokinesis, adhesion and aggregation, the specific natural humoral leukotaxin protein preparations only induce directional locomotion (chemotaxis) of leukocytes, without influencing their chemokinesis, adhesion, aggregations or phagocytosis responses.
All the mentioned and described preparations for influen-cing the chemokinesis and chemotaxis of leukocytes are humoral, serum protein-derived chemical substances. In add~tion, the existence of cellular (cell-secreted) chemotaxins has been shown. Furthermore, a migration-inhibiting activity of cellular origin has been found ("migration-inhibiting factor", MIF). However, the pre-parations which cause these activities have neither been characterized in detail nor have they been obtained in a orM acting in a biologically specific manner. Surveys on the variety of demonstra-ted biological activities are given in B.R. Bloom and J. R. David (ed.) "In Vitro Methods in Cell-mediated and Tumor Immunities", Academic Press, New York, 1976 and by J.I. Gallin and P.G. Quie (eds.) "Leukocyte Chemotaxis: Methods, Physiology and Clinical Applications", Raven Press, New York 197~.
The literature reveals that cellular chemokinesins have not been investigated or demonstrated so far. The migra-tion inhibition activity of the MIF preparation has not been clearly distinguished from chemotactic activities.
As far as they have been investigated, none of these pre-parations shows biological specificity. For instance, one chemotatic activity is said to be identical to the trans-fer factor activity; see J. I. Gallin and P.G. Quei, locO
cit. Morever, it is largely unknown whether or not such cellular activities can be differentiated from serum-derived humoral activities.
It is therefore a primary object of this invention to provide a new class of cellular chemokinesins and chemo-~a~ins from leukocytes.
It is another object of this invention to provide a new class of cellular chemokinesins and chemotaxins from leukocytes in highly purified form.
It is another object of this invention to provide a new ~8--class of cellular chemokinesins and chemotaxins from leuko-cytes in physical quantities for practical use.
It is another object of this invention to provide a new class of chemokinesins and chemotaxins from leukocytes, which represent biologically specific, active and nat-urally acting mediators for the promotion of the motility or the directional migration of leukocytes.
It is another object of this invention to provide a new class of chemokinesins and chemotaxins fron- leukocytes, which are suitable for specifically influencing inElam- !
matory p~rocesses in mammalian (e.g. human) organisms.
It is still another object of this invention to provide a process for producing and obtaining a new class of chemokinesins and chemotaxins from leukocytes in an eco-nomical, biotechnically useful and relatively simple manner.
Itis still another object of this invention to pxovide a process for producing and obtaining a new class of chemo-kinesins and chemotaxins from leukocytes in a highly puri-fied, molecularly homogenous form and in physical quan-tities for practical use.
It is still another object of this invention to provide a pharmaceutical composition for specifically influencing inflammatory processes in the body of mammalians.
These and other objects and advantages of the present invention will be evident from the followir.g description of the invention.
.. . . ...
8;~gtS
g SUMMARY OF THE INVENTION
The subject matter of the invention are chemokinesins and chemotaxins of leukocytes and inflamed tissue, which are characterized by the following properties:
a) biological activities in vivo and in vitro:
- selective reversible influence on the motility of leukocytes (chemokinesis~ or selective chemi-cal attraction of leukocytes (chemotaxis) in vitro;
- they are substantially free of other biological ef~ects;
b) physico-chemical properties:
- soluble in aqueous media including in 15% ethanol at a pH value of at least 4.0 to 10;
- insoluble in an ammonium sulfate solution at 90 saturation (3.6 mol/l~;
- electrophoretic migration in acrylamide matrices at a pH of 7.40 is anodic;
- they absorb reversibly in structure and biological activity on anion and cation exchangers, cal~ium phosphate gel and hydroxyapatite and can be sub-jected in native form to volume partition chroma-tography.
The chemokinesins and chemotaxins of leukocytes and in-flamed tissue which are evaluatQd for the first time and obtained in highly purified form in this invention are further characterized by the fact that they are substan-ti.ally free of other biological effects. More particu-larly the chemokinesins and chemotaxins of the invention do not show:
s - mobilization of adult and juvenile leukocytes from the bone marrow (leukocytosis or leftward shift reaction);
- spasmogenic effects on striated muscles;
- endotoxin contents and endotoxin-like or similar activities;
~ significant pyrogenic effects in vivo;
- lysis effects in vitro on erythrocytes, thrombocvtes and leukocytes;
- direct chemotropic mitogen effects on blood vessel cells;
- mitogenic effects on leukocytes.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the drawings which illustrate the invention:
Figures 1 to 7 show the W absorption spectra of the highly purified mediator proteinsf Figure 8 schematically shows a standard-pyrogen assay, and Figures 9 to 13 show negative chemokinetic effects and chemotactic effects.
The chemokinesins and chemotaxins of the invention have typical protein properties and protein reactions (folin and biuret reactions). Their melting point is at approximately 200C (decomposition in an air and oxygen-free atmosphere).
The chemokinesins and chemotaxins of the invention are structurally (antigenically) distinguishable from humoral chemokinesins and chemotaxins.
The chemokinesins and chemotaxins of the invention are cellu-lar inflammatory mediators with topobiochemically and bio-logically specific activity. Where in the following text both groups of substances are meant, they are referred to as "mediator proteins" for the sake of brevity. It is their biological task to regulate the emigration of mature and juvenile blood leukocytes. The mediator proteins are not moral independent blood or serum components. Apart from many other hormones and mediators, they are formed in vitro in leukocyte cultures or in vivo upon accumulation of leukocytes at the site of inflammation.
~ 38~5 From this it is apparent that the mediator proteins of the invention differ in many of their biological and chemical properties from structural and functional pro-perties of the bacterial endotoxins. An LD50 value can not be measured, since no lethal effectshave been obser-ved even with doses 10,000 times the amount of the phy-siologically active threshold dose.
The activity of he mediator proteins of the invention is measured in three different test systems. The first test is the direct microscopic observation of individual leukocytes and quantification of chemically-induced chan-ges of the parameters of the cell migration trace. These are speed, frequence, direction and length o the migra-tion steps of individual leukocytes. Said parameters are compared by means of the random walk theory under the influence of the substances to be tested; see S. ~. Peter-son and P.B. Noble, loc. cit. The test is performed wi-th and without the concentration gradient of the substance to ~e tested. I~ all parameters satisfy the random walk theory, no chemotaxis is involved. The type and intensity of a possible chemokinesis are decided by the parameters.
In the second test, chemokinesis and chemotaxis are de-termined by statistically measuring the migration of leukocyte populations. The negative chemokinesis o leu-kocytes is statistically measured by the third test based on the inhibition of the emigration of leukocyte popula-tions from capillaries; see A. R. Rich and M.R. Lewis, loc. cit.
The chemokines~s can be divided into two main groups.
According to Rothert, loc. cit., substances which reduce the random locomotion of the cells are called negative chemokinesins or apochemokinesins. If the random loco-motion of the cellsis increased, positive or proschemoki nesins are lnvolved. Where the actlvity is directed to ~8~45 a cell type in a selective or specific manner, the media-tor pro-teins are named by definition, for instance "mon~
apokinesin", "mono-proskinesin" or "granulo-apokinesin"
and "granulo-proskinesin". This means that the correspond-ing chemokinesin reduces or increases the random migration of monocytes or granulocytes.
Moreover, the chemokinesins ofthe invention can stem from different leukocyte types. This is also taken into con-sideration in the name by defintion.
Accordingly, the lymphocyto-monoapokinesin(LMAK~ is a chemo-kinesin which is produced by lymphocytes and specifically reduces the statistic locomotion of monocytes Imacrophages).
The monocyto-granuloapokinesin (MGAK) is a chemokinesin which is produced by monocytes and specifically reduces the locomotion of granulocytes. Analogously, the mono-cyto-granuloproskinesin (MGPK) is a protein which is pro-duced by monocytes and specifically increases the random locomotion of granulocytes. The lymphocyto-monoproskine-sin(LMPK) is a chemokinesin which is produced by lympho-cytes and specifically increases the locomotion of mono-cytes.
Apart from or in addition to the above-mentioned proper-ties which the mediator proteins of the invention have in common, the LMAK has the ~ollowing special properties:
a) biological effects:
- specific reversible lowering of the motility o~
macrophages ~monocytes) in vitro;
~ e~ective threshold dose in vitro: ~ 2nmol/l;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 14,000 dalton;
- absorption spectrum (W, visible and near IR-range) as given in Fig. 1;
- extinction coefficients according to the following Table I:
.
Table Wa~e length, nm E1 mg~ml, 1 cm ~H20, ~0C) 6 2~8 tmin) 0 43 277 ~ax) - ~.86 ~90 - -- 0-47 400 - 1000 - O .
E2 8 o ~ E~60 1.42 Apar-t from or in addition to the above-mentioned proper-.; ties which the mediator proteins of the invention have in common, the MGAK has the following special properties:
a) biological activities: ~
- specific reversible lowering of the motility of granulocytes in vitro;
- effective threshold dose in vitro: C 1 nmol~l;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approxlmately 9,000 dalton;
- no protein quaternary structure in the form of physi-cally bound peptide subunits: each of the native prote~ns consists of only one peptide unit;
- constant temperature coefficient of solubility in ammonium sulfate solutions between -10C and ~50C;
- absorption spectrum (W, visible and near IR-range) according to Fig. 2;
- extinction coefficient according to the following 8~5 Table II
Ta~le II
Wave length, nm1 mg/ml, 1 cm (~2~ 20C) 2~9 (min~ 0.39 260 Ø48 278 (max) . 0 71 2~0 - ~.7~ .
290 0.~8 400- 1000 .0 .
280 ~60 1.45 Apart from or in addition to the above-mentioned proper-ties which the mediator proteins of the invention have in common, the MGPK has the following special properties:
a) biological activities: -- specific reversible increase of the motility of granulocytes in vitro;
- effective threshold dose in vitro: C 2 nmol/l;
b) physico-chemical properties:
- molecular weight of the native pro-tein ~primary struc ture): approximately 16,000 dalton;
absorption spectrum (W, visible and near IR-range) as given in Fig. 3.;
- extinction coefficient according to the following Table III:
~88~5 Table III
Wave length, nm E1 mg/ml, 1 cm (H20, 20C) 25t ~min) 0 3Ç
260 U-~2 ~7~ (max) 0-56 ~80 0.5~.
~90 - 0.35 ~00-1000 0 E280/E26o 1.29 Apart from or in addition to the above-mentioned proper-ties which the mediator- proteins of the invention have in common, the LMPK has the following special properties:
a) biological activities:
- specific increase of the motility of macrophages ~monocytes) in vitro;
- effective threshold dose in vitro: C 10 nmol/l;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 22,000 dalton;
- absporption spectrum (W, visible and near IR-range) as given in Fig. 4;
- extinction coefficient according to the following Table IV:
1188;~5 Table IV
Wave length, nm El mg/ml, 1 cm (H20, 20C) i; ., ~ _ ._ ~49 (min) - 0 . 29 260 O . ~C~
2 79 ~max) D . ~
280 0 ~5 2gO ~-49 400- 1000 - 0 .
E280JE26o 1.62 The above-described chemokinesins of the invention, LMAK, MGAK, MGPK and LMPK are further characterized by the fact that they substantially do not display the following bio-logical activities:
.~ - capillary permeability-enhancing activity in the skin test;
- spasmogenic activity on smooth muscles;
- chemotaxis ofleukocytes in vitro;
- phagocytosis-stimulating effects on leukocytes in vitro;
- apparent shock or other systemically detrimental effects of the immediate or protracted type on the intact organism of mammals in vitro.
Furthermore, MGPK and LMPK do not show phlogistic activity in situ.
The ch~lotaxins of the invention are analogously named by de-finition after the speci f iG :leukocyte~type on which they ~ct and after that by which they are produced. Thus monocyto-granu-lotaxin (MGT) is a chemotaxin which is produced by monocytes and specifically influences the directional migration of : granulocytes. Granulocyto-monotaxin (GMT) is a chemotaxin which is produced by granulocytes and specifically influen-ces the directional migration of monocytes (macrophages3.
Finally, monocyto-eosinotaxin (MET) is a chemotaxinl which is produced by monocytes and specifically influences the directional migration of eosinophil leukocytes.
Apart from or in addition to the above-mentioned'properties which the mediator proteins of the invention have in com-mon, MGT has the following special properties:
a) biological activities:
- chemical attraction of neutrophilic granulocytes in vitro;
- accumulation of neutrophilic leukocytes in situ with indirect cell-induced angiogenesis and inflammation reaction;
- effective threshold dose in vitro: ~ 0.5 nmol/l;
b~ physico-chemical properties:
- molecular weight of the native pro~ein (primary structure): approximately 11, 000 dalton;
- absorption spectrum ~W, visible and near IR-range~
as given in Fig. 5;
- extinction coefficient ac¢ording to the following Table V:
Table V
Wave length, nm E1 mg/ml, 1 cm (H20, 20C) 6 253 - (min) 0.51 27~ (max) 0-66 280 ~.6 ~90 0.54 E280/E260 1.23 1~38;~9~5 Apart from or in addition to the above-mentioned properties which the media-tor proteins of the invention have in com-mon, GMT has the following special properti.es:
a) biological activities:
J q.
- chemical attraction of macrophages Imonoc~tes) in vitro;
- accumulation of monocytic leukocytes in situ with indirect, cell-induced angiogenesis and inflammation reaction;
- effective threshold dose in vitro: ~ 10 ~mol/l;
b) physico-chemical properties:
- molecular weight of the native protein (primary struc-ture): approximately 17,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 6;
- extinction coefficient according to the following Table VI:
Table VI
Wave length, nm E1 mg/ml, 1 cm (~2~ 20C~
249 ~min) 0.36-260 0.~3 278 ~max) 0.60 280. - . 0-59 290 0.34 280/E260 1.37 Apart from and in additi.on to the above-mentioned properties which the mediator prote~ns of the invention have in com-mon!~ET has the following special properties:
a) biological activities: .
- chemical attractlon of eosinophilic leukocytes ln 8~5 vitro;
- accumulation of eosinophilic leukocytes in situ;
- effective threshold dose in vitro; C5 nmol/l;
b) physico-chemical properties:
- molecular weight of the native protein tpr'imary structure): approximately S,000 dalton;
- no protein quaternary structure in the form of physically bound peptide subunits: each of the native proteins consists of only one peptide unit;
- absorption spec-trum (W, visible and near IR- range) as given in Fig. 7;
- extinction coefficient according to the following Table VII:
- Table VII
_. .
Wave length, nm E1 mg/01, 1 cm (H20, 20C) 6%
.
~52 (min~ 0.33 ~77 ~max) 0-~3 2$0 ~-~3 29~ ~-39 E280/E26o 1.36 The chemotaxins of the invention, MGT, GMT and MET do not show positive or negative chemokinetic effects on leiuko-cytes in vitro nor apparent shock or other systemically detrimental effects of the immedia-te or protracted type on the intac-t organism of mammals in vivo.
Vp to non-physiological concentrations of 10 ~mol/l, the mediator proteins of the invention have neither leucocy-tosis-inducing nor phagocytosis or mitosis-stimulating activities on neutrophil, eosinophil and mononuclear leuko-cytes of man, rabbit, pig, dog, gui.nea pig or rat. Finally, they have no apparent shock effect at threshold dose nor do they display a pyrogenic activity in rabbits (standardized method by measurement of rectal temperature according to Euro. Pharmacopoeia, vol. II (1975), p. 56 to 59).
Figures 1 to 7 show the W absortion spectra of the highly purified mediator proteins LMAK, MGAK, MGPK, LMPK, MGT, GMT and MET in water at 20C and extinction scale (0-100 E = 0-2 at a light path d = 1 cm~
Fig. 8 schematically shows a st~ndard-pyrogen assay according to Europ. Pharmacopoeia, vol. II 11975): The rectal,tempe-rature of 3 rabbits (a, b and c) having an average weight of 3 kg is measured prior IV,A~, during (*) and shortly after as well as 30 to 180 minutes after intravenous application of 10 ~g of LMAK (corresponding to about 0.4 nmol LMAK/kg animal) in 1 ml 0.9 (w/v~ ~ physiological saline.
The 1975 edi ion of the European Pharmacopoeia, the British (1973) and the American (USP3 (1975~ Standards allow the designation "pyrogen-free" to be applied to preparations for which the sum of the fluctuations of the rectal tempexature in a total of three experimental rabbits does not exceed the value of 2.6C and, in parti-cular, is below 1.15C. The experimental results given in Fig.8 fullfills these criteria~ According to these definitions,the ~M~K~-preparation is pyrogen-free and without febrile activity. This also applies to the o~her highly purified me~iator protein preparations. ~his extremely sensitive criterion for contamination of proteins with bacterial endotoxins and other ubiquitous pyrogens demon-strates the great efficacy of the process of the purifi-cation of the cellular m~ator proteins of the invention.
It is an obvious parameter for the biOl~qical specificity of the mediator proteins.
Figures 9 to 13 show the negatlve chemokinetic effect of LMAX and MGAK as well as the chemotactic effect of MGT
~L8~ 5 GMT and MET. Fig. 9 shows the negative chemokinetic effect of LMAK in the form of inhibition of mor.ocyte (macrophage) emigration from glass capillaries. Fig. 10 depicts the same for MGAK. Fig. 11 shows the chemotactic effect of MGT
in the form of chemical attraction and directional migra-tion of granulocytes through the pores of a membrane fil-ter. The same is depicted in figures 12 and 13 for GMT
and MET for monocytes (macrophages) ana eosinophilic leuko-cytes, respectively.
The mediator proteins prepared and obtained according to the invention are valuable, endogeous subs-tances. They can be used for specifically influencing the de~ence-state and inflammatory processes of ~he body, for instance the i~nune state~TheYare suitable to specifically influence the emigration and accumulation of leukocytes for inducing desired inflammatory reactions and controlling undesired onest for instance in tumors. Moreover, the mediator pro-teins can be used for producing their antibodies which are also suitable to specifically influence leukocyte aCcumulation processes.
The mediator proteins of the invention are applied locally alone or as a mixture to mammalians, for instance man, in the form of usual pharmaceutical compositions in a daily dose of ~50 fmol in a concentration of ~1 nmol/l.
-22~ 8~5 An~ther subjec-t matter of the invention is a proc'ess for the biotechnical preparation and isolation of mediator pro-teins ~rom leukocy-tes and from inflamed tissue sites. It is charac-terized in tha-t either the leukocytes or the in-flamed tissue are homogenized; or that leukocytes are cultured and the mediator proteinsformed or liberated are isolated from the homogenates or from the supernatant culture solution.
In principle, it is possible to prepare mediators from leukocytes directly without cell cultures. However, such a procedure is not economical: The leukocytes are destroyed by the process; the yields in mediators are low, since their synthesis and secretion is not stimulated prior to isolationj the mediators can be contaminated by intra-cellular structural constituents of leukocytes. Therefore, in the process of the invention, it is preferred to iso-late the llHdiator proteins from the supernatant solution of the leukocyte culture. In principle, the leukocytes can be cultured in any leukocyte-compatible medium.
For the culture of different cell types, such as bone marrow cells, heart muscle cells or leukocytes, different culture media are known. These media normally are aqueous solutions which contain numerous different compounds.
Main cons-tituents of these cul-ture media are salts, sugars and metabolites, amino acids and derivatives, nucleosides, vitamins, vitaminoids, coenzymes, steroids, antibiotlcs and other additives, such as tensides, heavy metal salts and indicator dyes. Special examples of known culture media are named "HAM"j"l~I~I 199" and "NCTC", see H.J.
Morton, In Vitro 6 (1970) p. 89 to 108.
When culturing cells for more than one hour, as in the case of leukocytes, mostly serum (e.g. fetal calf serum or horse serum) is added to the culture medium. The serum constituents are said to be favourable for the mainten-ance of cellular functions. ~.owever, if the serum-~ontain~
ing culture solution is to be subjected to processes for isolating proteins (mediators) which are o~med by cul~-turing cells, the preparation of trace protein products is difficult for reasons of the multipilicity of com-pounds making up the complex mixture of serum added to the culture. In addition, under sucn condi-tions, upon addi-tion of serum ~o a cell culture mediumt it is difficult if not at all imp,ossible to recognize the origin of the mediators: It is then an open question whether or not a distinct mediator is of humoral (serum) or cellular (leukocyte) origin and from which species this mPdiator stems~ Thus, the mediator may be derived from the species whosecells have been cultured; or , alternatively; it may be derived from the species from which thP added (mostly heterologous~ serum stems.
Besides serum-containing culture media, serum-ree, syn-thetic media are also known; see H.J~ Morton, loc. citi I. ~ayashi and G. H~ Satoj Nature 259 (1976) p. 132-134, N.N. Iscove and ~. ~elchers, J. Exp. Med. 1~7 ¦19783 p. 923-933.
However, these known media likewise have drawbacks for both the culture of cells and ror the preparation of the mediators formed from the culture supernatant. The tensides, heav~ metal salts and/or dyes contaLned therein may damage or irreversibly contaminate the txace mediator proteins.
-2~ 8~S
On the other hand, such known -serum-free media are devoid of essential constituents which are necessary for main-taining the structural and functional viability of leuko-cytes. Therefore, none of the culture media known so far can be suitably used for the culture of leukocytes and the biotechnical preparation of cellular trace components, such as mediator proteins.
For the culture of leukocytes, a new, fully synthetic chemically defined culture medium is preferably used.
It provides favourable conditions for cell culture and facilitates the preparation and isolation of the cellular mediator proteins from the culture supernatant.
The fully synthetic, chemically defined c~ll culture medium preferably used in this invention contains the normal groups of compounds, such as salts, sugars, polyols, uronic acids, and derivatives, amino acids and derivatives, nucleosides and nucleoside bases, vitamins, vitaminoids, phytyl derivatives, coenzymes and steroids in aqueous solu-tion. It is characterized in that it additionally contains one or a mixture of several compounds which so far have not been considered for use in cell culture media. These are especially valuable for expression of the life functions, for the proliferation of leukocytes and for promoting their capability to produce mediators. These substances include unsaturated fatty acids, flavanoids, ubiquinone, vitamin U, mevalolactone and L-carnosine.
In prolonged leukocyte culturinq, -the cell culture medium is preferably used without addition of serum. Instead, it contains at least one defined protein.
In further preferred embodiments of the invention, the syn-thetic, serum-free cell culture medium used in this inven-tion may contain additional compounds, e.g~ polyhydroxy S
compounds and sugars, amino acids, nucleosides, anionic compounds and/or vitamins which are not common in the known culture media. These compounds are useful in culturing leukocytes. The constituents in the culture medium used in this invention are equilibrated in their ratios so that their concentrations mainly correspond to the natural concentration ranges of the plasma; see Ciba-Geigy AG
(editor) (1969) in Documenta Geigy, Wissenschaftliche Tabellen, seventh edition, Geigy S.A. Basle.
Preferably, the cell culture medium is free of tensides, heavy metal salts and dye indicators which can damage the cells and may have a detrimental effect on the isolation of the desired cell products.
The cell culture medium with the composition given in Table VIII below is especially preferred in the process of the invention for culturing leukocytesO
The medium is prepared with water of ASTM l-quality; see ASTM D 1193-70 Standard Specification for Reagent Water 1970; Annual Book of ASTM - Standards, Easton, Maryland, ASTM 1970. In addition, it is freed from possible endotoxin-contaminations by ultrafiltration on tenside-free membranes with an exclusion limit of 10,000 dalton. The resulting medium is sterilized by filtration on tenside-free membranes with a pore size of 0.2 ~m.
26 ~ 5 Table VIII
No~ Component mol/l No. Component mol/l 1 Disodium 48 L-Alanine 0.2 m hydrogenphosphate 0.8 m 49 L-Arginine 0.1 m 2 Potassium 50 D,L-Carnithine dihydrogenphosphate 0.2 m chloride (BT) 50.0
Inflammatory mediators are soluble chemical substances which participate in the activation and regulation of the body's defence-system as carriers of specific information.
They are formed at the reaction site of inflammation by humoral or cellular mechanisms. The transmission of their specific information occurs systemically or locally to neigh~our or remote target cells. The mode of action of inflammatory mediators is often similar to that of the hormones of known endocrine glands.
The possible existence of inflammatory mediators with defined structure was first shown by Sir Thomas Lewis (1927) "The Vessels of the Human Skin and their Responses", Shaw, London. He showed that the so-called "triple res-ponse" can be mimicked by histamine, a substance of known structure. Today, many different kinds of inflammatory mediators are known. Inflammatory mediators can be simple or complex organic molecules, such as his-tamine, seroto-nin, prostaglandins, prostacyclins, thromboxans, leuko-triens, etc.
Chemotaxis is a reaction, by which the direction of loco-motion of cells or organisms is determined by chemical substances in their environment. This definition shows that chemotaxis is not a specific ability of leukocytes alone, but is a basic property of all living organisms.
Apart from directional locomotion to chemical stimuli (chemotaxis), cells can also be influenced chemically in their motility (chemokinesis). This may result in an in-hibition or a promotion of their random locomot~ion. ~ccord-ingly, chemokinesis is a reac-tion by which the motility of cells or organisms migrating randomly is determined by chemical substances in their environment; see W. Rothert, Flora, vol. 88, 1901, p. 371-421.
Positive and negative changes in the migration trace of a cell may result from alterations in cellular speed, pro-bability and frequency of migration or tumbling and direc-tion of locomotion. These changes in the random migration trace are called positive or negative chemokinesis. If all these changes cancel out each other, the substance inves-tigated has an indifferent chemokinetic activity.
Chemotaxis and chemokinesis of leukocytes (leukotaxis and leukokinesis~ can be objectively measured and differen-tiated only in well definedin vitro test systems. In vivo, such a measurement or differentiation of chemotaxis and chemokinesis is possible only with laborious and ingeneous device systems; see I.K. suckley,Exp. Mol Pathology, vol. II. p. 402 to 417.
Therefore, for a long time, a main research problem was the development of reproducible in vitro assay systems for chemokinesis and chemotaxis of leukocytes. The assess-ment in vitro occurs either by direct microscopic obser-vation ofsingle migration steps of single cells concomi-tantly to a measurement of locomotion speed of cells migrating in a concentration equilibrium or along a con-centration gradient of the substance to be investigated.
The results obtained have then to be examined as to whether or not they conform to or deviate from basic relations of random walk theory; see ~.C~ Peterson, s P.B. Noble, Biophys. J. vol. 12, (1972), p. 1048 to 1055.
When they conform to basic relations of random walk theory, the type of migration is called chemokinesis; If they deviate from the relations, the ~ype of migration of cells is called chemotaxis. Another test system for chemokinesis and chemotaxis has been developed by S.V.
Boyden in the form of a filter assay system consisting of a two compartment chamber. Today, many modifications of ~s assay system exist. In principle, the migration of many cells through the pores of a filter is measured;
see J.EI. Wissler et al.,Eur.~J. Immunol. vol. II, p. 90-96.
An alternative test system f or the measurement of negative chemokinetic activity of substances uses the inhibition of cell emigration from glass capillaries; see ~. R. ~ich and M.R. Lewis, Bull. John Hopkins Hosp. 7 vol. 50 (1932), p. 115 to 131. This inhibition is reversible, if the cells after the assay can be shown to be functionally viable~
This functional viability has to be demonstrated in a further assay system, i.e. whether or not they are still chemotactically responsive and motile. If the substances investigated are cytotoxic and therefore, the inhibi-tion o migration is positive, then the result of this second assay system is negative: The observed migration inhibition was not caused by reversible chemokinetic activity of the substance under investigation but the motility of the cells was inhibited by irreversible cyto-toxic actions and loss in the function of the cells.
V. ~lenkin, Biochemical Mechanisms in Inflammation, Char-les C. Thomas, ~pringfield, Illinois, 1956 has shown that soluble mediators are contributory factors in mecha-nisms which induce the emigration of blood leukocytes from blood vessels and their accumulation in tissues.
He isolated a crystallizable preparation of substances from inflammatory exudates whose nature has not been ~8~S
characterized in detail. However, with this preparation, he could induce an accumulation of leukocytes in tissues.
It has been assumed, however, that contamination with bacterial endoto~ins and other exogenous substances in the preparations obtained have contributed to the diffe-rent types of activities displayed. Such exogenous sub-stances, like endotoxins have a strong indirect biological action on blood plasma or blood cells. It is known that on the one hand, endotoxins may activate blood plasma pro-tein systems, such as kinine and complement protein systems. On the other hand, they have a mitogenic effect on mononuclear leukocytes (B-cell mitogens); see Ander-son et al. J. Exp. Med. 137 (1973), p. 943 to 953.
As a result of these findings, humoral serum protein pre-parations with chemotactic and/or chemokinetic activity on leukocytes have been prepared. However, these prepara-tions have been neither molecularly homogenous nor have they been biologically specific in their action. Nor have they been characterized in detail; see P.C. Wilkinson (ed.) Chemotaxis and Inflammation, Ch. Livingstone, ~din-burgh (1974).
Thus, some of these protein preparations also induce a leukocytosis reaction in vivo; see B. Damerau et al., Naunyn-Schmiedberg's Arch. Pharmacol. 302 (1978), p. 45 to 50. Detailed investigations of the mechanisms of for-mation of humoral chemotaxins for leukocytes derived from serum-proteins have shown their relationship with ana-phyJa:toxin activity which was detected by Friedberger in 1910; see J.A. Jensen, in ~ngram, D.G., (ed.): Biological Activities of Complement, Xarger, Basle, (1972), p. 132 to 157.
More recently, using modern chromatographical preparation techniques, such biological active humoral trace proteins could be isolated and characterized in molcularly homo-genous, crystalline and chemotactically acting form after .
~8~
about 5,000 to 20,000 fold purification; see J. H. Wissler, Eur. J. Immunol., vol. II, (1972), p. 73-96. These pro-tein preparations have neither a~leuko]cinetic activi~y nor can they mobilize and recruit leukoc~ytes from the bone marrow into blood circulation.
It is these molecular-biological properties, i e. the dis-tinct cell and action speci~icity, in which the natural humoral leukotaxin protein preparations prepared and highly purified from contact-activated serum basically differ from less purified natural and, especially, from synthetic low-mole~ular peptide leukotaxins (formyl-methio~
nyl deri~atives etc.). Taxis, kinesis, adhesion, aggre-gation and, in addition, phagocytosis of leukocytes are cbncomitantly and indiscriminately induced by such pre-parations and by the synthetic peptide.
Consequently, it has been postulated that a common recep-tor in the cell membrane of leukocytes exists whieh indis-criminately transmits information for taxis, kinesis, adhesion, aggregation and phagocytosis to the cell.
.
However, further investigation with highly purified, spe-cifically acting natural mediators show that these postu-~ates are not in conformity with reality. This can be dlrectly demonstrated by comparing the activity of the low-molecular synthetic peptide with the highly purified humoral, natural cell and reaction-specific leukotaxin preparatinS. While the synthetic peptides indiscriminate-ly activate cells to chemotaxis, chemokinesis, adhesion and aggregation, the specific natural humoral leukotaxin protein preparations only induce directional locomotion (chemotaxis) of leukocytes, without influencing their chemokinesis, adhesion, aggregations or phagocytosis responses.
All the mentioned and described preparations for influen-cing the chemokinesis and chemotaxis of leukocytes are humoral, serum protein-derived chemical substances. In add~tion, the existence of cellular (cell-secreted) chemotaxins has been shown. Furthermore, a migration-inhibiting activity of cellular origin has been found ("migration-inhibiting factor", MIF). However, the pre-parations which cause these activities have neither been characterized in detail nor have they been obtained in a orM acting in a biologically specific manner. Surveys on the variety of demonstra-ted biological activities are given in B.R. Bloom and J. R. David (ed.) "In Vitro Methods in Cell-mediated and Tumor Immunities", Academic Press, New York, 1976 and by J.I. Gallin and P.G. Quie (eds.) "Leukocyte Chemotaxis: Methods, Physiology and Clinical Applications", Raven Press, New York 197~.
The literature reveals that cellular chemokinesins have not been investigated or demonstrated so far. The migra-tion inhibition activity of the MIF preparation has not been clearly distinguished from chemotactic activities.
As far as they have been investigated, none of these pre-parations shows biological specificity. For instance, one chemotatic activity is said to be identical to the trans-fer factor activity; see J. I. Gallin and P.G. Quei, locO
cit. Morever, it is largely unknown whether or not such cellular activities can be differentiated from serum-derived humoral activities.
It is therefore a primary object of this invention to provide a new class of cellular chemokinesins and chemo-~a~ins from leukocytes.
It is another object of this invention to provide a new class of cellular chemokinesins and chemotaxins from leukocytes in highly purified form.
It is another object of this invention to provide a new ~8--class of cellular chemokinesins and chemotaxins from leuko-cytes in physical quantities for practical use.
It is another object of this invention to provide a new class of chemokinesins and chemotaxins from leukocytes, which represent biologically specific, active and nat-urally acting mediators for the promotion of the motility or the directional migration of leukocytes.
It is another object of this invention to provide a new class of chemokinesins and chemotaxins fron- leukocytes, which are suitable for specifically influencing inElam- !
matory p~rocesses in mammalian (e.g. human) organisms.
It is still another object of this invention to provide a process for producing and obtaining a new class of chemokinesins and chemotaxins from leukocytes in an eco-nomical, biotechnically useful and relatively simple manner.
Itis still another object of this invention to pxovide a process for producing and obtaining a new class of chemo-kinesins and chemotaxins from leukocytes in a highly puri-fied, molecularly homogenous form and in physical quan-tities for practical use.
It is still another object of this invention to provide a pharmaceutical composition for specifically influencing inflammatory processes in the body of mammalians.
These and other objects and advantages of the present invention will be evident from the followir.g description of the invention.
.. . . ...
8;~gtS
g SUMMARY OF THE INVENTION
The subject matter of the invention are chemokinesins and chemotaxins of leukocytes and inflamed tissue, which are characterized by the following properties:
a) biological activities in vivo and in vitro:
- selective reversible influence on the motility of leukocytes (chemokinesis~ or selective chemi-cal attraction of leukocytes (chemotaxis) in vitro;
- they are substantially free of other biological ef~ects;
b) physico-chemical properties:
- soluble in aqueous media including in 15% ethanol at a pH value of at least 4.0 to 10;
- insoluble in an ammonium sulfate solution at 90 saturation (3.6 mol/l~;
- electrophoretic migration in acrylamide matrices at a pH of 7.40 is anodic;
- they absorb reversibly in structure and biological activity on anion and cation exchangers, cal~ium phosphate gel and hydroxyapatite and can be sub-jected in native form to volume partition chroma-tography.
The chemokinesins and chemotaxins of leukocytes and in-flamed tissue which are evaluatQd for the first time and obtained in highly purified form in this invention are further characterized by the fact that they are substan-ti.ally free of other biological effects. More particu-larly the chemokinesins and chemotaxins of the invention do not show:
s - mobilization of adult and juvenile leukocytes from the bone marrow (leukocytosis or leftward shift reaction);
- spasmogenic effects on striated muscles;
- endotoxin contents and endotoxin-like or similar activities;
~ significant pyrogenic effects in vivo;
- lysis effects in vitro on erythrocytes, thrombocvtes and leukocytes;
- direct chemotropic mitogen effects on blood vessel cells;
- mitogenic effects on leukocytes.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the drawings which illustrate the invention:
Figures 1 to 7 show the W absorption spectra of the highly purified mediator proteinsf Figure 8 schematically shows a standard-pyrogen assay, and Figures 9 to 13 show negative chemokinetic effects and chemotactic effects.
The chemokinesins and chemotaxins of the invention have typical protein properties and protein reactions (folin and biuret reactions). Their melting point is at approximately 200C (decomposition in an air and oxygen-free atmosphere).
The chemokinesins and chemotaxins of the invention are structurally (antigenically) distinguishable from humoral chemokinesins and chemotaxins.
The chemokinesins and chemotaxins of the invention are cellu-lar inflammatory mediators with topobiochemically and bio-logically specific activity. Where in the following text both groups of substances are meant, they are referred to as "mediator proteins" for the sake of brevity. It is their biological task to regulate the emigration of mature and juvenile blood leukocytes. The mediator proteins are not moral independent blood or serum components. Apart from many other hormones and mediators, they are formed in vitro in leukocyte cultures or in vivo upon accumulation of leukocytes at the site of inflammation.
~ 38~5 From this it is apparent that the mediator proteins of the invention differ in many of their biological and chemical properties from structural and functional pro-perties of the bacterial endotoxins. An LD50 value can not be measured, since no lethal effectshave been obser-ved even with doses 10,000 times the amount of the phy-siologically active threshold dose.
The activity of he mediator proteins of the invention is measured in three different test systems. The first test is the direct microscopic observation of individual leukocytes and quantification of chemically-induced chan-ges of the parameters of the cell migration trace. These are speed, frequence, direction and length o the migra-tion steps of individual leukocytes. Said parameters are compared by means of the random walk theory under the influence of the substances to be tested; see S. ~. Peter-son and P.B. Noble, loc. cit. The test is performed wi-th and without the concentration gradient of the substance to ~e tested. I~ all parameters satisfy the random walk theory, no chemotaxis is involved. The type and intensity of a possible chemokinesis are decided by the parameters.
In the second test, chemokinesis and chemotaxis are de-termined by statistically measuring the migration of leukocyte populations. The negative chemokinesis o leu-kocytes is statistically measured by the third test based on the inhibition of the emigration of leukocyte popula-tions from capillaries; see A. R. Rich and M.R. Lewis, loc. cit.
The chemokines~s can be divided into two main groups.
According to Rothert, loc. cit., substances which reduce the random locomotion of the cells are called negative chemokinesins or apochemokinesins. If the random loco-motion of the cellsis increased, positive or proschemoki nesins are lnvolved. Where the actlvity is directed to ~8~45 a cell type in a selective or specific manner, the media-tor pro-teins are named by definition, for instance "mon~
apokinesin", "mono-proskinesin" or "granulo-apokinesin"
and "granulo-proskinesin". This means that the correspond-ing chemokinesin reduces or increases the random migration of monocytes or granulocytes.
Moreover, the chemokinesins ofthe invention can stem from different leukocyte types. This is also taken into con-sideration in the name by defintion.
Accordingly, the lymphocyto-monoapokinesin(LMAK~ is a chemo-kinesin which is produced by lymphocytes and specifically reduces the statistic locomotion of monocytes Imacrophages).
The monocyto-granuloapokinesin (MGAK) is a chemokinesin which is produced by monocytes and specifically reduces the locomotion of granulocytes. Analogously, the mono-cyto-granuloproskinesin (MGPK) is a protein which is pro-duced by monocytes and specifically increases the random locomotion of granulocytes. The lymphocyto-monoproskine-sin(LMPK) is a chemokinesin which is produced by lympho-cytes and specifically increases the locomotion of mono-cytes.
Apart from or in addition to the above-mentioned proper-ties which the mediator proteins of the invention have in common, the LMAK has the ~ollowing special properties:
a) biological effects:
- specific reversible lowering of the motility o~
macrophages ~monocytes) in vitro;
~ e~ective threshold dose in vitro: ~ 2nmol/l;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 14,000 dalton;
- absorption spectrum (W, visible and near IR-range) as given in Fig. 1;
- extinction coefficients according to the following Table I:
.
Table Wa~e length, nm E1 mg~ml, 1 cm ~H20, ~0C) 6 2~8 tmin) 0 43 277 ~ax) - ~.86 ~90 - -- 0-47 400 - 1000 - O .
E2 8 o ~ E~60 1.42 Apar-t from or in addition to the above-mentioned proper-.; ties which the mediator proteins of the invention have in common, the MGAK has the following special properties:
a) biological activities: ~
- specific reversible lowering of the motility of granulocytes in vitro;
- effective threshold dose in vitro: C 1 nmol~l;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approxlmately 9,000 dalton;
- no protein quaternary structure in the form of physi-cally bound peptide subunits: each of the native prote~ns consists of only one peptide unit;
- constant temperature coefficient of solubility in ammonium sulfate solutions between -10C and ~50C;
- absorption spectrum (W, visible and near IR-range) according to Fig. 2;
- extinction coefficient according to the following 8~5 Table II
Ta~le II
Wave length, nm1 mg/ml, 1 cm (~2~ 20C) 2~9 (min~ 0.39 260 Ø48 278 (max) . 0 71 2~0 - ~.7~ .
290 0.~8 400- 1000 .0 .
280 ~60 1.45 Apart from or in addition to the above-mentioned proper-ties which the mediator proteins of the invention have in common, the MGPK has the following special properties:
a) biological activities: -- specific reversible increase of the motility of granulocytes in vitro;
- effective threshold dose in vitro: C 2 nmol/l;
b) physico-chemical properties:
- molecular weight of the native pro-tein ~primary struc ture): approximately 16,000 dalton;
absorption spectrum (W, visible and near IR-range) as given in Fig. 3.;
- extinction coefficient according to the following Table III:
~88~5 Table III
Wave length, nm E1 mg/ml, 1 cm (H20, 20C) 25t ~min) 0 3Ç
260 U-~2 ~7~ (max) 0-56 ~80 0.5~.
~90 - 0.35 ~00-1000 0 E280/E26o 1.29 Apart from or in addition to the above-mentioned proper-ties which the mediator- proteins of the invention have in common, the LMPK has the following special properties:
a) biological activities:
- specific increase of the motility of macrophages ~monocytes) in vitro;
- effective threshold dose in vitro: C 10 nmol/l;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 22,000 dalton;
- absporption spectrum (W, visible and near IR-range) as given in Fig. 4;
- extinction coefficient according to the following Table IV:
1188;~5 Table IV
Wave length, nm El mg/ml, 1 cm (H20, 20C) i; ., ~ _ ._ ~49 (min) - 0 . 29 260 O . ~C~
2 79 ~max) D . ~
280 0 ~5 2gO ~-49 400- 1000 - 0 .
E280JE26o 1.62 The above-described chemokinesins of the invention, LMAK, MGAK, MGPK and LMPK are further characterized by the fact that they substantially do not display the following bio-logical activities:
.~ - capillary permeability-enhancing activity in the skin test;
- spasmogenic activity on smooth muscles;
- chemotaxis ofleukocytes in vitro;
- phagocytosis-stimulating effects on leukocytes in vitro;
- apparent shock or other systemically detrimental effects of the immediate or protracted type on the intact organism of mammals in vitro.
Furthermore, MGPK and LMPK do not show phlogistic activity in situ.
The ch~lotaxins of the invention are analogously named by de-finition after the speci f iG :leukocyte~type on which they ~ct and after that by which they are produced. Thus monocyto-granu-lotaxin (MGT) is a chemotaxin which is produced by monocytes and specifically influences the directional migration of : granulocytes. Granulocyto-monotaxin (GMT) is a chemotaxin which is produced by granulocytes and specifically influen-ces the directional migration of monocytes (macrophages3.
Finally, monocyto-eosinotaxin (MET) is a chemotaxinl which is produced by monocytes and specifically influences the directional migration of eosinophil leukocytes.
Apart from or in addition to the above-mentioned'properties which the mediator proteins of the invention have in com-mon, MGT has the following special properties:
a) biological activities:
- chemical attraction of neutrophilic granulocytes in vitro;
- accumulation of neutrophilic leukocytes in situ with indirect cell-induced angiogenesis and inflammation reaction;
- effective threshold dose in vitro: ~ 0.5 nmol/l;
b~ physico-chemical properties:
- molecular weight of the native pro~ein (primary structure): approximately 11, 000 dalton;
- absorption spectrum ~W, visible and near IR-range~
as given in Fig. 5;
- extinction coefficient ac¢ording to the following Table V:
Table V
Wave length, nm E1 mg/ml, 1 cm (H20, 20C) 6 253 - (min) 0.51 27~ (max) 0-66 280 ~.6 ~90 0.54 E280/E260 1.23 1~38;~9~5 Apart from or in addition to the above-mentioned properties which the media-tor proteins of the invention have in com-mon, GMT has the following special properti.es:
a) biological activities:
J q.
- chemical attraction of macrophages Imonoc~tes) in vitro;
- accumulation of monocytic leukocytes in situ with indirect, cell-induced angiogenesis and inflammation reaction;
- effective threshold dose in vitro: ~ 10 ~mol/l;
b) physico-chemical properties:
- molecular weight of the native protein (primary struc-ture): approximately 17,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 6;
- extinction coefficient according to the following Table VI:
Table VI
Wave length, nm E1 mg/ml, 1 cm (~2~ 20C~
249 ~min) 0.36-260 0.~3 278 ~max) 0.60 280. - . 0-59 290 0.34 280/E260 1.37 Apart from and in additi.on to the above-mentioned properties which the mediator prote~ns of the invention have in com-mon!~ET has the following special properties:
a) biological activities: .
- chemical attractlon of eosinophilic leukocytes ln 8~5 vitro;
- accumulation of eosinophilic leukocytes in situ;
- effective threshold dose in vitro; C5 nmol/l;
b) physico-chemical properties:
- molecular weight of the native protein tpr'imary structure): approximately S,000 dalton;
- no protein quaternary structure in the form of physically bound peptide subunits: each of the native proteins consists of only one peptide unit;
- absorption spec-trum (W, visible and near IR- range) as given in Fig. 7;
- extinction coefficient according to the following Table VII:
- Table VII
_. .
Wave length, nm E1 mg/01, 1 cm (H20, 20C) 6%
.
~52 (min~ 0.33 ~77 ~max) 0-~3 2$0 ~-~3 29~ ~-39 E280/E26o 1.36 The chemotaxins of the invention, MGT, GMT and MET do not show positive or negative chemokinetic effects on leiuko-cytes in vitro nor apparent shock or other systemically detrimental effects of the immedia-te or protracted type on the intac-t organism of mammals in vivo.
Vp to non-physiological concentrations of 10 ~mol/l, the mediator proteins of the invention have neither leucocy-tosis-inducing nor phagocytosis or mitosis-stimulating activities on neutrophil, eosinophil and mononuclear leuko-cytes of man, rabbit, pig, dog, gui.nea pig or rat. Finally, they have no apparent shock effect at threshold dose nor do they display a pyrogenic activity in rabbits (standardized method by measurement of rectal temperature according to Euro. Pharmacopoeia, vol. II (1975), p. 56 to 59).
Figures 1 to 7 show the W absortion spectra of the highly purified mediator proteins LMAK, MGAK, MGPK, LMPK, MGT, GMT and MET in water at 20C and extinction scale (0-100 E = 0-2 at a light path d = 1 cm~
Fig. 8 schematically shows a st~ndard-pyrogen assay according to Europ. Pharmacopoeia, vol. II 11975): The rectal,tempe-rature of 3 rabbits (a, b and c) having an average weight of 3 kg is measured prior IV,A~, during (*) and shortly after as well as 30 to 180 minutes after intravenous application of 10 ~g of LMAK (corresponding to about 0.4 nmol LMAK/kg animal) in 1 ml 0.9 (w/v~ ~ physiological saline.
The 1975 edi ion of the European Pharmacopoeia, the British (1973) and the American (USP3 (1975~ Standards allow the designation "pyrogen-free" to be applied to preparations for which the sum of the fluctuations of the rectal tempexature in a total of three experimental rabbits does not exceed the value of 2.6C and, in parti-cular, is below 1.15C. The experimental results given in Fig.8 fullfills these criteria~ According to these definitions,the ~M~K~-preparation is pyrogen-free and without febrile activity. This also applies to the o~her highly purified me~iator protein preparations. ~his extremely sensitive criterion for contamination of proteins with bacterial endotoxins and other ubiquitous pyrogens demon-strates the great efficacy of the process of the purifi-cation of the cellular m~ator proteins of the invention.
It is an obvious parameter for the biOl~qical specificity of the mediator proteins.
Figures 9 to 13 show the negatlve chemokinetic effect of LMAX and MGAK as well as the chemotactic effect of MGT
~L8~ 5 GMT and MET. Fig. 9 shows the negative chemokinetic effect of LMAK in the form of inhibition of mor.ocyte (macrophage) emigration from glass capillaries. Fig. 10 depicts the same for MGAK. Fig. 11 shows the chemotactic effect of MGT
in the form of chemical attraction and directional migra-tion of granulocytes through the pores of a membrane fil-ter. The same is depicted in figures 12 and 13 for GMT
and MET for monocytes (macrophages) ana eosinophilic leuko-cytes, respectively.
The mediator proteins prepared and obtained according to the invention are valuable, endogeous subs-tances. They can be used for specifically influencing the de~ence-state and inflammatory processes of ~he body, for instance the i~nune state~TheYare suitable to specifically influence the emigration and accumulation of leukocytes for inducing desired inflammatory reactions and controlling undesired onest for instance in tumors. Moreover, the mediator pro-teins can be used for producing their antibodies which are also suitable to specifically influence leukocyte aCcumulation processes.
The mediator proteins of the invention are applied locally alone or as a mixture to mammalians, for instance man, in the form of usual pharmaceutical compositions in a daily dose of ~50 fmol in a concentration of ~1 nmol/l.
-22~ 8~5 An~ther subjec-t matter of the invention is a proc'ess for the biotechnical preparation and isolation of mediator pro-teins ~rom leukocy-tes and from inflamed tissue sites. It is charac-terized in tha-t either the leukocytes or the in-flamed tissue are homogenized; or that leukocytes are cultured and the mediator proteinsformed or liberated are isolated from the homogenates or from the supernatant culture solution.
In principle, it is possible to prepare mediators from leukocytes directly without cell cultures. However, such a procedure is not economical: The leukocytes are destroyed by the process; the yields in mediators are low, since their synthesis and secretion is not stimulated prior to isolationj the mediators can be contaminated by intra-cellular structural constituents of leukocytes. Therefore, in the process of the invention, it is preferred to iso-late the llHdiator proteins from the supernatant solution of the leukocyte culture. In principle, the leukocytes can be cultured in any leukocyte-compatible medium.
For the culture of different cell types, such as bone marrow cells, heart muscle cells or leukocytes, different culture media are known. These media normally are aqueous solutions which contain numerous different compounds.
Main cons-tituents of these cul-ture media are salts, sugars and metabolites, amino acids and derivatives, nucleosides, vitamins, vitaminoids, coenzymes, steroids, antibiotlcs and other additives, such as tensides, heavy metal salts and indicator dyes. Special examples of known culture media are named "HAM"j"l~I~I 199" and "NCTC", see H.J.
Morton, In Vitro 6 (1970) p. 89 to 108.
When culturing cells for more than one hour, as in the case of leukocytes, mostly serum (e.g. fetal calf serum or horse serum) is added to the culture medium. The serum constituents are said to be favourable for the mainten-ance of cellular functions. ~.owever, if the serum-~ontain~
ing culture solution is to be subjected to processes for isolating proteins (mediators) which are o~med by cul~-turing cells, the preparation of trace protein products is difficult for reasons of the multipilicity of com-pounds making up the complex mixture of serum added to the culture. In addition, under sucn condi-tions, upon addi-tion of serum ~o a cell culture mediumt it is difficult if not at all imp,ossible to recognize the origin of the mediators: It is then an open question whether or not a distinct mediator is of humoral (serum) or cellular (leukocyte) origin and from which species this mPdiator stems~ Thus, the mediator may be derived from the species whosecells have been cultured; or , alternatively; it may be derived from the species from which thP added (mostly heterologous~ serum stems.
Besides serum-containing culture media, serum-ree, syn-thetic media are also known; see H.J~ Morton, loc. citi I. ~ayashi and G. H~ Satoj Nature 259 (1976) p. 132-134, N.N. Iscove and ~. ~elchers, J. Exp. Med. 1~7 ¦19783 p. 923-933.
However, these known media likewise have drawbacks for both the culture of cells and ror the preparation of the mediators formed from the culture supernatant. The tensides, heav~ metal salts and/or dyes contaLned therein may damage or irreversibly contaminate the txace mediator proteins.
-2~ 8~S
On the other hand, such known -serum-free media are devoid of essential constituents which are necessary for main-taining the structural and functional viability of leuko-cytes. Therefore, none of the culture media known so far can be suitably used for the culture of leukocytes and the biotechnical preparation of cellular trace components, such as mediator proteins.
For the culture of leukocytes, a new, fully synthetic chemically defined culture medium is preferably used.
It provides favourable conditions for cell culture and facilitates the preparation and isolation of the cellular mediator proteins from the culture supernatant.
The fully synthetic, chemically defined c~ll culture medium preferably used in this invention contains the normal groups of compounds, such as salts, sugars, polyols, uronic acids, and derivatives, amino acids and derivatives, nucleosides and nucleoside bases, vitamins, vitaminoids, phytyl derivatives, coenzymes and steroids in aqueous solu-tion. It is characterized in that it additionally contains one or a mixture of several compounds which so far have not been considered for use in cell culture media. These are especially valuable for expression of the life functions, for the proliferation of leukocytes and for promoting their capability to produce mediators. These substances include unsaturated fatty acids, flavanoids, ubiquinone, vitamin U, mevalolactone and L-carnosine.
In prolonged leukocyte culturinq, -the cell culture medium is preferably used without addition of serum. Instead, it contains at least one defined protein.
In further preferred embodiments of the invention, the syn-thetic, serum-free cell culture medium used in this inven-tion may contain additional compounds, e.g~ polyhydroxy S
compounds and sugars, amino acids, nucleosides, anionic compounds and/or vitamins which are not common in the known culture media. These compounds are useful in culturing leukocytes. The constituents in the culture medium used in this invention are equilibrated in their ratios so that their concentrations mainly correspond to the natural concentration ranges of the plasma; see Ciba-Geigy AG
(editor) (1969) in Documenta Geigy, Wissenschaftliche Tabellen, seventh edition, Geigy S.A. Basle.
Preferably, the cell culture medium is free of tensides, heavy metal salts and dye indicators which can damage the cells and may have a detrimental effect on the isolation of the desired cell products.
The cell culture medium with the composition given in Table VIII below is especially preferred in the process of the invention for culturing leukocytesO
The medium is prepared with water of ASTM l-quality; see ASTM D 1193-70 Standard Specification for Reagent Water 1970; Annual Book of ASTM - Standards, Easton, Maryland, ASTM 1970. In addition, it is freed from possible endotoxin-contaminations by ultrafiltration on tenside-free membranes with an exclusion limit of 10,000 dalton. The resulting medium is sterilized by filtration on tenside-free membranes with a pore size of 0.2 ~m.
26 ~ 5 Table VIII
No~ Component mol/l No. Component mol/l 1 Disodium 48 L-Alanine 0.2 m hydrogenphosphate 0.8 m 49 L-Arginine 0.1 m 2 Potassium 50 D,L-Carnithine dihydrogenphosphate 0.2 m chloride (BT) 50.0
3 Potassium chloride 5.0 m 51 L-Carnosine 5.0 ~
4 Sodium chloride120.0 m 52 L-Cysteine 0.2 m
5 Sodium sulfate0.2 m 53L-Glutathione reduced 3.0 ~
6 D-Glucose _ 5.0 m 54 Glycine 0.2 m
7 L-Ascorbic acid ~C) 0.2 m 55 L-Histidine 0~1 m
8 Choline chloride50.0 ~ 56 L-~ydroxyproline10.0
9 2-Deoxy-D-ribose5.0 ~ 57 L-Lysine-~Cl 0.2 m
10 D-G~lactose 0.5 m 58 L-Methionine 0.1 m
11 D-Glucurono-~-lactone 0.1 m59 D,L-Mevalolactone 5.0
12 Glyeerol 50.0 ~ 60 Nicotinie aeid amide 20.0
13 Myo-inositol 0.5 m 61 L-Ornithine-HCl 50.0
14 Sodium acetate0.2 m 62 D-Ca-pantothenate tB5) 5-0 ~
15 Sodium citrate50.0 ~ 63 L-Proline 0.1 m
16 Sodium pyruvate0.1 m 64 Pyridoxal-~Cl 5.0
17 D-Ribose 20.0 ~ 65 Pyridoxine-RCl (B6) 2.0
18 Suecinie aeid0.1 m 66 Sarcosine 50.0 ~
19 Xylitol 10.0 ~ 67 L-Serine 0.1 m
20 D-Xylose 20.0 ~ 68 Taurine 0.1 m
21 Calcium chloride2.0 m 69 Thiamine-HCl (B1)5.0 ~
22 Magnesium chloride 1.0 m 70 L-Threonine 0.2 m
23 Sodium 71 Vitamin B 12 0.5 hydroqencarbonate 10.0 m 72 Vitamin U 1.0
24 Sërum albumin (human) 7.7 ~ 73 Adenine 50.0
25 LrAsparagine0.1 m 74 Folie acid (Bc) 5.0
26 L-Glutamine 1.0 m 75 Guanine 5.0
27 Adenosine 50.0 ~ 76 Guanosine 20.0
28 4-Aminobenzoic acid 2.0 ~77 ~ypoxanthine 5.0
29 L-Aspartic acid0.1 m 78 Rutin (Vitamin P) 5.0
30 D-Biotine (Vitamin ~) 1.0 ~ 79 Xanthine 5.0
31 Cytidine 50.0 ~ 80 Ethanol (60 ~l/l) 1.0 m
32 ~rGlutamic acid0.1 m 81 Cholesterol 1.0
33 L-Isoleueine 0.2 m 82 Ergocalciferol (D2) 0.5
34 5-Methylcytosine5.0 ~ 83 D,L-~-Lipoie aeid 2.0
35 L-Phenylalanine0.1 m 84 Menadione tK3) 0.2
36 Riboflavine- (B2)1.0 ~ 85 D,L ff-Tocopherol
37 Thymine t5-methyluraeil)5.0 ~ acetate (E) __ 1.0 U
38 L-Tryptophane50.0 ~ 86 Coenzyme
39 L-Tyrosine 0.1 m Q 10 ubiquinone 50 0.1
40 Uraeil 5.0 ~ 873-Phytylmenadione (K1) 0.2
41 Uridine 20.0 ~ 88 Retinol aeetate (A) 1.0
42 L-Leucine 0.2 m 89 Linolenie aeid (F) 5.0
43 L,Valine _0.2 m 90 Linoleie aeid (F) 1.O
44 Thymidine 20~0 ~ 91 Oleie aeid 5.0
45 Water 55.4 92 Penicillin G 80.0 ~
46 Hydrogen ions (p~ 7.1) 79.4 n 93 Streptomycin 80.0 ~_
47 Oxygen (air saturation) 0.2 m94 Activator(s) (CON A) 50.0 n _ -27~ 5 Dependent on the type of desired product, either mixed populations of leukocytes or homogenous leukocyte types are cultured. The preparation and culture of leukocytes must be performed under sterile conditions. Culturing is performed for a period sufficiently long to obtai'n a satisfactory medlator level. A suitable period of time is 10 to 50 hours. Shorter periods resul~ in lower mediator yields and the process is thus not economical. On the other hand, the medium is used up after a culture period oE 50 hours and the cells begin to die. An increase of the yield can therefore not be obtained in this case, except in the case of subculturing of cells and renewal of the culture medi~m.
The leukocytes are cultured at a temperature of about 30 to 42C, preferably at about 37C. At lower temperatures the culture process is not satisfactory, while at tempe-ratures of above 42C the leukocytes are damaged.
Culturing is carried out at a concentration of about 106 to 5 x 1 o8 cells/ml, preferably 107 to 10~ cells/ml. At lower cell concentrations the mediator yield per volume unit of the culture solution is too low. With too large culture volumes, the process is not economical. ~t cell concentrations cf above 5 x 108 cells/ml, nutrition of the cells in the medium becomes rapidly inefficient.
Culturing can be carried out in normal atmosphere. Pre-ferably increased carbon dioxide partial pressure is maintained during culturing. This presssure can amount to about 10 vol%. 2 vol% are preferred. The oxygen sup-ply to the culture is of great importance Oxygen can be supplied e~g. by bubbling air through the culture To avoid contamination of the culture, the air is pre-ferably sterilized and heat-decontaminated, i.e. it is freed of endotoxins and other organic constituents. The ~;
,:
cell suspension is stirred or agitated during culturing.
Certain types of the inventive ~ator prote~ are already obtained in satisfacto~y yields by normal culture of leu-kocytes or certain leukocyte types. The ~ , for instance, is obtained in high yields by culturing mixed populations of leukocytes or homogenous populations of granulocytes under the above-indicated conditions.
Other types of mediator proteins of the invention are how-ever only formed in small amounts by normal culture of leukocytes or certain leukocyte types. This applies for instance to the m~ator prote~ls of mononuclear cells~
To produce them in higher yields, it is necessary to stimulate the cells in culture to mitosis.
Possible mitosis-inducing influences are the addition of polyvalent mitogens, endotoxin-mitogens and immune reac-tions on the cel~l surface of sensitized cells. Examples of suitable mitogens are lectins, in particular those of Canavalia ensiformis (Concanavalin A = CON). The mito-sis-inducing factor CON is added as a solution to the culture medium.
To terminate culturing, the leukocytes are centrifuged from the supernatant culture solution which is subsequently processed for the resulting mediator proteins . To avoid damaging the cells and thus contamination of the culture solution with cell particles, the culture is centrifuged at relatively low speed, i.e. at about 300 to 400 x g.
After removal of the major part of the cells from the supernatant, it is expedient to centrifuge the lat-ter again at a higher speed. In this way, the remaining float-ing particles are removed. The separated leukoc~tes can either be cultured again, cryo-preserved or used for other biotechnical purposes.
~ y ~ s The supernatant culture solution freed from the c~lls contains the secretion products of the cultured leuko-cytes. ~hese include the me_iator proteins of the invention and a number of othex proteins and other substances. Their concentration in the culture solution is approximately within the nanomolar range~ Consequently, a yield of about 1 to 1G mg of a defined mediator requires a culture solution volume of about l,OOU 1 with respect to a 10~
recovery after purification. As regards the number of cells to be used, it can be calculated that in view of the molecular efficiency of the cells, about 1014 leukocytes are necessary ~or obtaining a quantity of about 100 nmol proteins.~This corresponds to about 1 mg of a mediator with the molecular weight of 10,000 dalton. This means that for the isolation of mediators in physical am~unts about 50 kg of leukocytes are necessary for the culture.
For reasons of availability, leukocytes of man, cow, horse, pig, she~p, dog, cat, rabbit, rat, mouse or guinea pig are preferred. The process described in the German unexamined patent publication DE ~ 30 09 12~ is especially suitable for the preparation of large amounts of leuko~
cytes; see also J.H. Wissler et al., Hoppe Seylerls z. f. Physiol. Che 361 (1980), p. 351 to 352~
Apart form leukocyte cultures, the mediator proteins of the invention ca~ also be obtained from inflamed tissue sites.
There, they are formed by the accumulation of leukocytes in the course of inflammatory processes induced by tissue injuries. The inflamed tissue can be obtained in the usual manner and used for the preparation of the mediator ~roteins.
Inflamed tissues are homogenized in bufEer solution and soluble constituents or exudates are separated from insol-uble structural components by means of centrifugation.
Preferably, inflamed, infarcted heart mucle tissue is used which was formed by ligatlon of 24 hours of the left '~3 ~8~L~5 anterior descendent branch of the left coronary artery by a transfemOral catheter technique. The leukocyte-contain-ing inflamed heart muscle site is separated at 0 to 4C
from the remaining non-infracted tissue.
As shown above, the preparation and isolation of the mediator proteins of the invention requires the processing of a very large culture solution volume. Therefore, at the be-ginning of the purification process effective reduction of the s~lution volume to be processed is necessary. In addi-tlon to the small amounts of the proteins produced, the cul-ture solution contains the mixture of the components of the medium. Preferably in the first step of the purifi-cation process a separation of the formed proteins from the medium components with a concomitant reduction of the large volume of aqueous solution is achieved. This can be effec-ted by selective salting-out precipitation of the proteins from the supernatant culture solution, for instance by adding a sulfate or a phosphate. In the following, the salting-out precipitation of proteins is exemplified b~
adding ammonium sulfate to the culture solution.
By saturation of the *upernatant culture solution-with ammo-nium sulfate, a major portion of the pro'eins formed is precipitated together with serum albumin present as medium component. The proteins precipitated are recovered e.g.
~y oentr~fu~ation. They`are then separated into the individual components of the mixture as described below. Thereby, some me~iator proteins are obtai~ed. On the other hand, some other mediator proteins are salt-soluble and remain in the supernatant solution of the salting-out precipi-tation process. This supernatant also contains all soluble components of the medium. It is concentrated and the pro-teins obtained are processed in the manner described below.
~f the protein-containing supernatant culture solution is saturated with ammonium sulate, a major portion of pro-teins is precipitated. In this way, a protein mixture is obtained consisting of numerous different proteins. Their separation into the individual protein components is obviously laborious. Therefore, in a preferred embodiment of the inventive process the protein mixture of the super-natant culture solution is already separated into several fractions by the salting-out of precipitation step. The separation into several crude protein fractions is possible, since groups of individual proteins precipitate at different ammonium sulfate concentrations. Preferably, in the process of the invention, ammonium sulfate is therefore added step-wise to the culture solution up to a specific degree of saturation. Each fraction contains a group of proteins, the solubility product of which corresponds to the range of salt saturation. Hence, in the process according to the invention a crude separation into groups of proteins can be achieved in this first step by suitable choice of the saturation limits.
For instance, the supernatant culture solution is first brought to a 35% saturation with ammonium sulfate. The protein precipitate obtained is separated off. The 35%
saturation of the supernatant solution is then increased to 45% by further addition of ammonium sulfate. A protein precipitate is again formed which is separated off. There-after, the 45% salt-saturated supernatant solution is brought to a 90~ ammonium sulfate saturation. The protein precipit-ate formed is again separated off. The supernatant solution of this precipitate is concentrated e.g. by dehydration dialysis or ultrafiltration.
The salting-out precipitation of proteins is preferably carried out at a temperature of about 0 to 10C, espec-ially of about 0 to 4C. The subsequent purification steps are performed under the same conditions. The sol--32~ 5 utions used for the purification have a pH value oE
between 5 and 9, in particular between 6 and 8. In order to achieve a constant pH-value of the solution, a strong buffer, for instance 0.1 mol/l of phosphate buffer is preferably add~d prior to the salting-out precipitation.
To maintain the redox potential of the proteins, cysteine is preferably added in an amount of 0.001 mol/l to all solutions throughout the process. The protein purification does not require sterile conditions.
After dissolution in a protein-compatible medium, the pro-teins obtained by salting-out precipitation can be directly subjected to purification and separation in the manner described below. The 90% salt-saturated supernatant of the last precipitation step is concentrated. For instance, by dehydration dialysis or ultrafiltration, all compounds having a molecular weight higher than about 300 to 500 aaltons are obtained as a retentate fraction. They can also be further processed for purlfication of salt-soluble chemo-recruitins.
The protein fractions obtained in the step described above contain the mediator proteins of the invention in admixture with numerous foreign proteins, e.g. other secreted pro-teins, in part serum albumins and in part CON. These foreign proteins form the major part of the c~nstituents of this mixture. The med;~tor r,roteins must be further purified by a sequence of further purification steps. Foreign proteins must be removed to avoid interference with the molecular-biological specifity of mediator proteins .
In addition, mediator proteins themselves form a class of protein compounds which must be separated into indi-vidual, specifically acting structures.
In general, purification processes for proteins and other natural substances comprise sequences of combined sepa-ration techniques. Subtle differences in molecular si~e, charge, form/structure stabili-t~ and nature of the mole-cular s ~ aces between the desired natural substance and the accompanying inactive foreign materials are used in such purification steps for -their separation. Accord.ingly~
a large number of combinations of various modifications of preparation techniques can be devised for the puriEi-cation of a protein. The nature and the conditions of the preparation steps usedj but also their sequential combination, are of paramount significance for operational properties, technical practicability, possibility of optional automatization and for the economical.performance of a purification process and also for the yield and mole-cular quality oE a natural product investigated~ Parti-cular attention has to be given to the optimum form of separation steps and on their ingenious combination.into a purification sequence within the framework of structural and functional stability and other molecular parameters of the substancç under investigation. This implies that the use ofidentical or similar separation principles (molecular sieve filtr~tion, dialysis, ion exchange adsorp-tion, etc.) - however in a different combination ~ can be of specific and paramount importance for the practice and economical performance of the purification process as w~ll as for the yield and quality of the product obtained.
In some cases, the use or o~ission of a single technique (.e.g hydroxyapaptite chromatography, zone precipitat.ion chromatography, etc.) at a certain point in the purifi-cation sequence or within a partial sequence~ i.s of decisive significance for the yield and quality of the desired natural product as well as for the practice anfl economical performance of the purification process. These general relationships and basic principles inherent to the puri-fication processes of natural products are clearly illus-trated e.g. by some well known facts. Thus, within an eco-nomically and technically operable process for the puri-fication of a natural product, initial dialysis, ultra-~B;~5 ! filtration or lyophilization steps are not recommended prior to reduction of orginial volumes of crude starting extracts by a factor of at least 500 to 1000 through other techniques.
For the purification of the individual protein fractions,a plurality of purification steps so far known in bio-chemistry can beused. Examples of such purification steps are: Preparative and analytical molecular sieve chroma-tography, anion and cation exchange chromatography and batch adsorption techniques, chromatography on hydroxy-apatite, zone precipitation chromatography and recycling or cascade molecular sieve filtration.
It is possible to remove a considerable amount of accom-panying foreign proteins from mediator proteins by only one performance of these p~rification methods. However, proteins contained in the fractions tend to adhere together very strongly. Therefore, for example, in spite of different molecular weights of proteins, using m~lecu~r sieve filtration, no complete~ideal) separation of pro-tein polyelectrolytes according to their exact mole-cular weight is obtained immediately. Hence it is neces-sary to perform at least two of the mentioned separation processes in sequence. A particularly preferred embodi-ment of the process in accordance with the invention uses three of the mentioned purification steps in sequence for the purification of mediator protein activity from the protein fractions.
All combinations of the mentioned separation steps consti-tute objeGts of the invention. It is evident, that certain sequences of separation steps are of less advantage than other combinations. Thus, for example, it is imperative to perform a preparative molecular sieve filtration before an analytical molcular sieve filtration: In reverse order of performance, difficulties in handling, economic effi-ciency and yield are obvious.
s Molecular sieve filtration achieves separation of proteins according to their molecular weights. Since the bulk of the foreign proteins have molecular weights differen-t from those of mediator proteins they can be separated off in this manner. A hydrophilic water-swelling molecular sieve as matri~ is used for separation of the proteins by molecular weight. Examples of suitable molecular sieve matrice~ are dextrans cross-linked with epichlorohydrin (SephadeX), agaroses cross-linked with acrylamides (Ultrogels), and three-dimensionally cross-linked acrylamides (Biogels).
The exclusion limits of the matrices used are higher thanthe separation limits.
If several separation steps are used, the molecular sieve filtration is preferably carried out as one of the first separation steps. Depending on the length-to-diameter ratio of the column used and the particle diameter of the gel matrix, molecular sieve filtration is termed "preparative"
or "analytical". A molecular sieve filtration is "prepara-tive" when the chromatography is performed on columns with a length-to-diameter ratio of up to 10:1 and a charge of the column of up to 1/3 of its capacity in terms of the total separation volume of the matrix. "Analytical n mole-cular sieve filtration means a length-to-diameter ratio larger than 10:1, and preferably about 50:1, and a maxi-mum charge of the column of up to 3~ of its capacity.
In preparative molecular sieve chromatography, gel matrices with the largest possible particle size are used for maxi-mum flow-through rates of mostly viscous protein solutions applied at reasonably low pressures. In analytical mole-cular sieve filtration the particle size ranges of the gel matrix are selected as small as possible, to obtain a maximum number of theoretical plates, a flow rate of the mobile phase in the range of 2 to 4 cm/h combined with a pressure which is limited to technical and safe-ty aspects.
These parameters are dependent on the structure of the gel I ro~de~
-36~ 8~S
matrix and may vary from gel to gel.
If several preparative molecular sieve filtrations are per-formed in sequence, graduated separation limits can be selected. This can be followed by an analytical molecular sieve filtration with correspondingly graduated separation limits. The exclusion limit of ~he gel used must in all cases be higher than about 10,000 daltons to allow-a volume distribution of mediator proteins between the stationary gel matrix phase and the mobile aqueous buffer phase.
..
The "exclusion limit" is a hydrodynamic parameter of a dis-solved particle, which corresponds to the pore size of the gel matrix. Particles with a greater hydrodynamic para-meter cannot penetrate the gel matrix (volume distribution coefficient KD = )- The "separation limit" refers to a hydrodynamic parameter which has been chosen for the sepa-ration of dissolved particles from others and which has a value of between the volume distribution coefficient = 0 and KD = 1-~or molecular sieve filtration, the proteins are appliedto the molecular sieve after dissolution in a protein-compatible liquid. A special example of a suitable solvent is 0.003 mol/l sodium-potassium phosphate solution contain-ing 0.3 mol/l NaCl and 0.001 mol/l cysteine and having a pH of 7.~. After filtration, the mediator protein -contain-ing fractions are concentrated in the manner described below and optionally subjected to a further purification step.
Examples of suitable anion exchangers are dextran matrices cross-linked with epichlorohydrin (Sephadex) or cellulose matrices carrying functional groups with anion exchanger capacity. These exchangers can be regenerated for repeated further use. It is preferable to use a weak anion exchan-ger in the Cl form such as DEAE-Sephadex A-50, pre-swollen ~3 _37- ~8~45 and equilibrated in a buffer. Swelling and equilibration is preferably carried out at a pH of 8 to 10. A special example of such a buffer solution is 0.01 mol/l tris-HCl contain-ing 0.04 mol~l NaCl and 0.001 mol/l cysteine and having a pH value of 8Ø
The anion exchanger is added to the protein fraction in an amount sufficient for complete adsorption of the mediator proteins and of the other positively adsorbing accompany-ing proteins. Two volume parts of swollen anion exchanger per volume of concentrated protein solution are normally sufficient. The reaction can be carried out either as chromatographic process or as an easy and fast batch adsorp-tion technique~. In the latter case, the supernatant liquid containing negatively adsorbed proteins is separated from the anion exchanger which is charged with the positively adsorbed mediator prvteins or other proteins, e.g. by fil-tration in a chromatographic column, by decantation or centrifugation. The charged anion exchanger is freed f~om adhering negatively adsorbing compounds by washing with water or a salt solution having a maximum ionic strength equivalent to 0.04 mol/l NaCl, preferably at a pH of 8 to 10.
The maximum preferred temperature is about 15C. A special example of salt solution suitable for the washing-out process is the tris-HCI buffer of pH 8Ø
The anion exchanger on which the mediator prote;ns and other pro-teins are adsorbed and which is freed from the negatively adsorbed compounds is eluted with a protein-compatible aqueous salt solution having an ionic strength higher than 0.04 mol/l NaCl and a pH of between 4.0 and 10Ø A salt solution of high ionic strength and a pH of between 5.0 ar-d 7.0 is preferably used. A special example of such a salt solution is a 2.0 mol/l NaCl solution buffered to a pH of 6.5 with 0.01 mol/l pipera~ine-HCl and containing ~ .
-38- ~ 188~5 0.001 mol/l cys-teine.
If the anion exchange reaction is carried out as a chroma-tographic process, elution of the mediator pro-teins and o-ther positively adsorbed proteins can also be done by a, linear NaCl concentration gradient.
~xamples of cation exchange matrices suitable for the puri-fication of the protein fraction are dextrans crosslin~ed with epichlorohydrin (Sephadex) or cellulose ma-trices carry-ing functional groups with cation exchange capacity. These can be readily regenerated after use and employed again.
It is preferable to use a weakly acidic cation exchanger such as CM-Sephadex C-50 having Na as mobile counter-ion, and to perform the exchange reaction at a pH between 4 and 6. To facilitate the charge process and to approach more idealequilibria conditions prior to treatment with the cation exchanger the protein fractions should be diluted with a protein-compatible salt solution hav~g a maximum ionic strength equivalent to 0.04 mol/l NaCl. This salt solution can be used at the same time to adjusi the pH.
A special example of a salt solution for this purpose is a 0.001 mol/l potassium phosphate-acetate buffer containing 0.04 mol/l NaCl and 0.001 mol/l cysteine and having a pH
of 4 to 6. This cation-exchange reaction may be performed as a chromatographic process, or technically easier, as a batch process.
The swollen cation exchanger is added to the protein frac-tion in a quantity sufficient to adsorb it. As a rule, about 2 volume parts of swollen ion exchanger per volume par-t of protein solution is sufficient for this purpose.
The supernatant is -then separated from the cation exchanger charged with proteins, for example by decantation or cen-tri~ugation. The charged cation exchanger is freed from adhering, negatively adsorbed compounds by washing with water or a salt solution, having a maximum ionic strength -39~ 1~8~5 equivalent to 0.04 mol/l NaCl. Preferably a pH of about 4 to 6 and a maximum temperature of about 15C is used. A
special example of a salt solution suitable for the washing out process is the mentioned potassium phosphate-acetate buffer having a pH of 5Ø
The washed protein-charged cation exchanger is now eluted with a protein-compatible aqueous salt solution. ~ salt solution of high ionic strength with a p~ of about 4 to 10 is preferably used for tihis purpose. Special examples of such salt solu-tions are aqueous 0.5 mol/l potassium phos-phate with a pH of 6.5 to 7.5 or a 2 to 5 mol/l NaCl with the same pH.
For chromatography on hydroxyapatite, salts, e.g. ammonium sulfate and especially phosphates, possibly present from preceding steps are removed from the protein solution, preferably by dialysis or ultrafiltration at membranes with an exclusion limit of 500daltons prior to the appli-cation of the proteins to hydroxyapatite. Apart from visco-sity increase by accompanying salts, however, only the phosphate concentration of the protein solution is critical for the chromatography on hydroxyapatite. The mediator pro-teins are eluted by a potassium phosphate concentration gradient which is preferably linear. The mediator protein containing fractions are collected and then concentrated in the manner described below.
The use of hydroxyapatite is of essential significance for the structure-conserving isolation of pure mediator pro-teins. However, in general, for technical and economic reasons, considerable difficulties arise from chromato-graphy of larger volumes of protein solutions on hydroxy-apatite columns. On the one hand, larger protein amounts contribute to the strong tendency of hydroxyapatite to clog, thus becoming unusable as stationary matrix in chromatography. On the other hand, hydroxyapatite is very -'I 0- 1~8B~S
expensive. Its use on larger scales is not economi.cal. For these reasons, in the process oE the invention, the sepa-ration of a large part of the accompanying foreign pro-teins by appropriate biotechnical purification steps from the mediator protein -containing protein fractions is preferred for considerably reducing the volume of the protein solu-tion prior to its chromatography on hydroxyapatite.
In the zone precipitation chromatography (cf. J. Porath, Nature, vol. 196 (1962); p. 47-48), residual protein con-taminations in the mediator proteins are separated by salt-ing-out fractionation o the proteins by means and along a salt concentration gradient. The basic principle of separation of proteins in zone precipitation chromato-graphy are differen-t, structure-related, reversible sol-ubility characteris-tics of proteins. They belong to the most sensitve molecular separation criteria and are often used for demonstration of molecular homogeneity of a pro-tein. Two variants of this technique or development o the chromatogram are known: Fractional precipitation zone chromatography and ractional elution zone chromatography.
Both types o techniques may have s~lective advantages in speciic cases as described for fractional precipitation and fractional elution methods in protein separation. Tem-perature and pH, column charac-teristics can all be varied within relatively wide limits.
The temperature or zone precipitation chromatography can be bet~eeen O and 40C. Preerably, a temperature range from about O to 10C is used, especially from about 4 to 6C. The pH canbe between 4 and 10; preferably, a pH range of 6 to 8 is used, especially a pH of about 7. The length~
to-diameter ratio of the column used should be greater than about 10:1. A ratio of 30 to 100:1 and especially of about 50:1 is preferred. All protein-compatible salts having salting-out proper-ties for proteins are suitable.
8~S
Examples of such salts are sodium-potassium phosphate, ammonium sulfate, and sodium sulfate. Ammonium sulfate is preferred.
The salt concentra-tion gradient can have any desired shape provided that salting-out criteria of proteins achieve protein separation. Linear concentration gradients are preferred, especailly an ascendent linear concen-tration gradient from 25 to 100% ammonium sulfate satu-ration. The maximum column charge is about 5% and pre-ferably about 1% of total column volume.
~he recycling or cascade molecular sieve filtration can be performed under -the conditions described above for the analytical molecular sieve filtration. The same mole-cular sieves and the same column conditions can be used.
Sephadex G 50 as stationary matrix is preferred in a column of a length-to-diameter ratio of at least about 50:1 and a maximum charge of about 3% of the column volume. rrhe solvents used in the analytical molecular sieve filtration are also preferred as solvents for the elution in this method.
In recycling molecular sieve filtration, the distribution equilibria are disturbed continuously and the eluate is recycled onto the same column with fixed separation limits.
In this way, the separation length of the migrating pro-tein distribution bands are differentially extended.
Alternatively, in cascade molecular sieve filtration, distribution equilibria are disturbed by continous trans-fer of the eluate into a new second column with the same or similar, defined pa,rameters at fixed separation limits.
~etween the above-described purification steps, and if necessary at any stage ~or special purposes, protein solu-tions can be separated and fre~d from unwanted salts and s water as well as concomitantly concentrated. The concen-tration (separation of a major portion of aqueous salt solution of the protein) can be achieved in different ways. Dehydration dialysis or ultrafiltration against protein-compatible liquid, preferably a sodium po~assium phosphate buffer, are such methods. Dehydration dialysis is carrried out preferably a~ainst polyethylene glycol (molecular weight 20,000 daltons~ at membranes with exclu-sion limites of preferably SOOdaltons. Ultrafiltration is preferably achieved at membranes with an exclusion limit of about 500 daltons Small amounts of protein ~recipi-tates formed are removed by intermediary centrifugation to result in a clear protein solution. A desalting mole-cular sieve filtration on matrices with appropriate separation and exclusion limits can as well be used for this purpose, e.g. on Sephadex G 10, G 15 or G 20 as matrices. Furthermore, by selecting an appropriate mobile phase in the usual way, a usual molecular sieve filtration step can also be used concomitantly for this purpose.
To prevent sulfhydryl group oxidation-, about 0.001 mol/l of cysteine is preferably added to protein solutions through-out.
In the molecular sieve ,iltration purification steps about 0.~ mol/l ammonium sulfate is preferably added to the pro-tein solution. In contrast to higher concentrations of thi.s salt, at this concentration ammonium sulfate exerts a strong salting-ineffect on proteins. Thus, proteins are better kept in solution during the molecular sieve fil-tration. Moreover, ammonium sulfate prevents growth of microorganisms and inhibits certain enzymes. Hence, it contributes to stabilization of the mediator protein struc-ture which is important when chromatography is performed at higher temperature (above about 20C~ and under non-sterile conditions.
-~r .~
88~S
-~3-Mediator proteins which can be salted ou-t are preferably completely precipitated alone or -toge-ther with accompany-ing proteins by adding ammonium sulfa-te up to a concentra-tion of about 3.25 to 3.7 mol/l (80 to 90% saturation). For this purpose 630 g/l ammonium sulfate are added (about 90% saturation). The pH value is preferably kept between 4 and 9 and the temperature up to 40C, preferably between O and BC.~he m~ia-tor protein-con-taining protein precipi.tate is separated from the protein-free supernatant solution by filtration, decantation or centrifugation. Unless otherwise stated, centrifugation is preferably carried out at least at 10,000 x g for a minimum of 45 min, and preferably for 1 h, in a one-step process. Or it can be carried out in two stages, at lower forces in the first stage for removal of the bulk of precipitated proteins;
and then, for the supernatant of the first stage contain-ing residual fine protein particles at higher forces, e.g.
20,000 to 50,000 x g, by flow-through centrifugation.
The temperature and pH conditions during performance of the purification steps are not particularly critical.
~f the native conformation of the protein is to be pre-served, an optimum temperature range is abou-t O to 8C, and preferably about O to 4~C. Moreover, the separation and purification steps must be carried out under essen-tially physiological pH and salt conditions. An essential advantage of the process of the invention consists in tha-t these condi-tions are for the first time easy to adhere to.
The mediator proteins ob-tained can be stored in a buffered physiological saline, e.g. in 0.0015 mol/l sodium-potas-sium phosphate solution containing 0.15 mol/l (0.9 w/v~) NaC1, O.OG1 mol/1 cysteine and having a pFI of 7.4. After usual sterilization by filtration (pore diameter 0.2 ~m), the protein preparation remains native and biologically -4~-active a-t room tempera-ture for at least 200 h or frozen a-t -25C for atleas-t 5 years. This stability of the pro-tein can be considered, among others, to be one of the criteria of molecular homogeneity. Medj~tor Proteln solutions are safely s-tored at temperatures of between -20 and +50C i.n the presence oE 2.0 to 3.6 mol/l ammonium sulfate (50 to 90 % saturation). At this high osmoti.c pressure mediator protei.n solutions are protected against infection and degradation by microorganisms and bacterial growth.
For their physiological, therapeutical and any other use, the mediator proteins are again freed from salts by dia-lysis or ultrafiltration against an appropri.ate saline as described above.
The invention will now be given in detail by examples describing the isolation of the mediator protein preparation starting from leukocytes of porcine blood.
However, the invention is not restricted to this embodi-ment. Leukocytes and inflanedtissues of other mammalians can be used too.
Example A
PREPARATION OF CHEMOKINESINS AND CHEMOTAXINS FROM SUPERNA-NATANTS OF CULTURES OF A MIXED POPULATION OF VIABLE LEUKOCYTES
The production of chemo]cinesins and chemotaxins in a culture solution of a mixed population of leukocytes and the separation of lymphocyto-monoapokinesin (L~), monocyto-granuloapokinesin (MGAK), monocyto-granuloproskinesin (MGPK), lymphocyto-monoproskinesin (LMPK), monocyto- ;
granulotaxin (MGT), granulocyto-monotaxin.(GMT) and monocyto-eosinotc~xin (MET) from the o-ther components of the culture supernatant are descri.bed. All process steps are carri.ed out at 0 to ~C
in the presence of 0.001 mol/l cysteine, unless otherwise -~5~ ~ l 882 ~ 5 speci~ied. The centrifugation is carried out in the manner described , either as a one or two step procedure (as flow-through centrifugation).
A 1 Preparation and cul-ture of a mixed population of ~iable leukocytes 50 kg (about 10 ) leukocytes are isolated as mlxed cell population of physiological composition from 10,000 l of porcine blood and cultured in 20 batches of 2.5 kg (about S x 1012 cells) under sterile conditions. The medium indi-cated in tableVIII is used as culture solution. 50 1 of cul-ture medium are used per batch. Culturing is performed in glass vessels (Duran 50 or Pyrex glass). Initially, the cell density is a~out 1o8 cells/ml. -~he culture is maintained at 37C in an atmosphere of 1 v/v % CO2 over ~0 hours. During this period, the cell suspension is slowly stirred (to r.p.m.) and flooded with sterile, water-washed and heat-decontaminated air bubbles (C1mm).
The heat~decontamination of air is performed at about 500C by flowing through a silica tube- In addition to the partial oxygen pressue, the pH value (7.1) and the D-glucose level are measured and maintained constant. During culturing, the cells are induced to mitosis by the polyvalent mitogen eontent (CON) of the culture medium. The number, differential and morpholo-gical viability (dye exclusion test) of the cells are continously determined by usual methods of hematology and cell culture techniques. The functional viability of cells is measured by their motility and their ability to respond to chemokinetic and chemotactic proteins.
Mitoses are determined by chromosome count. The morpho-logical viability of the cells after their biotechnical culturing is 95%. The entire loss in cells (mainly granu-ocytes) during eulturing is at most 20~ which is normal ~or primary cell cultures.
- 4 6 ~ 5 The culture is terminated by separating the cells from the supernatant solution by centrifugation for 10 minutes at 400 x g and 10C. The cells are washed twice in a salt solution containing 0.15 mol/l NaCl, 0.0015 mol/l sodium potassium phosphate and having the pH-value 7.1. They can be used for ano~her purpose.
The culture supernatant solution is then centrifuged again for 1 hour at 10,000 x g and at 4C to remove sus-pended particles. The resuLtant clear supernatant culture solution which has a total volume of 1000 liters and con-tains about 1,400 g protein as well as other macromole-cules and salts is directly subjected to salting-out frac-tionation with ammonium sulfate (A2). Unless other~ise stated, all further steps are carried out at 0-4C.
.
A.2. First purification step (salting-out fractionation):
Preparation of crude protein concentrate fractions.
-0.5 mol/l sodium-potassium phosphate buffer solution with a pH value of 6.7 is added to the supernatant culture solution (~ 1) up to a final concentration cf 0.1 mol/l.
Furthermore, solid L-cysteine is added up to a concen-tration of 0.001 mol/l.
This buffered supernatant culture solution is then adjusted to 35% saturation of ammonium sulfate by addition of 199 g of ammonium sulfate/l solution. During the addition, the pH-value of the protein solution is continuously controlled and maintained at 6.7 by the addition of 2 n ammonia. Part of the proteins is precipitated from the solution. The protein precipitate formed is separated from the super-natant containing salt-soluble proteins by centrifugation for 1 hour at 10,000 x g. The precipitated crude protein -~7-fraction I is obtained as ammonium sulfate-containing pro-tein sludge which contains about 100 g protein. This crude protein concentrate fraction I may separately be processcd for its constituents according to the procedure described below for the crude protein concentrate fraction III.
t Then the 35% salt-saturated supernatant cul~ure solution is adjusted to ~5~ saturation of ammonium sulfate by add-ing 60 g of ammonium sulfate/l solution. The pH ~alue of the protein solution is continuously controlled-and maintained constant at 6.7 by 2 n ammonia. Another portion of proteins is precipitated from the solution. The protein precipitate is separated from the supernatant containing salt-soluble proteins by centrifugation for 1 hour at 10,000 x g. The precipitated crude protein concentrate fraction II is obtained as ammonium sulfate-containing protein sludge, ~he protein content of which is about 60 g.
This crude protein concentrate fraction II may be processed separately for its constituents , according to the procedure described below for the crude protein concentrate frac-tion III.
The 45~ salt-saturated supernatant culture solution is then adjustedt 90~ saturation of ammonium sulfate by add-ing 323 g ofam~onium sulfate/l of solution. The pH-value of the protein solution is again continuously controlled and maintained constant at 6.7 by 2 n ammonia. Another portion of the proteins is precipitated from the solution.
The protein precipitate is separated from the supernatant containing salt-soluble proteins by centrifugation for 1 hour at 10,000 x g. The precipitated crude protein con-centrate fraction III is obtained as ammonium sulfate-containing protein sludge the protein content of which is approximately 1,080 g. This fraction also contains the bul~ of the serum albumin as component of the culture medium. This crude protein concentrate fraction III
~"`3 .......
-~8- ~ 45 contains the mediator proteins of the invention and is processed according to the procedure described below.
The 90~ salt saturated supernatant fraction IV of the crude fraction III con-tains 160 g of salt-soluble pro-teins and other macro molecules ( ~ 500 daltons)~ It may also be processed for its constituen-ts.
A.3. Fine purification of mediator prote~s in the c~ude ~ tein concentrate fraction III
A~3.1.Anion exchange chromatography The crude protein concentrate fraction III obtained above ~A 2) is dissolved in a minimum volume of buffer solution B (0.01 mol/l of tris~HCl solution coniaining 0.04 mol/l NaCl and 0.001 mol/l cys~ine and having a pH value of 8.0). The resultant slightly turbid solution (20 l) is clarifled by centrifugation and then freed of salts by dialysis at a membrane with the exclusion limit of 500 dalton against buffer solution B until no sulfate ions are detectable. The clear solution obtained is then applied to a column of a swollen regenerated anion exchan- -ger (Cl as mobile exchangeable ion). It has a dextran matrix cross-linked with epichlorohydrin (DEAE-Sephadex A 50) which is equilibrated in the above-mentioned buffer system B.
:
8;~S
_~9_ The column has four -times the volume of the protein solu-tion and a length-to-diameter ratio of 10 : 1. The gel column is then washed with the above-mentioned adsorp-- tion buffer solution B until the extinction of the fil~
trate at 280 nm is c 1 0 .
In this puriication step, chemokinesins are separated rom chemotaxins, because the former are adsorbed where-as the latter flow through.
For elution of the chemok~nesins and the adsorbed pro-teins, the charged ion exchanger gel is eluted with a NaCl-concentratio~ gradient during 2 days. he gradient is linearly ascending from 0.04 to 2.0 mol/l ~aCl, whereas the pH value, the tris/HCl and tne cysteine concen- -tra~ions are maintained constant. The same shape of gra-dient is then used for lowering the pH from 8 to 6.5 for fur~her elution of the compounds. It is made up by 0.01 mol/l piperacine-HCl-buffer containing 200 mol/l NaCl and 0.001 mol/l cysteine and having the-pH 6.5.
~he chemokinesin or chemotaxin-containing fractions are collected separately. They are separately processed in further purification steps described below (A~3.2 - A.3.6)~
A.3.2. Preparative molecular sieve filtration After concentration of the proteins in the fractions ~A.3.1l by salting-out precipitation with ammonium sul-ate, the protein precipitate containing either chemokinesins or chemotaxins is dissolved in a minimum volume o buffer solution C
(0.003 mol/l sodium-potassium phosphate containing .
.
-50- ~ 3~ 5 0.3 mol/l NaCl and 0~001 mol/l cysteine and having a p~
value of 7.4). A~ter removal of a small amount of insol-uble compounds by centrifugation., the solution is applied to a column of a molecular sieve matrix of agarose cross-linked with acrylamide (Ultrogel AcA 34, particle size 60 to 160 ~m) for preparative molecular sieve filtration:
The column has 10 times the volume of the protein solution and a length-to-diameter ratio of 20:1. The column is then eluted with an upward flow (3 cm/h) of the mentianed buffer solution C For chemokinesins, the frac-tion with the separation lu~ts of 25,000 and 6,000 daltons and for chemotaxins, the fraction with the separation limits of 20,000 and 3,000 daltons are collected. For the concentration of the proteins, the frac-tions are lyophilized and ultrafiltered at ane~brane with the exclusion limit of 500 dalton or are adjusted to an ammonium sulfate concentration of 3.7 mol/l. In this case, the protein precipitates are separated from the super-natant by centrifugation and f~rther processed.as des-cribed below (~.3.3).
A.3.3 Cation.exchange chromatography The resultant che~.o~inesins or chemotaxIns-containing protein precipitates (A 3.2) are dissol~ed in 1.5 volume parts of buffer sol-ution D (0.01 mol~ sodium-potassium phosphate, 0.04 mol/l NaCl, 0.001 mol/l cysteine~pH 6.0). The.solutions are cen-trifuged at 10,000 x g for 1 hour for removal of a small amount of insoluble material.
The clear solution is dialyzed against the bu~fer solution D
at a membrane wi~h the exlusion limit of 500 daltons until no sulfate îons are detectable. The clear solution obtained is then applied to a column of swollen, regenerated cation exchanger based on a dextran matxix cross-linked with epichlorahydrin (CM-Sephadex C 50j. The exchanger is equili-brated in the above-mentioned buffer system D (Na as mobile ~38~5 exchangeable ion).
The column has four times the volume of theprotein solution and a length-to-diame-ter ratio of 10 : 1. The gel~column is then washed with the above-mentioned adsorption buffer solution D, ~til the extinction of the filtrate at 280 nm is - 1Ø
For elution of the me~iator proteins and the adsorbed pro-teins, the charged ion exchange gel is eluted with an NaCl-concentration g~adient during 2 days. The gradient is linearly ascending from 0.04 to 2.0 mol/l NaCl whereas the pH-value and the phosphate and cysteme concentrations are maintained constant. For further elution, the same shape of gradient is then used for increasing the phos-phate concentration from 0.01 to 0.5 mol/l at a p~ of 8.0,whereas the NaCl (2 rlol/l) and~cysteine concentrations are kept constant.
The chemokinesins or chemotaxins-containing fractions are collected and concentrated in the usual manner and further processed as described below (Ao3~4~
A.3.4 Chromato~_aphy on hydrox~vapatite The chemokinesins or chemotaxins-containing protein preci-pitates (A.3.3) are dissolved in a minimum volume of 0.0001 mol/l sodium-po-tassium p~losphate bufEer solution E containing 0.001 mol/l cysteine and having a pH of 7.20. The solutions are then desalted with this buffer by molecular sieve fil-tration, ultrafiltration or dialysis (exclusion limit 500 dalton), until no sulfate is detectable in the dia-lysis buffer. Thereafter, a small portion of insoluble material is removed by centrifugation a~ 10,000 x g for 1 hour.
' -52- ~882~5 The clear chemo~inesins or c~lemotaxins-containing protein solutions ~bbtained are separately applied to a column of hydroxyapatite. The l~ngth-to-diameter ratio of the column is 10 : 1 and it has four times the volume of theprotein volume to be applied.
The column has been equilibrated with the mentionea buffer E u~ed in an amount five times the column volume (flow 3 cm/h).
The negatively adsorbed proteins are washed out with the buffer solution E used for equilibrating:the column. The elution of the mediator pro-tein -containing fractions is '!
caxried out with a phosphate concentration gradient for 4 days~ The gradient is linearly ascending from 0.0001 mol/l to 0.5 mol/l sodium-potassium phosphate having a constant pH value of 7.4 and constant cysteine concentration.
The different mediator proteins are separated in this step.
LMAK is eluted at an average phosphate concentration of about 0.04 mol/l, MGAK at about 0.001 mol/l, MGPK at about 0~005 mol/l~ LMPX at about O.OB mol/l. In the chromatogram of the chemotaxins MGT appears at an average phosphate concentration of about 0.004 mol/l, MET at about 0,05 mol/l and GMT at about 0,2 mol/l. The elution gradient is measured and con~
trolled by means of conductivity. The mediator protein -con-taining fractions are concentrated in the usual manner and further processed as described below (A.3.5).
~18~5 A.3 5. Zone precipitation chromatography The mediator protein-containing fractions (A.3.4) are dissolved in 0.1 mol/l sodium-potassium phosphate solution ~ con-taining 0.1 mol/l NaCl, 0.001 mol/l cysteine and 1 mol~l ammonium sulfate and having a pH value of 7.4. The resultant solution is applied at a tempera-ture of 4C to a column of swollen m~lecular sieve matrix of dextran cross-linked with epichlorhydrin tSephadex G-25). In the matrix, an ascendent, linear ammonium sulfate concentration gradient is estab-lished with the mobile buffer phase from 1.0 to ~.0 mol/l ammonium sulfate ~25 to 100% saturation). The slope of the gradient is +2% of the ammonium sulfate saturation/cm of column height (0.08 mol/l (NH432SO4/cm). The range of the gradient ex~ends over approximately half the length of the column.
. .
The len~th-to-diameter ratio of the column is 50 : 1, the column volume is 100 times higher than the protein solution volume to be applied~ The flow rate is 2 cm~h.
The elution is carried ou~ with the above-mentioned sodium-potassium phosphate solution F containing 1 mol/l o~ ~mmo~
nium sulfate. The mediator prOtein-containing fractions which are ~luted at 65% l~), 77% (MGAK), 72 % (M~PK), 62% I~K), 70~ IMGT~, 57~ (GMT) and 80% (MET) ammonium sulfate sa-turation, respect~ively, are collec-ted. The proteins are concentrated in the usual manner and further processed as described below (A.3.63.
-5~ 4~
A.3.6. Analytical recycling molecular sieve filtration The medi.a-tor protein-containing fractions (A.3.5) are dissolved in buffer C (0.003 mol/l sodium-potassium phospha~e con-taining 0,3 mol/l NaCl and 0.001 mol/l casteine:.and having a pH value of 7.4). Removal of a small portion of insol-uble substances is achieved by centrifugation for 3 minutes at 48,000 x g.
The resultant clear solu-tion is then subjected to . ana-lytical recycling molecular sieve chromatographyO For this purpose, the solution is applied at a temperature of 4C
to a column o Ultrogel AcA 44 having a particle size of 60 to 140 ~m. The column has 50 times the volume of the pro~
tein solution and a length-to-diameter ratio of 50 : 1 The elution is carried out with the mentioned buffer C~
The eluates are recycled three times at separation limits of either 17,000 dalton (LMAK), 12rOOO dalton (MGAK~, 19,000 dalton (MGPK~, 25/OOO dalton (LMPK), 13,000 dalton (MGT), 20,000 dalton (GMT~, or 8,000 dalton (MET).
After usual protein concentration, approximately 3 mg of LMAK, 5 mg of MGAK, 5 mg of MGPK, 4 mg of LMPX, 6 mg of I MGT, 9 mg of GMT and 5 mg of MET are ob-tained. The chemo-i kinesins and chemotaxins have a molecular homogeneity of 95%, as indicated by conventional methods.
In the following flow sheet the above-described process for preparing and isolating the chemokinesins and chemotaxins of the invention is schematjcally represented.
~î8~245 X
W
U~
H
tn z K
0~
O
p:; ~ p, H
a ~ ~ z ~ z O ~ O O ~ z H Z
~; C) S~ ~ Z
~ D '3 ~ ~ ~ O ~ C~ H ~ ~ Z ~
O W ~ O ~ ~ ~ V H . ~ Z Z ~; Z Z Z
o ~, P; o ~: ~ x H ~ r 2 ~c ~ a ¦ Z ~ ~ 2 ~ o A
W H CL~ E~ V O ~ ~1 0 c~
. v ~ ~ ''¢ m ~ ~ O zO ~ O ~ O
H , Z ~
O ' ' m ~ u~.... . .
;I~ , ' .
u~ . i ,~
.
4~
E x a m p 1 e B
PREPARATION OF ~ I~R PRarEINS FROM SUPERNATANTS OY CUL--TURES OF VIABLE ~YMPHOC~3 ~ 5 kg (about 3 x 1013~ lymDhocytes obtained from po,rcine blood are cultured unde~ the conditions described in example A. During culture, the polyvalent mitogen (CON) in the medium induces the mitosis of the cells.
The chemokinesins LMAK and LMPK secreted ~o the cul~ure -solution are isolated according to the procedure des~
cribed in example A. They are thereby ob;tained in a highly purified state. The yields obtained are comparable to those of example A.
E x a m p l e C
PREPARATION OF MEDIATOR PROTEINS FROM SUPERNATANTS OF
CULTURES OF VIABLE MONOCYTES
Example s is repeated with a culture of 3,5 kg (about 7 x 1o12) monocytes. The chemokinesins and chemotaxins of the monocytes are obtained in yields as in example A.
E x a m p l e D
PREPARATION OF ~DIA~OR PRCTEI~S FROM INFLAMED TISSUE SITES
The preparation and isolation of chem~kinesins and ch~mo~ins from inflamed tissue are described. 500 g of infarcted, inflamed canine heart muscle tissue are used. The heart muscle tissue is ground at 0-4C. 0.0~ mol/l sodium potassium phos-phate buffer solution containing 0.001 mol/l cysteine and having a pH of 6.~ is added in a quantity three times the amount of the tissue. The resultant suspension is homoge-4~
nized in a homogenizer (ultraturax). Thereafter, the super-slatant containiny the soluble compounds of the inflamed tissue is separated from the insoluble constituents by cen-triguation at 10,000 x g and 4C. The resultant supernatant solution is then centrifuged ~or 3 hours at 100,000 x ~.
The clear supernatant solution obtained is siphoned of~ from the flotating lipid layer.
The rnedi.ator protein-con-taining clear supernatant protein solution is then subjected to fractional salting-out preci-pitation with ammonium sulfate according -to example A. The resultant protein fracti.on III is then processed as des-cribed in example A. The yields, as compared to example A, are about 50% with the pro-teins from monocytes and granu-locytes and only about 10% with the proteins from lympho-cytes.
E x a m p l e E
PREPARATION OF 1~'0~ PROTEINS FROM LEUKOCYTE HOMO-GENATES
Leukocytes are prepared from blood according to example A. A homogenate of 500 g of leukocytes is prepared as shown in example D for muscle tissue. The isolation of the protein mediators contained in -the leukocy-tes is performed according to example A. The leukocytes cultured w.ithout stimulation contain only relatively small (about 1~) amounts of monocyte- and lymphocyte- chemo-kinesins and -chemotaxins, whereas the GMT is contained in a yield of about 10 % compared with example A.
The leukocytes are cultured at a temperature of about 30 to 42C, preferably at about 37C. At lower temperatures the culture process is not satisfactory, while at tempe-ratures of above 42C the leukocytes are damaged.
Culturing is carried out at a concentration of about 106 to 5 x 1 o8 cells/ml, preferably 107 to 10~ cells/ml. At lower cell concentrations the mediator yield per volume unit of the culture solution is too low. With too large culture volumes, the process is not economical. ~t cell concentrations cf above 5 x 108 cells/ml, nutrition of the cells in the medium becomes rapidly inefficient.
Culturing can be carried out in normal atmosphere. Pre-ferably increased carbon dioxide partial pressure is maintained during culturing. This presssure can amount to about 10 vol%. 2 vol% are preferred. The oxygen sup-ply to the culture is of great importance Oxygen can be supplied e~g. by bubbling air through the culture To avoid contamination of the culture, the air is pre-ferably sterilized and heat-decontaminated, i.e. it is freed of endotoxins and other organic constituents. The ~;
,:
cell suspension is stirred or agitated during culturing.
Certain types of the inventive ~ator prote~ are already obtained in satisfacto~y yields by normal culture of leu-kocytes or certain leukocyte types. The ~ , for instance, is obtained in high yields by culturing mixed populations of leukocytes or homogenous populations of granulocytes under the above-indicated conditions.
Other types of mediator proteins of the invention are how-ever only formed in small amounts by normal culture of leukocytes or certain leukocyte types. This applies for instance to the m~ator prote~ls of mononuclear cells~
To produce them in higher yields, it is necessary to stimulate the cells in culture to mitosis.
Possible mitosis-inducing influences are the addition of polyvalent mitogens, endotoxin-mitogens and immune reac-tions on the cel~l surface of sensitized cells. Examples of suitable mitogens are lectins, in particular those of Canavalia ensiformis (Concanavalin A = CON). The mito-sis-inducing factor CON is added as a solution to the culture medium.
To terminate culturing, the leukocytes are centrifuged from the supernatant culture solution which is subsequently processed for the resulting mediator proteins . To avoid damaging the cells and thus contamination of the culture solution with cell particles, the culture is centrifuged at relatively low speed, i.e. at about 300 to 400 x g.
After removal of the major part of the cells from the supernatant, it is expedient to centrifuge the lat-ter again at a higher speed. In this way, the remaining float-ing particles are removed. The separated leukoc~tes can either be cultured again, cryo-preserved or used for other biotechnical purposes.
~ y ~ s The supernatant culture solution freed from the c~lls contains the secretion products of the cultured leuko-cytes. ~hese include the me_iator proteins of the invention and a number of othex proteins and other substances. Their concentration in the culture solution is approximately within the nanomolar range~ Consequently, a yield of about 1 to 1G mg of a defined mediator requires a culture solution volume of about l,OOU 1 with respect to a 10~
recovery after purification. As regards the number of cells to be used, it can be calculated that in view of the molecular efficiency of the cells, about 1014 leukocytes are necessary ~or obtaining a quantity of about 100 nmol proteins.~This corresponds to about 1 mg of a mediator with the molecular weight of 10,000 dalton. This means that for the isolation of mediators in physical am~unts about 50 kg of leukocytes are necessary for the culture.
For reasons of availability, leukocytes of man, cow, horse, pig, she~p, dog, cat, rabbit, rat, mouse or guinea pig are preferred. The process described in the German unexamined patent publication DE ~ 30 09 12~ is especially suitable for the preparation of large amounts of leuko~
cytes; see also J.H. Wissler et al., Hoppe Seylerls z. f. Physiol. Che 361 (1980), p. 351 to 352~
Apart form leukocyte cultures, the mediator proteins of the invention ca~ also be obtained from inflamed tissue sites.
There, they are formed by the accumulation of leukocytes in the course of inflammatory processes induced by tissue injuries. The inflamed tissue can be obtained in the usual manner and used for the preparation of the mediator ~roteins.
Inflamed tissues are homogenized in bufEer solution and soluble constituents or exudates are separated from insol-uble structural components by means of centrifugation.
Preferably, inflamed, infarcted heart mucle tissue is used which was formed by ligatlon of 24 hours of the left '~3 ~8~L~5 anterior descendent branch of the left coronary artery by a transfemOral catheter technique. The leukocyte-contain-ing inflamed heart muscle site is separated at 0 to 4C
from the remaining non-infracted tissue.
As shown above, the preparation and isolation of the mediator proteins of the invention requires the processing of a very large culture solution volume. Therefore, at the be-ginning of the purification process effective reduction of the s~lution volume to be processed is necessary. In addi-tlon to the small amounts of the proteins produced, the cul-ture solution contains the mixture of the components of the medium. Preferably in the first step of the purifi-cation process a separation of the formed proteins from the medium components with a concomitant reduction of the large volume of aqueous solution is achieved. This can be effec-ted by selective salting-out precipitation of the proteins from the supernatant culture solution, for instance by adding a sulfate or a phosphate. In the following, the salting-out precipitation of proteins is exemplified b~
adding ammonium sulfate to the culture solution.
By saturation of the *upernatant culture solution-with ammo-nium sulfate, a major portion of the pro'eins formed is precipitated together with serum albumin present as medium component. The proteins precipitated are recovered e.g.
~y oentr~fu~ation. They`are then separated into the individual components of the mixture as described below. Thereby, some me~iator proteins are obtai~ed. On the other hand, some other mediator proteins are salt-soluble and remain in the supernatant solution of the salting-out precipi-tation process. This supernatant also contains all soluble components of the medium. It is concentrated and the pro-teins obtained are processed in the manner described below.
~f the protein-containing supernatant culture solution is saturated with ammonium sulate, a major portion of pro-teins is precipitated. In this way, a protein mixture is obtained consisting of numerous different proteins. Their separation into the individual protein components is obviously laborious. Therefore, in a preferred embodiment of the inventive process the protein mixture of the super-natant culture solution is already separated into several fractions by the salting-out of precipitation step. The separation into several crude protein fractions is possible, since groups of individual proteins precipitate at different ammonium sulfate concentrations. Preferably, in the process of the invention, ammonium sulfate is therefore added step-wise to the culture solution up to a specific degree of saturation. Each fraction contains a group of proteins, the solubility product of which corresponds to the range of salt saturation. Hence, in the process according to the invention a crude separation into groups of proteins can be achieved in this first step by suitable choice of the saturation limits.
For instance, the supernatant culture solution is first brought to a 35% saturation with ammonium sulfate. The protein precipitate obtained is separated off. The 35%
saturation of the supernatant solution is then increased to 45% by further addition of ammonium sulfate. A protein precipitate is again formed which is separated off. There-after, the 45% salt-saturated supernatant solution is brought to a 90~ ammonium sulfate saturation. The protein precipit-ate formed is again separated off. The supernatant solution of this precipitate is concentrated e.g. by dehydration dialysis or ultrafiltration.
The salting-out precipitation of proteins is preferably carried out at a temperature of about 0 to 10C, espec-ially of about 0 to 4C. The subsequent purification steps are performed under the same conditions. The sol--32~ 5 utions used for the purification have a pH value oE
between 5 and 9, in particular between 6 and 8. In order to achieve a constant pH-value of the solution, a strong buffer, for instance 0.1 mol/l of phosphate buffer is preferably add~d prior to the salting-out precipitation.
To maintain the redox potential of the proteins, cysteine is preferably added in an amount of 0.001 mol/l to all solutions throughout the process. The protein purification does not require sterile conditions.
After dissolution in a protein-compatible medium, the pro-teins obtained by salting-out precipitation can be directly subjected to purification and separation in the manner described below. The 90% salt-saturated supernatant of the last precipitation step is concentrated. For instance, by dehydration dialysis or ultrafiltration, all compounds having a molecular weight higher than about 300 to 500 aaltons are obtained as a retentate fraction. They can also be further processed for purlfication of salt-soluble chemo-recruitins.
The protein fractions obtained in the step described above contain the mediator proteins of the invention in admixture with numerous foreign proteins, e.g. other secreted pro-teins, in part serum albumins and in part CON. These foreign proteins form the major part of the c~nstituents of this mixture. The med;~tor r,roteins must be further purified by a sequence of further purification steps. Foreign proteins must be removed to avoid interference with the molecular-biological specifity of mediator proteins .
In addition, mediator proteins themselves form a class of protein compounds which must be separated into indi-vidual, specifically acting structures.
In general, purification processes for proteins and other natural substances comprise sequences of combined sepa-ration techniques. Subtle differences in molecular si~e, charge, form/structure stabili-t~ and nature of the mole-cular s ~ aces between the desired natural substance and the accompanying inactive foreign materials are used in such purification steps for -their separation. Accord.ingly~
a large number of combinations of various modifications of preparation techniques can be devised for the puriEi-cation of a protein. The nature and the conditions of the preparation steps usedj but also their sequential combination, are of paramount significance for operational properties, technical practicability, possibility of optional automatization and for the economical.performance of a purification process and also for the yield and mole-cular quality oE a natural product investigated~ Parti-cular attention has to be given to the optimum form of separation steps and on their ingenious combination.into a purification sequence within the framework of structural and functional stability and other molecular parameters of the substancç under investigation. This implies that the use ofidentical or similar separation principles (molecular sieve filtr~tion, dialysis, ion exchange adsorp-tion, etc.) - however in a different combination ~ can be of specific and paramount importance for the practice and economical performance of the purification process as w~ll as for the yield and quality of the product obtained.
In some cases, the use or o~ission of a single technique (.e.g hydroxyapaptite chromatography, zone precipitat.ion chromatography, etc.) at a certain point in the purifi-cation sequence or within a partial sequence~ i.s of decisive significance for the yield and quality of the desired natural product as well as for the practice anfl economical performance of the purification process. These general relationships and basic principles inherent to the puri-fication processes of natural products are clearly illus-trated e.g. by some well known facts. Thus, within an eco-nomically and technically operable process for the puri-fication of a natural product, initial dialysis, ultra-~B;~5 ! filtration or lyophilization steps are not recommended prior to reduction of orginial volumes of crude starting extracts by a factor of at least 500 to 1000 through other techniques.
For the purification of the individual protein fractions,a plurality of purification steps so far known in bio-chemistry can beused. Examples of such purification steps are: Preparative and analytical molecular sieve chroma-tography, anion and cation exchange chromatography and batch adsorption techniques, chromatography on hydroxy-apatite, zone precipitation chromatography and recycling or cascade molecular sieve filtration.
It is possible to remove a considerable amount of accom-panying foreign proteins from mediator proteins by only one performance of these p~rification methods. However, proteins contained in the fractions tend to adhere together very strongly. Therefore, for example, in spite of different molecular weights of proteins, using m~lecu~r sieve filtration, no complete~ideal) separation of pro-tein polyelectrolytes according to their exact mole-cular weight is obtained immediately. Hence it is neces-sary to perform at least two of the mentioned separation processes in sequence. A particularly preferred embodi-ment of the process in accordance with the invention uses three of the mentioned purification steps in sequence for the purification of mediator protein activity from the protein fractions.
All combinations of the mentioned separation steps consti-tute objeGts of the invention. It is evident, that certain sequences of separation steps are of less advantage than other combinations. Thus, for example, it is imperative to perform a preparative molecular sieve filtration before an analytical molcular sieve filtration: In reverse order of performance, difficulties in handling, economic effi-ciency and yield are obvious.
s Molecular sieve filtration achieves separation of proteins according to their molecular weights. Since the bulk of the foreign proteins have molecular weights differen-t from those of mediator proteins they can be separated off in this manner. A hydrophilic water-swelling molecular sieve as matri~ is used for separation of the proteins by molecular weight. Examples of suitable molecular sieve matrice~ are dextrans cross-linked with epichlorohydrin (SephadeX), agaroses cross-linked with acrylamides (Ultrogels), and three-dimensionally cross-linked acrylamides (Biogels).
The exclusion limits of the matrices used are higher thanthe separation limits.
If several separation steps are used, the molecular sieve filtration is preferably carried out as one of the first separation steps. Depending on the length-to-diameter ratio of the column used and the particle diameter of the gel matrix, molecular sieve filtration is termed "preparative"
or "analytical". A molecular sieve filtration is "prepara-tive" when the chromatography is performed on columns with a length-to-diameter ratio of up to 10:1 and a charge of the column of up to 1/3 of its capacity in terms of the total separation volume of the matrix. "Analytical n mole-cular sieve filtration means a length-to-diameter ratio larger than 10:1, and preferably about 50:1, and a maxi-mum charge of the column of up to 3~ of its capacity.
In preparative molecular sieve chromatography, gel matrices with the largest possible particle size are used for maxi-mum flow-through rates of mostly viscous protein solutions applied at reasonably low pressures. In analytical mole-cular sieve filtration the particle size ranges of the gel matrix are selected as small as possible, to obtain a maximum number of theoretical plates, a flow rate of the mobile phase in the range of 2 to 4 cm/h combined with a pressure which is limited to technical and safe-ty aspects.
These parameters are dependent on the structure of the gel I ro~de~
-36~ 8~S
matrix and may vary from gel to gel.
If several preparative molecular sieve filtrations are per-formed in sequence, graduated separation limits can be selected. This can be followed by an analytical molecular sieve filtration with correspondingly graduated separation limits. The exclusion limit of ~he gel used must in all cases be higher than about 10,000 daltons to allow-a volume distribution of mediator proteins between the stationary gel matrix phase and the mobile aqueous buffer phase.
..
The "exclusion limit" is a hydrodynamic parameter of a dis-solved particle, which corresponds to the pore size of the gel matrix. Particles with a greater hydrodynamic para-meter cannot penetrate the gel matrix (volume distribution coefficient KD = )- The "separation limit" refers to a hydrodynamic parameter which has been chosen for the sepa-ration of dissolved particles from others and which has a value of between the volume distribution coefficient = 0 and KD = 1-~or molecular sieve filtration, the proteins are appliedto the molecular sieve after dissolution in a protein-compatible liquid. A special example of a suitable solvent is 0.003 mol/l sodium-potassium phosphate solution contain-ing 0.3 mol/l NaCl and 0.001 mol/l cysteine and having a pH of 7.~. After filtration, the mediator protein -contain-ing fractions are concentrated in the manner described below and optionally subjected to a further purification step.
Examples of suitable anion exchangers are dextran matrices cross-linked with epichlorohydrin (Sephadex) or cellulose matrices carrying functional groups with anion exchanger capacity. These exchangers can be regenerated for repeated further use. It is preferable to use a weak anion exchan-ger in the Cl form such as DEAE-Sephadex A-50, pre-swollen ~3 _37- ~8~45 and equilibrated in a buffer. Swelling and equilibration is preferably carried out at a pH of 8 to 10. A special example of such a buffer solution is 0.01 mol/l tris-HCl contain-ing 0.04 mol~l NaCl and 0.001 mol/l cysteine and having a pH value of 8Ø
The anion exchanger is added to the protein fraction in an amount sufficient for complete adsorption of the mediator proteins and of the other positively adsorbing accompany-ing proteins. Two volume parts of swollen anion exchanger per volume of concentrated protein solution are normally sufficient. The reaction can be carried out either as chromatographic process or as an easy and fast batch adsorp-tion technique~. In the latter case, the supernatant liquid containing negatively adsorbed proteins is separated from the anion exchanger which is charged with the positively adsorbed mediator prvteins or other proteins, e.g. by fil-tration in a chromatographic column, by decantation or centrifugation. The charged anion exchanger is freed f~om adhering negatively adsorbing compounds by washing with water or a salt solution having a maximum ionic strength equivalent to 0.04 mol/l NaCl, preferably at a pH of 8 to 10.
The maximum preferred temperature is about 15C. A special example of salt solution suitable for the washing-out process is the tris-HCI buffer of pH 8Ø
The anion exchanger on which the mediator prote;ns and other pro-teins are adsorbed and which is freed from the negatively adsorbed compounds is eluted with a protein-compatible aqueous salt solution having an ionic strength higher than 0.04 mol/l NaCl and a pH of between 4.0 and 10Ø A salt solution of high ionic strength and a pH of between 5.0 ar-d 7.0 is preferably used. A special example of such a salt solution is a 2.0 mol/l NaCl solution buffered to a pH of 6.5 with 0.01 mol/l pipera~ine-HCl and containing ~ .
-38- ~ 188~5 0.001 mol/l cys-teine.
If the anion exchange reaction is carried out as a chroma-tographic process, elution of the mediator pro-teins and o-ther positively adsorbed proteins can also be done by a, linear NaCl concentration gradient.
~xamples of cation exchange matrices suitable for the puri-fication of the protein fraction are dextrans crosslin~ed with epichlorohydrin (Sephadex) or cellulose ma-trices carry-ing functional groups with cation exchange capacity. These can be readily regenerated after use and employed again.
It is preferable to use a weakly acidic cation exchanger such as CM-Sephadex C-50 having Na as mobile counter-ion, and to perform the exchange reaction at a pH between 4 and 6. To facilitate the charge process and to approach more idealequilibria conditions prior to treatment with the cation exchanger the protein fractions should be diluted with a protein-compatible salt solution hav~g a maximum ionic strength equivalent to 0.04 mol/l NaCl. This salt solution can be used at the same time to adjusi the pH.
A special example of a salt solution for this purpose is a 0.001 mol/l potassium phosphate-acetate buffer containing 0.04 mol/l NaCl and 0.001 mol/l cysteine and having a pH
of 4 to 6. This cation-exchange reaction may be performed as a chromatographic process, or technically easier, as a batch process.
The swollen cation exchanger is added to the protein frac-tion in a quantity sufficient to adsorb it. As a rule, about 2 volume parts of swollen ion exchanger per volume par-t of protein solution is sufficient for this purpose.
The supernatant is -then separated from the cation exchanger charged with proteins, for example by decantation or cen-tri~ugation. The charged cation exchanger is freed from adhering, negatively adsorbed compounds by washing with water or a salt solution, having a maximum ionic strength -39~ 1~8~5 equivalent to 0.04 mol/l NaCl. Preferably a pH of about 4 to 6 and a maximum temperature of about 15C is used. A
special example of a salt solution suitable for the washing out process is the mentioned potassium phosphate-acetate buffer having a pH of 5Ø
The washed protein-charged cation exchanger is now eluted with a protein-compatible aqueous salt solution. ~ salt solution of high ionic strength with a p~ of about 4 to 10 is preferably used for tihis purpose. Special examples of such salt solu-tions are aqueous 0.5 mol/l potassium phos-phate with a pH of 6.5 to 7.5 or a 2 to 5 mol/l NaCl with the same pH.
For chromatography on hydroxyapatite, salts, e.g. ammonium sulfate and especially phosphates, possibly present from preceding steps are removed from the protein solution, preferably by dialysis or ultrafiltration at membranes with an exclusion limit of 500daltons prior to the appli-cation of the proteins to hydroxyapatite. Apart from visco-sity increase by accompanying salts, however, only the phosphate concentration of the protein solution is critical for the chromatography on hydroxyapatite. The mediator pro-teins are eluted by a potassium phosphate concentration gradient which is preferably linear. The mediator protein containing fractions are collected and then concentrated in the manner described below.
The use of hydroxyapatite is of essential significance for the structure-conserving isolation of pure mediator pro-teins. However, in general, for technical and economic reasons, considerable difficulties arise from chromato-graphy of larger volumes of protein solutions on hydroxy-apatite columns. On the one hand, larger protein amounts contribute to the strong tendency of hydroxyapatite to clog, thus becoming unusable as stationary matrix in chromatography. On the other hand, hydroxyapatite is very -'I 0- 1~8B~S
expensive. Its use on larger scales is not economi.cal. For these reasons, in the process oE the invention, the sepa-ration of a large part of the accompanying foreign pro-teins by appropriate biotechnical purification steps from the mediator protein -containing protein fractions is preferred for considerably reducing the volume of the protein solu-tion prior to its chromatography on hydroxyapatite.
In the zone precipitation chromatography (cf. J. Porath, Nature, vol. 196 (1962); p. 47-48), residual protein con-taminations in the mediator proteins are separated by salt-ing-out fractionation o the proteins by means and along a salt concentration gradient. The basic principle of separation of proteins in zone precipitation chromato-graphy are differen-t, structure-related, reversible sol-ubility characteris-tics of proteins. They belong to the most sensitve molecular separation criteria and are often used for demonstration of molecular homogeneity of a pro-tein. Two variants of this technique or development o the chromatogram are known: Fractional precipitation zone chromatography and ractional elution zone chromatography.
Both types o techniques may have s~lective advantages in speciic cases as described for fractional precipitation and fractional elution methods in protein separation. Tem-perature and pH, column charac-teristics can all be varied within relatively wide limits.
The temperature or zone precipitation chromatography can be bet~eeen O and 40C. Preerably, a temperature range from about O to 10C is used, especially from about 4 to 6C. The pH canbe between 4 and 10; preferably, a pH range of 6 to 8 is used, especially a pH of about 7. The length~
to-diameter ratio of the column used should be greater than about 10:1. A ratio of 30 to 100:1 and especially of about 50:1 is preferred. All protein-compatible salts having salting-out proper-ties for proteins are suitable.
8~S
Examples of such salts are sodium-potassium phosphate, ammonium sulfate, and sodium sulfate. Ammonium sulfate is preferred.
The salt concentra-tion gradient can have any desired shape provided that salting-out criteria of proteins achieve protein separation. Linear concentration gradients are preferred, especailly an ascendent linear concen-tration gradient from 25 to 100% ammonium sulfate satu-ration. The maximum column charge is about 5% and pre-ferably about 1% of total column volume.
~he recycling or cascade molecular sieve filtration can be performed under -the conditions described above for the analytical molecular sieve filtration. The same mole-cular sieves and the same column conditions can be used.
Sephadex G 50 as stationary matrix is preferred in a column of a length-to-diameter ratio of at least about 50:1 and a maximum charge of about 3% of the column volume. rrhe solvents used in the analytical molecular sieve filtration are also preferred as solvents for the elution in this method.
In recycling molecular sieve filtration, the distribution equilibria are disturbed continuously and the eluate is recycled onto the same column with fixed separation limits.
In this way, the separation length of the migrating pro-tein distribution bands are differentially extended.
Alternatively, in cascade molecular sieve filtration, distribution equilibria are disturbed by continous trans-fer of the eluate into a new second column with the same or similar, defined pa,rameters at fixed separation limits.
~etween the above-described purification steps, and if necessary at any stage ~or special purposes, protein solu-tions can be separated and fre~d from unwanted salts and s water as well as concomitantly concentrated. The concen-tration (separation of a major portion of aqueous salt solution of the protein) can be achieved in different ways. Dehydration dialysis or ultrafiltration against protein-compatible liquid, preferably a sodium po~assium phosphate buffer, are such methods. Dehydration dialysis is carrried out preferably a~ainst polyethylene glycol (molecular weight 20,000 daltons~ at membranes with exclu-sion limites of preferably SOOdaltons. Ultrafiltration is preferably achieved at membranes with an exclusion limit of about 500 daltons Small amounts of protein ~recipi-tates formed are removed by intermediary centrifugation to result in a clear protein solution. A desalting mole-cular sieve filtration on matrices with appropriate separation and exclusion limits can as well be used for this purpose, e.g. on Sephadex G 10, G 15 or G 20 as matrices. Furthermore, by selecting an appropriate mobile phase in the usual way, a usual molecular sieve filtration step can also be used concomitantly for this purpose.
To prevent sulfhydryl group oxidation-, about 0.001 mol/l of cysteine is preferably added to protein solutions through-out.
In the molecular sieve ,iltration purification steps about 0.~ mol/l ammonium sulfate is preferably added to the pro-tein solution. In contrast to higher concentrations of thi.s salt, at this concentration ammonium sulfate exerts a strong salting-ineffect on proteins. Thus, proteins are better kept in solution during the molecular sieve fil-tration. Moreover, ammonium sulfate prevents growth of microorganisms and inhibits certain enzymes. Hence, it contributes to stabilization of the mediator protein struc-ture which is important when chromatography is performed at higher temperature (above about 20C~ and under non-sterile conditions.
-~r .~
88~S
-~3-Mediator proteins which can be salted ou-t are preferably completely precipitated alone or -toge-ther with accompany-ing proteins by adding ammonium sulfa-te up to a concentra-tion of about 3.25 to 3.7 mol/l (80 to 90% saturation). For this purpose 630 g/l ammonium sulfate are added (about 90% saturation). The pH value is preferably kept between 4 and 9 and the temperature up to 40C, preferably between O and BC.~he m~ia-tor protein-con-taining protein precipi.tate is separated from the protein-free supernatant solution by filtration, decantation or centrifugation. Unless otherwise stated, centrifugation is preferably carried out at least at 10,000 x g for a minimum of 45 min, and preferably for 1 h, in a one-step process. Or it can be carried out in two stages, at lower forces in the first stage for removal of the bulk of precipitated proteins;
and then, for the supernatant of the first stage contain-ing residual fine protein particles at higher forces, e.g.
20,000 to 50,000 x g, by flow-through centrifugation.
The temperature and pH conditions during performance of the purification steps are not particularly critical.
~f the native conformation of the protein is to be pre-served, an optimum temperature range is abou-t O to 8C, and preferably about O to 4~C. Moreover, the separation and purification steps must be carried out under essen-tially physiological pH and salt conditions. An essential advantage of the process of the invention consists in tha-t these condi-tions are for the first time easy to adhere to.
The mediator proteins ob-tained can be stored in a buffered physiological saline, e.g. in 0.0015 mol/l sodium-potas-sium phosphate solution containing 0.15 mol/l (0.9 w/v~) NaC1, O.OG1 mol/1 cysteine and having a pFI of 7.4. After usual sterilization by filtration (pore diameter 0.2 ~m), the protein preparation remains native and biologically -4~-active a-t room tempera-ture for at least 200 h or frozen a-t -25C for atleas-t 5 years. This stability of the pro-tein can be considered, among others, to be one of the criteria of molecular homogeneity. Medj~tor Proteln solutions are safely s-tored at temperatures of between -20 and +50C i.n the presence oE 2.0 to 3.6 mol/l ammonium sulfate (50 to 90 % saturation). At this high osmoti.c pressure mediator protei.n solutions are protected against infection and degradation by microorganisms and bacterial growth.
For their physiological, therapeutical and any other use, the mediator proteins are again freed from salts by dia-lysis or ultrafiltration against an appropri.ate saline as described above.
The invention will now be given in detail by examples describing the isolation of the mediator protein preparation starting from leukocytes of porcine blood.
However, the invention is not restricted to this embodi-ment. Leukocytes and inflanedtissues of other mammalians can be used too.
Example A
PREPARATION OF CHEMOKINESINS AND CHEMOTAXINS FROM SUPERNA-NATANTS OF CULTURES OF A MIXED POPULATION OF VIABLE LEUKOCYTES
The production of chemo]cinesins and chemotaxins in a culture solution of a mixed population of leukocytes and the separation of lymphocyto-monoapokinesin (L~), monocyto-granuloapokinesin (MGAK), monocyto-granuloproskinesin (MGPK), lymphocyto-monoproskinesin (LMPK), monocyto- ;
granulotaxin (MGT), granulocyto-monotaxin.(GMT) and monocyto-eosinotc~xin (MET) from the o-ther components of the culture supernatant are descri.bed. All process steps are carri.ed out at 0 to ~C
in the presence of 0.001 mol/l cysteine, unless otherwise -~5~ ~ l 882 ~ 5 speci~ied. The centrifugation is carried out in the manner described , either as a one or two step procedure (as flow-through centrifugation).
A 1 Preparation and cul-ture of a mixed population of ~iable leukocytes 50 kg (about 10 ) leukocytes are isolated as mlxed cell population of physiological composition from 10,000 l of porcine blood and cultured in 20 batches of 2.5 kg (about S x 1012 cells) under sterile conditions. The medium indi-cated in tableVIII is used as culture solution. 50 1 of cul-ture medium are used per batch. Culturing is performed in glass vessels (Duran 50 or Pyrex glass). Initially, the cell density is a~out 1o8 cells/ml. -~he culture is maintained at 37C in an atmosphere of 1 v/v % CO2 over ~0 hours. During this period, the cell suspension is slowly stirred (to r.p.m.) and flooded with sterile, water-washed and heat-decontaminated air bubbles (C1mm).
The heat~decontamination of air is performed at about 500C by flowing through a silica tube- In addition to the partial oxygen pressue, the pH value (7.1) and the D-glucose level are measured and maintained constant. During culturing, the cells are induced to mitosis by the polyvalent mitogen eontent (CON) of the culture medium. The number, differential and morpholo-gical viability (dye exclusion test) of the cells are continously determined by usual methods of hematology and cell culture techniques. The functional viability of cells is measured by their motility and their ability to respond to chemokinetic and chemotactic proteins.
Mitoses are determined by chromosome count. The morpho-logical viability of the cells after their biotechnical culturing is 95%. The entire loss in cells (mainly granu-ocytes) during eulturing is at most 20~ which is normal ~or primary cell cultures.
- 4 6 ~ 5 The culture is terminated by separating the cells from the supernatant solution by centrifugation for 10 minutes at 400 x g and 10C. The cells are washed twice in a salt solution containing 0.15 mol/l NaCl, 0.0015 mol/l sodium potassium phosphate and having the pH-value 7.1. They can be used for ano~her purpose.
The culture supernatant solution is then centrifuged again for 1 hour at 10,000 x g and at 4C to remove sus-pended particles. The resuLtant clear supernatant culture solution which has a total volume of 1000 liters and con-tains about 1,400 g protein as well as other macromole-cules and salts is directly subjected to salting-out frac-tionation with ammonium sulfate (A2). Unless other~ise stated, all further steps are carried out at 0-4C.
.
A.2. First purification step (salting-out fractionation):
Preparation of crude protein concentrate fractions.
-0.5 mol/l sodium-potassium phosphate buffer solution with a pH value of 6.7 is added to the supernatant culture solution (~ 1) up to a final concentration cf 0.1 mol/l.
Furthermore, solid L-cysteine is added up to a concen-tration of 0.001 mol/l.
This buffered supernatant culture solution is then adjusted to 35% saturation of ammonium sulfate by addition of 199 g of ammonium sulfate/l solution. During the addition, the pH-value of the protein solution is continuously controlled and maintained at 6.7 by the addition of 2 n ammonia. Part of the proteins is precipitated from the solution. The protein precipitate formed is separated from the super-natant containing salt-soluble proteins by centrifugation for 1 hour at 10,000 x g. The precipitated crude protein -~7-fraction I is obtained as ammonium sulfate-containing pro-tein sludge which contains about 100 g protein. This crude protein concentrate fraction I may separately be processcd for its constituents according to the procedure described below for the crude protein concentrate fraction III.
t Then the 35% salt-saturated supernatant cul~ure solution is adjusted to ~5~ saturation of ammonium sulfate by add-ing 60 g of ammonium sulfate/l solution. The pH ~alue of the protein solution is continuously controlled-and maintained constant at 6.7 by 2 n ammonia. Another portion of proteins is precipitated from the solution. The protein precipitate is separated from the supernatant containing salt-soluble proteins by centrifugation for 1 hour at 10,000 x g. The precipitated crude protein concentrate fraction II is obtained as ammonium sulfate-containing protein sludge, ~he protein content of which is about 60 g.
This crude protein concentrate fraction II may be processed separately for its constituents , according to the procedure described below for the crude protein concentrate frac-tion III.
The 45~ salt-saturated supernatant culture solution is then adjustedt 90~ saturation of ammonium sulfate by add-ing 323 g ofam~onium sulfate/l of solution. The pH-value of the protein solution is again continuously controlled and maintained constant at 6.7 by 2 n ammonia. Another portion of the proteins is precipitated from the solution.
The protein precipitate is separated from the supernatant containing salt-soluble proteins by centrifugation for 1 hour at 10,000 x g. The precipitated crude protein con-centrate fraction III is obtained as ammonium sulfate-containing protein sludge the protein content of which is approximately 1,080 g. This fraction also contains the bul~ of the serum albumin as component of the culture medium. This crude protein concentrate fraction III
~"`3 .......
-~8- ~ 45 contains the mediator proteins of the invention and is processed according to the procedure described below.
The 90~ salt saturated supernatant fraction IV of the crude fraction III con-tains 160 g of salt-soluble pro-teins and other macro molecules ( ~ 500 daltons)~ It may also be processed for its constituen-ts.
A.3. Fine purification of mediator prote~s in the c~ude ~ tein concentrate fraction III
A~3.1.Anion exchange chromatography The crude protein concentrate fraction III obtained above ~A 2) is dissolved in a minimum volume of buffer solution B (0.01 mol/l of tris~HCl solution coniaining 0.04 mol/l NaCl and 0.001 mol/l cys~ine and having a pH value of 8.0). The resultant slightly turbid solution (20 l) is clarifled by centrifugation and then freed of salts by dialysis at a membrane with the exclusion limit of 500 dalton against buffer solution B until no sulfate ions are detectable. The clear solution obtained is then applied to a column of a swollen regenerated anion exchan- -ger (Cl as mobile exchangeable ion). It has a dextran matrix cross-linked with epichlorohydrin (DEAE-Sephadex A 50) which is equilibrated in the above-mentioned buffer system B.
:
8;~S
_~9_ The column has four -times the volume of the protein solu-tion and a length-to-diameter ratio of 10 : 1. The gel column is then washed with the above-mentioned adsorp-- tion buffer solution B until the extinction of the fil~
trate at 280 nm is c 1 0 .
In this puriication step, chemokinesins are separated rom chemotaxins, because the former are adsorbed where-as the latter flow through.
For elution of the chemok~nesins and the adsorbed pro-teins, the charged ion exchanger gel is eluted with a NaCl-concentratio~ gradient during 2 days. he gradient is linearly ascending from 0.04 to 2.0 mol/l ~aCl, whereas the pH value, the tris/HCl and tne cysteine concen- -tra~ions are maintained constant. The same shape of gra-dient is then used for lowering the pH from 8 to 6.5 for fur~her elution of the compounds. It is made up by 0.01 mol/l piperacine-HCl-buffer containing 200 mol/l NaCl and 0.001 mol/l cysteine and having the-pH 6.5.
~he chemokinesin or chemotaxin-containing fractions are collected separately. They are separately processed in further purification steps described below (A~3.2 - A.3.6)~
A.3.2. Preparative molecular sieve filtration After concentration of the proteins in the fractions ~A.3.1l by salting-out precipitation with ammonium sul-ate, the protein precipitate containing either chemokinesins or chemotaxins is dissolved in a minimum volume o buffer solution C
(0.003 mol/l sodium-potassium phosphate containing .
.
-50- ~ 3~ 5 0.3 mol/l NaCl and 0~001 mol/l cysteine and having a p~
value of 7.4). A~ter removal of a small amount of insol-uble compounds by centrifugation., the solution is applied to a column of a molecular sieve matrix of agarose cross-linked with acrylamide (Ultrogel AcA 34, particle size 60 to 160 ~m) for preparative molecular sieve filtration:
The column has 10 times the volume of the protein solution and a length-to-diameter ratio of 20:1. The column is then eluted with an upward flow (3 cm/h) of the mentianed buffer solution C For chemokinesins, the frac-tion with the separation lu~ts of 25,000 and 6,000 daltons and for chemotaxins, the fraction with the separation limits of 20,000 and 3,000 daltons are collected. For the concentration of the proteins, the frac-tions are lyophilized and ultrafiltered at ane~brane with the exclusion limit of 500 dalton or are adjusted to an ammonium sulfate concentration of 3.7 mol/l. In this case, the protein precipitates are separated from the super-natant by centrifugation and f~rther processed.as des-cribed below (~.3.3).
A.3.3 Cation.exchange chromatography The resultant che~.o~inesins or chemotaxIns-containing protein precipitates (A 3.2) are dissol~ed in 1.5 volume parts of buffer sol-ution D (0.01 mol~ sodium-potassium phosphate, 0.04 mol/l NaCl, 0.001 mol/l cysteine~pH 6.0). The.solutions are cen-trifuged at 10,000 x g for 1 hour for removal of a small amount of insoluble material.
The clear solution is dialyzed against the bu~fer solution D
at a membrane wi~h the exlusion limit of 500 daltons until no sulfate îons are detectable. The clear solution obtained is then applied to a column of swollen, regenerated cation exchanger based on a dextran matxix cross-linked with epichlorahydrin (CM-Sephadex C 50j. The exchanger is equili-brated in the above-mentioned buffer system D (Na as mobile ~38~5 exchangeable ion).
The column has four times the volume of theprotein solution and a length-to-diame-ter ratio of 10 : 1. The gel~column is then washed with the above-mentioned adsorption buffer solution D, ~til the extinction of the filtrate at 280 nm is - 1Ø
For elution of the me~iator proteins and the adsorbed pro-teins, the charged ion exchange gel is eluted with an NaCl-concentration g~adient during 2 days. The gradient is linearly ascending from 0.04 to 2.0 mol/l NaCl whereas the pH-value and the phosphate and cysteme concentrations are maintained constant. For further elution, the same shape of gradient is then used for increasing the phos-phate concentration from 0.01 to 0.5 mol/l at a p~ of 8.0,whereas the NaCl (2 rlol/l) and~cysteine concentrations are kept constant.
The chemokinesins or chemotaxins-containing fractions are collected and concentrated in the usual manner and further processed as described below (Ao3~4~
A.3.4 Chromato~_aphy on hydrox~vapatite The chemokinesins or chemotaxins-containing protein preci-pitates (A.3.3) are dissolved in a minimum volume of 0.0001 mol/l sodium-po-tassium p~losphate bufEer solution E containing 0.001 mol/l cysteine and having a pH of 7.20. The solutions are then desalted with this buffer by molecular sieve fil-tration, ultrafiltration or dialysis (exclusion limit 500 dalton), until no sulfate is detectable in the dia-lysis buffer. Thereafter, a small portion of insoluble material is removed by centrifugation a~ 10,000 x g for 1 hour.
' -52- ~882~5 The clear chemo~inesins or c~lemotaxins-containing protein solutions ~bbtained are separately applied to a column of hydroxyapatite. The l~ngth-to-diameter ratio of the column is 10 : 1 and it has four times the volume of theprotein volume to be applied.
The column has been equilibrated with the mentionea buffer E u~ed in an amount five times the column volume (flow 3 cm/h).
The negatively adsorbed proteins are washed out with the buffer solution E used for equilibrating:the column. The elution of the mediator pro-tein -containing fractions is '!
caxried out with a phosphate concentration gradient for 4 days~ The gradient is linearly ascending from 0.0001 mol/l to 0.5 mol/l sodium-potassium phosphate having a constant pH value of 7.4 and constant cysteine concentration.
The different mediator proteins are separated in this step.
LMAK is eluted at an average phosphate concentration of about 0.04 mol/l, MGAK at about 0.001 mol/l, MGPK at about 0~005 mol/l~ LMPX at about O.OB mol/l. In the chromatogram of the chemotaxins MGT appears at an average phosphate concentration of about 0.004 mol/l, MET at about 0,05 mol/l and GMT at about 0,2 mol/l. The elution gradient is measured and con~
trolled by means of conductivity. The mediator protein -con-taining fractions are concentrated in the usual manner and further processed as described below (A.3.5).
~18~5 A.3 5. Zone precipitation chromatography The mediator protein-containing fractions (A.3.4) are dissolved in 0.1 mol/l sodium-potassium phosphate solution ~ con-taining 0.1 mol/l NaCl, 0.001 mol/l cysteine and 1 mol~l ammonium sulfate and having a pH value of 7.4. The resultant solution is applied at a tempera-ture of 4C to a column of swollen m~lecular sieve matrix of dextran cross-linked with epichlorhydrin tSephadex G-25). In the matrix, an ascendent, linear ammonium sulfate concentration gradient is estab-lished with the mobile buffer phase from 1.0 to ~.0 mol/l ammonium sulfate ~25 to 100% saturation). The slope of the gradient is +2% of the ammonium sulfate saturation/cm of column height (0.08 mol/l (NH432SO4/cm). The range of the gradient ex~ends over approximately half the length of the column.
. .
The len~th-to-diameter ratio of the column is 50 : 1, the column volume is 100 times higher than the protein solution volume to be applied~ The flow rate is 2 cm~h.
The elution is carried ou~ with the above-mentioned sodium-potassium phosphate solution F containing 1 mol/l o~ ~mmo~
nium sulfate. The mediator prOtein-containing fractions which are ~luted at 65% l~), 77% (MGAK), 72 % (M~PK), 62% I~K), 70~ IMGT~, 57~ (GMT) and 80% (MET) ammonium sulfate sa-turation, respect~ively, are collec-ted. The proteins are concentrated in the usual manner and further processed as described below (A.3.63.
-5~ 4~
A.3.6. Analytical recycling molecular sieve filtration The medi.a-tor protein-containing fractions (A.3.5) are dissolved in buffer C (0.003 mol/l sodium-potassium phospha~e con-taining 0,3 mol/l NaCl and 0.001 mol/l casteine:.and having a pH value of 7.4). Removal of a small portion of insol-uble substances is achieved by centrifugation for 3 minutes at 48,000 x g.
The resultant clear solu-tion is then subjected to . ana-lytical recycling molecular sieve chromatographyO For this purpose, the solution is applied at a temperature of 4C
to a column o Ultrogel AcA 44 having a particle size of 60 to 140 ~m. The column has 50 times the volume of the pro~
tein solution and a length-to-diameter ratio of 50 : 1 The elution is carried out with the mentioned buffer C~
The eluates are recycled three times at separation limits of either 17,000 dalton (LMAK), 12rOOO dalton (MGAK~, 19,000 dalton (MGPK~, 25/OOO dalton (LMPK), 13,000 dalton (MGT), 20,000 dalton (GMT~, or 8,000 dalton (MET).
After usual protein concentration, approximately 3 mg of LMAK, 5 mg of MGAK, 5 mg of MGPK, 4 mg of LMPX, 6 mg of I MGT, 9 mg of GMT and 5 mg of MET are ob-tained. The chemo-i kinesins and chemotaxins have a molecular homogeneity of 95%, as indicated by conventional methods.
In the following flow sheet the above-described process for preparing and isolating the chemokinesins and chemotaxins of the invention is schematjcally represented.
~î8~245 X
W
U~
H
tn z K
0~
O
p:; ~ p, H
a ~ ~ z ~ z O ~ O O ~ z H Z
~; C) S~ ~ Z
~ D '3 ~ ~ ~ O ~ C~ H ~ ~ Z ~
O W ~ O ~ ~ ~ V H . ~ Z Z ~; Z Z Z
o ~, P; o ~: ~ x H ~ r 2 ~c ~ a ¦ Z ~ ~ 2 ~ o A
W H CL~ E~ V O ~ ~1 0 c~
. v ~ ~ ''¢ m ~ ~ O zO ~ O ~ O
H , Z ~
O ' ' m ~ u~.... . .
;I~ , ' .
u~ . i ,~
.
4~
E x a m p 1 e B
PREPARATION OF ~ I~R PRarEINS FROM SUPERNATANTS OY CUL--TURES OF VIABLE ~YMPHOC~3 ~ 5 kg (about 3 x 1013~ lymDhocytes obtained from po,rcine blood are cultured unde~ the conditions described in example A. During culture, the polyvalent mitogen (CON) in the medium induces the mitosis of the cells.
The chemokinesins LMAK and LMPK secreted ~o the cul~ure -solution are isolated according to the procedure des~
cribed in example A. They are thereby ob;tained in a highly purified state. The yields obtained are comparable to those of example A.
E x a m p l e C
PREPARATION OF MEDIATOR PROTEINS FROM SUPERNATANTS OF
CULTURES OF VIABLE MONOCYTES
Example s is repeated with a culture of 3,5 kg (about 7 x 1o12) monocytes. The chemokinesins and chemotaxins of the monocytes are obtained in yields as in example A.
E x a m p l e D
PREPARATION OF ~DIA~OR PRCTEI~S FROM INFLAMED TISSUE SITES
The preparation and isolation of chem~kinesins and ch~mo~ins from inflamed tissue are described. 500 g of infarcted, inflamed canine heart muscle tissue are used. The heart muscle tissue is ground at 0-4C. 0.0~ mol/l sodium potassium phos-phate buffer solution containing 0.001 mol/l cysteine and having a pH of 6.~ is added in a quantity three times the amount of the tissue. The resultant suspension is homoge-4~
nized in a homogenizer (ultraturax). Thereafter, the super-slatant containiny the soluble compounds of the inflamed tissue is separated from the insoluble constituents by cen-triguation at 10,000 x g and 4C. The resultant supernatant solution is then centrifuged ~or 3 hours at 100,000 x ~.
The clear supernatant solution obtained is siphoned of~ from the flotating lipid layer.
The rnedi.ator protein-con-taining clear supernatant protein solution is then subjected to fractional salting-out preci-pitation with ammonium sulfate according -to example A. The resultant protein fracti.on III is then processed as des-cribed in example A. The yields, as compared to example A, are about 50% with the pro-teins from monocytes and granu-locytes and only about 10% with the proteins from lympho-cytes.
E x a m p l e E
PREPARATION OF 1~'0~ PROTEINS FROM LEUKOCYTE HOMO-GENATES
Leukocytes are prepared from blood according to example A. A homogenate of 500 g of leukocytes is prepared as shown in example D for muscle tissue. The isolation of the protein mediators contained in -the leukocy-tes is performed according to example A. The leukocytes cultured w.ithout stimulation contain only relatively small (about 1~) amounts of monocyte- and lymphocyte- chemo-kinesins and -chemotaxins, whereas the GMT is contained in a yield of about 10 % compared with example A.
Claims (39)
1. Process for production and isolation of chemokinesins and chemotaxins of leukocytes and inflamed tissue, having the following properties:
a) biological activities in vivo and in vitro:
- selective reversible influence on the motility of leukocytes (chemokinesis) or selective chemical attraction of leukocytes (chemotaxis) in vitro;
b) physico-chemical properties:
- soluble in aqueous media including in 15% ethanol at a pH value of at least 4.0 to 10;
- insoluble in ammonium sulfate solution at 90%
saturation (3.6 mol/1) - electrophoretic migration in acrylamide matrices at a pH of 7.40 is anodic;
- adsorb reversibly in structure and biological activ-ity on anion and cation exchangers, calcium phosphate gel and hydroxyapatite and can be subjected in native form to volume partition chromatography, character-ized by homogenizing leukocytes or inflamed tissue or culturing leukocytes and isolating the resultant chemokinesins and chemotaxins from the homogenates or the supernatant culture solution.
a) biological activities in vivo and in vitro:
- selective reversible influence on the motility of leukocytes (chemokinesis) or selective chemical attraction of leukocytes (chemotaxis) in vitro;
b) physico-chemical properties:
- soluble in aqueous media including in 15% ethanol at a pH value of at least 4.0 to 10;
- insoluble in ammonium sulfate solution at 90%
saturation (3.6 mol/1) - electrophoretic migration in acrylamide matrices at a pH of 7.40 is anodic;
- adsorb reversibly in structure and biological activ-ity on anion and cation exchangers, calcium phosphate gel and hydroxyapatite and can be subjected in native form to volume partition chromatography, character-ized by homogenizing leukocytes or inflamed tissue or culturing leukocytes and isolating the resultant chemokinesins and chemotaxins from the homogenates or the supernatant culture solution.
2. Process according to claim 1, characterized by cultur-ing a mixed leukocyte population.
3. Process according to claim 1, characterized by cultur-ing a specific leukocyte type.
4. Process according to claim 1, 2 or 3, characterized by culturing the leukocytes in a fully synthetic serum-free cell culture medium containing serum albumin as the only protein.
5. Process according to claim 1, characterized by inducing the mitosis of the leukocytes during the culture.
6. Process according to claim 5, characterized by adding a polyvalent mitogen or endotoxin-mitogen or promoting an immune reaction on the cell surface so as to induce the mitosis of the leukocytes.
7. Process according to claim 6, characterized by induc-ing the mitosis of the leukocytes by the addition of a lectin.
8. Process according to claim 7, characterized by using a lectin from Canavalia ensiformis (Concanavalin A = CON).
9. Process according to claim 1, 2 or 3, characterized by culturing the leukocytes in a cell culture medium having the composition given in Table VIII.
10. Process according to claim 1, 2 or 3, characterized by culturing the leukocytes for approximately 40 hours at about 37°C and a concentration of about 107 to 108 cells/ml culture solution at a CO2-partial pressure of about 1%
while sufficient oxygen is supplied to the culture.
while sufficient oxygen is supplied to the culture.
11. Process according to claim 1, characterized in that after termination of culturing by separating the leuko-cytes, the protein portion contained in the culture solution which becomes insoluble upon salt addition is obtained by salting out from the solution, and in that the protein portion which is soluble in the saturated salt solution is obtained by concentrating this solution.
12. Process according to claim 11, characterized by using ammonium sulfate for salting out the proteins.
13. Process according to claim 12, characterized by step-wise increasing the ammonium sulfate concentration of the culture solution, separating the proteins precipitated after each ammonium sulfate addition and by thus obtaining several crude protein fractions having graduated solubility at different ammonium sulfate concentration.
14. Process according to claim 13, characterized by adjust-ing the ammonium sulfate concentration of the culture solution stepwise to 35%, 45% and 90% saturation.
15. Process according to claim 11, 12 or 13, characterized by concentrating the supernatant of the salting-out precip-itation after separation of the protein precipitate by ultrafiltration or dialysis.
16. Process according to claim 11, 12 or 13, characterized by processing the crude protein fractions isolated by stepwise salting out and the concentrated supernatant of the salting-out precipitation separately to obtain chemokinesins and chemotaxins.
17. Process according to claim 1, characterized by perform-ing the processing of the crude protein fractions and the isolation of the chemokinesins and chemotaxins by prepar-ative and analytical molecular sieve filtration, anion and cation exchange chromatography and batch adsorption proces-ses, respectively, chromatography on hydroxyapaptite, zone precipitation chromatography and/or recycling or cascade molecular sieve filtration.
18. Process according to claim 17, characterized by performing at least two of the said purification steps in sequence.
19. Process according to claim 18, characterized by performing at least three of the said purification steps in sequence.
20. Process according to claim 1, 2 or 3, characterized in that for obtaining lymphocyto-monoapokinesin a mixed leuko-cyte population or only lymphocytes are cultured, the mitosis of the cells is induced by CON during culturing, after termination of culturing ammonium sulfate is added to the culture solution up to a 90% saturation, the precip-itated proteins are separated from the ammonium sulfate-containing supernatant, are redissolved and purified by an anion exchange chromatography step, a preparative molecular sieve filtration, a cation exchange chromatography step, a chromatography on hydroxyapatite, a zone precipitation chromatography and a cascade molecular sieve filtration and in that the lymphocyto-monoapokinesin is isolated in highly purified form in the eluate of the cascade molecular sieve filtration after separation of the accompanying foreign proteins.
21. Process according to claim 1, 2 or 3, characterized in that for obtaining monocyto-granuloapokinesin a mixed leukocyte population or only monocytes are cultured, the mitosis of the cells is induced by CON during culturing, after termination of culturing ammonium sulfate is added to the culture solution up to a 90% saturation, the precip-itated proteins are separated from the ammonium sulfate-containing supernatant, are redissolved and purified by an anion exchange chromatography step, a preparative molecular sieve filtration, a cation exchange chromatography step, a chromatography on hydroxyapatite, a zone precipitation chromatography and a cascade molecular sieve filtration and in that the monocyto-granuloapokinesin is isolated in highly purified form in the eluate of the cascade molec-ular sieve filtration after separation of the accompanying foreign proteins.
22. Process according to claim 1, 2 or 3, characterized in that for obtaining monocyto-granuloproskinesin a mixed leukocyte population or only monocytes are cultured, the mitosis of the cells is induced by CON during culturing, after termination of culturing ammonium sulfate is added to the culture solution up to a 90% saturation, the precip-itated proteins are separated from the ammonium sulfate-containing supernatant, are redissolved and purified by an anion exchange chromatography step, a preparative molecular sieve filtration, a cation exchange chromatography step, a chromatography on hydroxyapatite, a zone precipitation chromatography and a cascade molecular sieve filtration and in that the monocyto-granuloproskinesin is isolated in highly purified form in the eluate of the cascade molecular sieve filtration after separation of the accompanying foreign proteins.
23. Process according to claim 1, 2 or 3, characterized in that for obtaining lymphocyto-monoproskinesin a mixed leukocyte population or only lymphocytes are cultured, the mitosis of the cells is induced by CON during culturing, after termination of culturing ammonium sulfate is added to the culture solution up to a 90% saturation, the precip-itated proteins are separated from the ammonium sulfate-containing supernatant, are redissolved and purified by an anion exchange chromatography step, a preparative molecular sieve filtration, a cation exchange chromatography step, a chromatography on hydroxyapatite, a zone precipitation chromatography and a cascade molecular sieve filtration and in that the lymphocyto-monoproskinesin is isolated in highly purified form in the eluate of the cascade molecular sieve filtration after separation of the accompanying foreign proteins.
24. Process according to claim 1, 2 or 3, characterized in that for obtaining monocyto-granulotaxin a mixed leukocyte population or only lymphocytes are cultured, the mitosis of the cells is induced by CON during culturing, after termination of culturing ammonium sulfate is added to the culture solution up to a 90% saturation, the precip-itated proteins are separated from the ammonium sulfate-containing supernatant, are redissolved and purified by an anion exchange chromatography step, a preparative molecular sieve filtration, a cation exchange chromatography step, a chromatography on hydroxyapatite, a zone precipitation chromatography and a cascade molecular sieve filtration and in that the monocyto-granulotaxin is isolated in highly purified form in the eluate of the cascade molecular sieve filtration after separation of the accompanying foreign proteins.
25. Process according to claim 1, 2 or 3, characterized in that for obtaining granulocyto-monotaxin a mixed leuko-cyte population or only granulocytes are cultured, the mitosis of the cells is optionally induced by CON during culturing, after termination of culturing ammonium sulfate is added to the culture solution up to a 90% saturation, the precipitated proteins are separated from the ammonium sulfate-containing supernatant, are redissolved and purified by an anion exchange chromatography step, a preparative molecular sieve filtration, a cation exchange chromatography step, a chromatography on hydroxyapatite, a zone precipitation chromatography and a cascade molecular sieve filtration and in that the granulocyto-monotaxin is isolated in highly purified form in the eluate of the cascade molecular sieve filtration after separation of the accompanying foreign proteins.
26. Process according to claim 1, 2 or 3, characterized in that for obtaining monocyto-eosinotaxin a mixed leukocyte population or only lymphocytes are cultured, the mitosis of the cells is induced by CON during culturing, after termin-ation of culturing ammonium sulfate is added to the culture solution up to a 90% saturation, the precipitated proteins are separated from the ammonium sulfate-containing super-natant, are redissolved and purified by an anion exchange chromatography step, a preparative molecular sieve filtration, a cation exchange chromatography step, a chromatography on hydroxyapatite, a zone precipitation chromatography and a cascade molecular sieve filtration and in that the monocyto-eosinotaxin is isolated in highly purified form in the eluate of the cascade molecular sieve filtration after separation of the accompanying foreign proteins.
27. Process according to claim 1, 2 or 3, characterized by using the soluble portion of a leukocyte or inflamed tissue homogenate instead of the culture solution of the leukocytes.
28. Chemokinesins and chemotaxins of leukocytes and inflamed tissue, having the following properties:
a) biological activities in vivo and in vitro:
- selective reversible influence on the motility of leukocytes (chemokinesis) or selective chemical attraction of leukocytes (chemotaxis) in vitro;
b) physico-chemical properties:
- soluble in aqueous media including in 15% ethanol at a pH value of at least 4.0 to 10;
- insoluble in ammonium sulfate solution at 90%
saturation (3.6 mol/1) - electrophoretic migration in acrylamide matrices at a pH of 7.40 is anodic;
- adsorb reversibly in structure and biological activity on anion and cation exchangers, calcium phosphate gel and hydroxyapatite and can be subjected in native form to volume partition chromatography, whenever prepared by the process according to claim 1, or an obvious chemical equivalent.
a) biological activities in vivo and in vitro:
- selective reversible influence on the motility of leukocytes (chemokinesis) or selective chemical attraction of leukocytes (chemotaxis) in vitro;
b) physico-chemical properties:
- soluble in aqueous media including in 15% ethanol at a pH value of at least 4.0 to 10;
- insoluble in ammonium sulfate solution at 90%
saturation (3.6 mol/1) - electrophoretic migration in acrylamide matrices at a pH of 7.40 is anodic;
- adsorb reversibly in structure and biological activity on anion and cation exchangers, calcium phosphate gel and hydroxyapatite and can be subjected in native form to volume partition chromatography, whenever prepared by the process according to claim 1, or an obvious chemical equivalent.
29. Chemokinesins and chemotaxins according to claim 28, obtainable from leukocytes, by culturing leukocytes and isolation from the supernatant culture solution or from inflamed tissue, whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
30. Chemokinesins according to claim 28, characterized in that they reversibly lower the motility (statistical cell locomotion) of the leukocytes (negative chemokinesis =
apochemokinesis), whenever prepared by the process accord-ing to claim 1, or an obvious chemical equivalent.
apochemokinesis), whenever prepared by the process accord-ing to claim 1, or an obvious chemical equivalent.
31. Chemokinesin (lymphocyto-monoapakinesin) according to claim 28, characterized in that it is obtainable from lymphocytes and possesses the following additional properties:
a) biological effects:
- specific reversible lowering of the motility of macrophages (monocytes) in vitro;
- effective threshold dose in vitro <2nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 14,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 1;
- extinction coefficients according to the following Table I:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
a) biological effects:
- specific reversible lowering of the motility of macrophages (monocytes) in vitro;
- effective threshold dose in vitro <2nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 14,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 1;
- extinction coefficients according to the following Table I:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
32. Chemokinesin (monocyto-granuloapokinesin) according to claim 28, characterized in that it is obtainable from mononuclear leukocytes and possesses the following additional properties:
a) biological activities:
- specific reversible lowering of the motility of granulocytes in vitro;
- effective threshold dose in vitro < 1 nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 9,000 dalton;
- no protein quaternary structure in the form of physically bound peptide subunits; each of the native proteins consists of only one peptide unit;
- constant temperature coefficient of solubility in ammonium sulfate solutions between -10°C and +50°C;
- absorption spectrum (UV, visible and near IR-range) according to Fig. 2;
- extinction coefficient according to the following Table II:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
a) biological activities:
- specific reversible lowering of the motility of granulocytes in vitro;
- effective threshold dose in vitro < 1 nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 9,000 dalton;
- no protein quaternary structure in the form of physically bound peptide subunits; each of the native proteins consists of only one peptide unit;
- constant temperature coefficient of solubility in ammonium sulfate solutions between -10°C and +50°C;
- absorption spectrum (UV, visible and near IR-range) according to Fig. 2;
- extinction coefficient according to the following Table II:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
33. Chemokinesins according to claim 28, characterized in that they reversibly increase the motility (statistical cell locomotion) of the leukocytes (positive chemokinesis = proschemokinesis), whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
34. Chemokinesin (monocyto-granuloproskinesin) according to claim 28, characterized in that it is obtainable from mononuclear leukocytes and possesses the following additional properties:
a) biological activities:
- specific reversible increase of the motility of granulocytes in vitro;
- effective threshold dose in vitro < 2 nmol/1 b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 16,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 3;
- extinction coefficient according to the following Table III:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
a) biological activities:
- specific reversible increase of the motility of granulocytes in vitro;
- effective threshold dose in vitro < 2 nmol/1 b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 16,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 3;
- extinction coefficient according to the following Table III:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
35. Chemokinesin (lymphocyto-monoproskinesin) according to claim 28, characterized in that it is obtainable from lymphocytes and possesses the following additional properties:
a) biological activities:
- specific increase of the motility of macrophages (monocytes) in vitro;
- effective threshold dose in vitro < 10 nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 22,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 4;
- extinction coefficient according to the following Table IV:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
a) biological activities:
- specific increase of the motility of macrophages (monocytes) in vitro;
- effective threshold dose in vitro < 10 nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 22,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 4;
- extinction coefficient according to the following Table IV:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
36. Chemotaxins according to claim 28, characterized in that they chemically attract leukocytes and produce a directed cell migration along their concentration gradient (chemotaxis), whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
37. Chemotaxin (monocyto-granulotaxin) according to claim 28, characterized in that it is obtainable from mono-nuclear leukocytes and possesses the following additional properties:
a) biological activities:
- chemical attraction of neutrophilic granulocytes in vitro;
- accumulation of neutrophilic leukocytes in situ with indirect cell-induced angiogenesis and inflammation reaction;
- effective threshold dose in vitro < 0.5 nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 11,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 5;
- extinction coefficient according to the following Table V:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
a) biological activities:
- chemical attraction of neutrophilic granulocytes in vitro;
- accumulation of neutrophilic leukocytes in situ with indirect cell-induced angiogenesis and inflammation reaction;
- effective threshold dose in vitro < 0.5 nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 11,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 5;
- extinction coefficient according to the following Table V:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
38. Chemotaxin (granulocyto-monotaxin) according to claim 28, characterized in that it is obtainable from granulo-cytes and possesses the following additional properties:
a) biological activities:
- chemical attraction of macrophages (monocytes) in vitro;
- accumulation of monocytic leukocytes in situ with indirect cell-induced angiogenesis and inflammation reaction;
- effective threshold dose in vitro < 10 nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 17,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 6;
- extinction coefficient according to the following Table VI:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
a) biological activities:
- chemical attraction of macrophages (monocytes) in vitro;
- accumulation of monocytic leukocytes in situ with indirect cell-induced angiogenesis and inflammation reaction;
- effective threshold dose in vitro < 10 nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 17,000 dalton;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 6;
- extinction coefficient according to the following Table VI:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
39. Chemotaxin (monocyto-eosinotaxin) according to claim 28, characterized in that it is obtainable from mono-nuclear leukocytes and possesses the following additional properties:
a) biological activities:
- chemical attraction of eosinophilic leukocytes in vitro;
- accumulation of eosinophilic leukocytes in situ;
- effective threshold dose in vitro < 5 nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 5,000 dalton;
- no protein quaternary structure in the form of physically bound peptide subunits: each of the native proteins consists of only one peptide unit;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 7;
- extinction coefficient according to the following Table VII:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
a) biological activities:
- chemical attraction of eosinophilic leukocytes in vitro;
- accumulation of eosinophilic leukocytes in situ;
- effective threshold dose in vitro < 5 nmol/1;
b) physico-chemical properties:
- molecular weight of the native protein (primary structure): approximately 5,000 dalton;
- no protein quaternary structure in the form of physically bound peptide subunits: each of the native proteins consists of only one peptide unit;
- absorption spectrum (UV, visible and near IR-range) as given in Fig. 7;
- extinction coefficient according to the following Table VII:
whenever prepared by the process according to claim 1 or an obvious chemical equivalent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000398628A CA1188245A (en) | 1981-03-18 | 1982-03-17 | Chemokinesins and chemotaxins of leukocytes and inflamed tissues and process of their preparation |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3110610.2 | 1981-03-18 | ||
| CA000398628A CA1188245A (en) | 1981-03-18 | 1982-03-17 | Chemokinesins and chemotaxins of leukocytes and inflamed tissues and process of their preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1188245A true CA1188245A (en) | 1985-06-04 |
Family
ID=4122327
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000398628A Expired CA1188245A (en) | 1981-03-18 | 1982-03-17 | Chemokinesins and chemotaxins of leukocytes and inflamed tissues and process of their preparation |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1188245A (en) |
-
1982
- 1982-03-17 CA CA000398628A patent/CA1188245A/en not_active Expired
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1188244A (en) | Angiotropins of leukocytes and inflamed tissue and process for their preparation | |
| Saklatvala et al. | Purification to homogeneity of pig leucocyte catabolin, a protein that causes cartilage resorption in vitro | |
| Klagsbrun | Human milk stimulates DNA synthesis and cellular proliferation in cultured fibroblasts. | |
| US4514387A (en) | Chemokinesins and chemotaxins of leukocytes and inflamed tissues | |
| Moscatelli et al. | Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis, and migration. | |
| US4350687A (en) | Platelet derived cell growth factor | |
| US4002602A (en) | Ubiquitous immunopoietic polypeptide (UBIP) and methods | |
| Duc-Goiran et al. | Unusual apparently constitutive interferons and antagonists in human placental blood. | |
| JPS5813397A (en) | Immune interferon and production of mrna | |
| EP0260708A2 (en) | Tumor egress factor and processes for producing the same | |
| US5540923A (en) | Interferon proteins | |
| IE56089B1 (en) | Lymphocyte growth factor | |
| US4167557A (en) | Ubiquitous immunopoietic polypeptide (UBIP) and methods | |
| CA1188242A (en) | Mitogens of leukocytes and inflamed tissues and process for their preparation | |
| Csejtey et al. | Purification of human macrophage colony Stimulatingv factor (CSF-1) from medium conditioned by pancreatic carcinoma cells | |
| CA1188245A (en) | Chemokinesins and chemotaxins of leukocytes and inflamed tissues and process of their preparation | |
| CA1188243A (en) | Chemorecruitins of leukocytes and inflamed tissues and process for their preparation | |
| EP0550756A1 (en) | Interleukin 6 composition and production thereof | |
| Wolf et al. | Soluble inhibitory factor (SIF) in normal human serum | |
| US4610815A (en) | Process for producing and obtaining anaphylatoxin- and cocytotaxin-containing leucotaxine preparations and of anaphylatoxin and cocytotaxin proteins in molecularly homogeneous, biologically active form | |
| EP0417129B1 (en) | Lung growth stimulatory and inhibitory factors for carcinoma tumor cells | |
| EP0047979B1 (en) | Serum-derived "leukorecruitin": a protein mediator of inflammation from mammalian serum for specific induction of a leukocytosis reaction, a process for its biotechnical production and isolation in molecularly homogenous, crystallizable, biologically specific, active form, and leukorecruitin-containing pharmaceutical compositions | |
| Green et al. | Connective tissue activation. XXX: Isoelectric point microheterogeneity of CTAP-III, a human platelet-derived growth factor | |
| JPH02308799A (en) | Production of cancer metastasis-inhibitor | |
| EP0137691A1 (en) | Production of immune interferon and its mRNA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |