HK1071291B - Usr of the long pentraxin ptx3 for the preparation of medicament for the prevention and cure of autoimmune pathologies - Google Patents

Description

Use of long pentraxin PTX3 for preparing a medicament for the prevention and treatment of autoimmune diseases

The invention relates to application of long pentraxin PTX3(PTX3) or functional derivatives thereof in preparing medicaments for preventing and treating autoimmune diseases.

PTX3(Bottazzi et al, J., biol. chem.1997, 272: 32817-32823), particularly mononuclear phagocytes and endothelial cells, was expressed in a variety of cell types following exposure to the inflammatory cytokines interleukin 1 β (IL-1 β) and tumor necrosis factor α (TNF- α).

The biological function of PTX3 has not been understood to date.

PTX3 consists of two domains, the N-terminal domain is independent of any known molecule, and the C-terminal domain is similar to a short pentraxin, such as C-reactive protein (CRP) (breviariof et al, j.biol. chem.267: 22190, 1992).

A high degree of similarity has been found between human PTX3(hPTX3) and animal PTX 3. In particular, mouse PTX3(mPTX3) has a DNA sequence and gene organization and localization very similar to hPTX 3. The degree of identity between the human and mouse PTX3 genes was 82%, and 90% if conservative substitutions were considered (Introna M. et al, Blood87(1996) 1862-1872). The mouse PTX3 gene is located in a region on mouse chromosome 3 similar to human 3q (q24-28), consistent with the documented location of hPTX3 in the 3q 25 region (Introna M. et al, Blood87(1996) 1862-1872).

The high similarity of the sequences of hPTX3 and mPTX3 indicates that the pentraxin is highly conserved during evolution (Pepys m.b., Baltz m.l.: adv.immunol.34: 141, 1983).

For a review of pentraxin, see h.gewurz et al, Current Opinionin Immunology, 1995, 7: 54-64.

The long pentraxin PTX3 is produced in tissues exposed to pro-inflammatory signals provided by cytokines and Lipopolysaccharide (LPS).

These pro-inflammatory compounds promote apoptosis, which is involved in the inflammatory process, and promote physiological maturation of Dendritic Cells (DCs).

Cell death typically occurs during inflammation in vivo.

The organism removes apoptotic cells from living tissue. In an excessive, chaotic apoptotic process, the large number of dead cells hinders the ability of an organism to clear a cell population. These cells accumulate in tissues and increase the likelihood of promoting autoimmune processes through phagocytosis by non-conventional phagocytic cells (e.g., immature DCs) (Cell Death Diff.1998; 5: 563-568).

The development and maintenance of the autoimmune process requires the recognition of self-antigens (autoantigens) presented by immature DCs as antigens that are harmful (foreign or non-self) to the organism. This recognition error occurs during the immature DC removal of apoptotic cell populations.

Intracellular self-antigens are modified during apoptosis and protein esterification due to kinase activation caused by apoptotic stress, resulting in new conformational epitopes (1998J. exp. Med.187: 547-560).

Cell death and nuclear rupture, with autoantigens destroyed by specific proteases (caspases) 1996 j.exp.med.184: 765-770; 1996 j.exp.med.183: 1957-.

Some autoantigens are normally associated with the endoplasmic reticulum and plasma membrane of the cytoskeleton; these antigens segregate early in apoptosis.

Nuclear autoantigens isolated from apoptotic cell populations generated late in apoptosis excluded 1994 j. exp. med.179: 1317-1330; 1997 exp.cell res.234: 512-520.

In contrast, processing of internalized dead (apoptotic) cells produces the T cell epitope 1997 j. immunol.159: 5391-5399.

The lack of specific or semi-specific phagocytic clearance of the ability to elicit an immune response, the clearance of the majority of dead cells in vivo 1998 Cell Death diff.5: 563-568. However, the most potent Antigen Presenting Cell (APC) is DC 1997 curr. 10-16; 1998Nature 392: 245-252, which internalize the dead cells and present their processed epitopes to MHC class I and class II restricted T lymphocytes 1998Nature 392: 86-89; 1998 j.immunol.159: 5391-5399; 1998 j.exp.med.188: 2163-2173; 1999Nat Med 5: 1249 and 1255; 1999Nat Med 5: 1232-1233.

DCs were derived from bone marrow CD34+ progenitor cells 1992 nature.360: 258-261.

DC precursors enter the blood, reach peripheral organs, where they develop into immature DCs.

Immature DC capture soluble antigen via megacytopathic action (macropinocytosis) 1994 j. exp. med.179: 1109-: 479-488.

The basic pro-inflammatory signals, such as IL- β and TNF- α, promote DC physiological maturation and antigen presentation function 1999 curr. 308-313.

Because cells (defined as the "self" antigen depot) die continuously during development and normal tissue turnover, DC presentation of dead cell antigens is tightly regulated.

Normal cell death via apoptosis occurs in the absence of inflammatory signals that lead to DC maturation.

Dead cells and mature DCs therefore coexist, affecting tolerance to peripheral antigens 1999 nat. med.5: 1232-1233. Autoimmunity is generally not associated with inflammation.

Under physiological conditions, only a few apoptotic cells are phagocytosed by immature DC cells.

Environmental factors, possibly supplemented by inflammatory signals themselves, must prevent immature DCs from phagocytosing dead cells.

Only during severely deregulated inflammation is cell death increased and the non-conventional phagocytic cells increase their phagocytic activity, which increases the likelihood that the organism will recognize self antigens as non-self antigens.

The mechanism by which immunocompetent cells recognize self-antigens as harmful or foreign antigens to organisms is the basis of the onset of many autoimmune diseases, such as Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), arthritis, diabetes, thyroiditis, hemolytic anemia, atrophic orchitis, Goodpasture's disease, autoimmune retinopathy, autoimmune thrombocytopenia, myasthenia gravis, primary biliary cirrhosis (primary biliarlity cirhosis), aggressive chronic hepatitis, ulcerative colitis, dermatitis, chronic glomerulonephritis, sjogrens syndrome (Sjogrensyndrome), Reiter syndrome (Reiter syndrome), myositis (miositis), systemic sclerosis, and polyarthritis.

Systemic lupus erythematosus is an inflammatory systemic autoimmune disease characterized by the production of high avidity autoimmune antibodies from T cells.

SLE patients in active disease stage show particularly low plasma levels of C-reactive protein CRP.

It has been found that patients suffering from SLE are congenital lacking the complement factor Clq (1998Nature Genetics 19: 56-59; 1998Nature Genetics 19: 3-4), the factor recognized by PTX3 (1997 J.biol.chem.272: 32817-32823).

In addition, PTX3 binds to autoantigens that are very characteristic of SLE, such as histones and the like (1990 J.exp.Med.172: 13-18; 1997 J.biol.chem.272: 32817-32823).

Previous uses of PTX3 are known, for example in WO99/32516 filed in the name of the applicant, describing the use of the long pentraxin PTX3 in the treatment of neoplastic, inflammatory and infectious diseases.

US 5767252 describes neuronal cell growth factors belonging to the pentraxin family (see also the cited publications). This patent relates to the field of neurobiology.

To date, there has been no satisfactory therapy for most of the autoimmune diseases known. There remains a great need for new drugs that both prevent the recognition of self-antigens as foreign antigens and alleviate the symptoms of these diseases without causing further side effects.

This inhibition determines the blocking of the onset of autoimmune diseases, which underlies the above-mentioned diseases.

It has now been found, surprisingly, that the long pentraxin PTX3 can be used to prevent and alleviate the symptoms of autoimmune diseases.

Indeed, the long pentraxin PTX3 is able to bind apoptotic cells (and not living cells), preventing these cells from being phagocytosed by immature DCs during excessive or deregulated apoptosis.

As mentioned above, during inflammation and increased cell death, the large number of dead cells blocks the ability of the organism to remove these cells, which accumulate in the tissue and can be phagocytized by non-conventional phagocytic cells (e.g., immature DC cells), with an increased likelihood of initiating the autoimmune process.

The combination of apoptotic cells and PTX3 helps macrophages to clear apoptotic cells, preventing apoptotic cells from being phagocytosed by immature DC cells and promoting autoimmune disease.

The autoimmune disease that can be treated by the present invention is selected from the group consisting of Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), arthritis, diabetes, thyroiditis, hemolytic anemia, atrophic orchitis, goodpasture's disease, autoimmune retinopathy, autoimmune thrombocytopenia, myasthenia gravis, primary biliary cirrhosis, aggressive chronic hepatitis, ulcerative colitis, dermatitis, chronic glomerulonephritis, sjogren's syndrome, reiter's syndrome, myositis, systemic sclerosis, and polyarthritis.

"Long pentraxin PTX 3" means any long pentraxin PTX3 or functional analogue thereof, respectively from its origin (human or animal) or synthetic.

The long pentraxin is preferably human PTX3, the sequence of which is described in WO 99/32516.

Some examples illustrating the invention are reported below.

Example 1

PTX3 binding to human apoptotic cells and modulationIts ability to clear

Cell type

1. Leukemia cells

Human leukemia Jurkat T cells were purchased from ATCC (Rockville, Md.) and grown in RPMI (Gibco BRL, Grand Island, NY) (Tissue Culture Medium, TCM) or 1% Nutridoma-SP (Boehringer Mannhein, Germany) (TCM-nut) containing 100U/ml penicillin, 0.1mg/ml streptomycin, 2mM L-glutamine and 10% FCS (Hyclone, Logan, UT).

2. Dendritic cells

Immature human DCs derived from monocytes in circulation were purified from recombinant GM-CSF and IL-4[ (1999) j.leuk.biol., 66: 345-349; (1997) exp.med.185: 317-328], which results in a homogeneous population of immature DC cells.

Apoptosis induction

Jurkat cells were subcultured in TCM-nut to avoid atypical serum cofactor interference, in TCM-nut anti-CD 95 moAb (CH-11, 100 ng/10) was used at 37 deg.C⁶Cells) or 20 "under a u.v. source, to cause apoptosis. Apoptosis was confirmed using flow cytometry and morphological characterization as described (Arthritis Rheum.1998; 41: 205-.

Exposed Phosphatidylserine (PS) was determined by flow cytometry and focused microscopy (see below) after staining with FITC-labeled Annexin V (Bender MedSystems, Prodotti Gianni, Milano, Italy) (0.5ml) in PBS (PBS + +) containing 0.1mM MgCl2 and 0.1mM CaCl2 at room temperature for 10 minutes.

DNA content was detected using flow cytometry.

Some experiments used different drug treatments (5. mu.M BAPTA-AM, 10. mu.g/ml cycloheximide, 20. mu.M staurosporine, 100. mu.M and 50mM pyrrolidine dithiocarbamate PDTC, 100nM dexamethasone) to induce apoptosis.

Cells were necrotized by heating the cells (95 ℃ for 5 min), freeze-thawing or hypertonic shock (10 min in 150mM NaP, pH 7.5, 1.4M NaCl buffer).

Protein

Such as j.biol.chem.1997; 272: 32817 Across 32823, human PTX3 was purified from CHO cells stably and persistently expressing the protein. Such as EMBO j.1992; 11: 813-89, carrying out biotinylation of PTX 3; subsequently as in j.biol.chem.1997; 272: 32817 Across 32823 biotinylated PTX3 was analyzed in native state on 5-10% gradient PAGE.

Purified human C-reactive protein (CRP), serum amyloid P fraction (SAP), and Bovine Serum Albumin (BSA) were purchased from Sigma (st.

PTX3 binding to apoptotic cells

Cells were challenged with biotinylated PTX3 (test range 0.1-500. mu.g/ml) for 30 min at room temperature.

After addition of FITC-conjugated streptavidin (Pierce, Rockford, IL), cell-bound cofactors were visualized by flow cytometry. In the presence of FITC-conjugated streptavidin alone, the fluorescence background was calculated.

The concentration of biotinylated PTX3 used was increased and binding was observed to increase linearly, reaching a plateau at approximately 100. mu.g/ml.

Apoptotic cells were preincubated with PTX3, SAP, CRP and BSA (500g/ml) before adding biotinylated PTX3 (10. mu.g/ml), FITC streptavidin and FACS analysis to verify the specificity of binding.

The binding of PTX3 to dead cells was also characterized using focused imaging: in this case, the stained cell species were fixed on polylysine-coated coverslips for 20 minutes at room temperature in 4% (w/v) paraformaldehyde for 15 minutes at room temperature and mounted in Mowiol medium.

Focused laser scanning microscopy was performed using a Leica TCS-NT (Leica microsystems, Heidelberg, Germany) focused laser scanning microscope equipped with an argon/krypton laser, 75mW multiline.

Jurkat cells triggered apoptosis by CD95(FAS) are readily identified based on nuclear features such as chromatin marginalization, condensation, and fragmentation.

Phagocytosis

Amplified log phase Jurkat cells were apoptotic by u.v. irradiation and labeled with green fluorescent aliphatic dye PKH2-gl (sigma).

After washing, the cells were incubated with or without PTX3 (100. mu.g/ml for 30 minutes at room temperature) and then incubated with DCs for 90 minutes at 37 ℃ or 4 ℃.

Both immature and mature DCs treated with TNF- α (1997 J.exp. Med.185: 317-. As a control, FITC-labeled ovalbumin (Sigma) or green fluorescent latex beads (2 μm diameter, Polysciences, Warrington, Pa.) internalized by immature and mature DCs were determined in the presence or absence of PTX 3. After phagocytosis, DCs were incubated in PBS containing EDTA and trypsin for 15 minutes at 37 ℃ and analyzed by flow cytometry (FACS Scan, Becton and Dickinson, san jose, CA).

The percentage of cells that bound or internalized apoptotic cells was compared in independent samples.

Example 1/1

Jurkat cells were challenged with biotinylated PTX3 and FITC-streptavidin as described above, and their binding was then determined using flow cytometry.

The results (representative of 4 independent experiments) are shown in table 1 below, expressed as the Relative Fluorescence Intensity (RFI) calculated as the average fluorescence intensity divided by the mean fluorescence intensity in the presence of PTX3 and FITC-streptavidin and in the presence of FITC-streptavidin alone.

TABLE 1

Cells	Apoptosis treatment	RFI**
			Living cell	Is free of	1
Apoptotic cells	Cycloheximide	10
			Apoptotic cells	PDTC	12.3
Apoptotic cells	Dexamethasone	17.9
			Apoptotic cells	Staurosporine	19.8
Apoptotic cells	U.V. irradiation (16h)	22.4
			Post-apoptotic cells	U.V. irradiation (48h)	2.8
Necrotic cells	Freezing device	7.3
			Necrotic cells	Boiling by boiling	2.4
Necrotic cells	High osmotic shock	5.6

Jurkat cells; relative fluorescence intensity.

These results indicate that PTX3 has the ability to bind late apoptotic cells and that PTX3 recognition is independent of the initiating stimulus used to trigger apoptosis. Furthermore, PTX3 was unable to bind to cells that evolved to late apoptotic stages.

The results shown in table 1 indicate that only apoptotic cells were able to bind biotinylated PTX3, demonstrating the specificity of PTX3 binding.

Example 1/2

The apoptotic cells bound by PTX3 described above escape internalization of immature DCs.

Immature DCs or TNF-. alpha.treated mature DCs were used in this experiment.

As a control, FITC-labeled ovalbumin or green fluorescent latex beads were internalized by immature as well as mature DCs, in the presence or absence of PTX 3.

TABLE 2

	DC% of internalized Green fluorescent apoptotic cells or latex beads
					Substrate	Immature DC		Mature DC (TNF-alpha treated DC)
	No PTX3	Having PTX3	No PTX3	Having PTX3
					Apoptotic cells	56±8	21±6	6±5	8±6
Latex bead	30±9	28±12	11±5	11±2

The results shown in table 2 (average of 3 independent experiments) indicate that PTX3 does not affect phagocytosis of inert substrates by immature DCs.

Claims (6)

1. Use of the long pentraxin PTX3(PTX3) for the preparation of a medicament for the prevention or treatment of autoimmune diseases.

2. The use of claim 1, wherein the autoimmune disease is selected from the group consisting of Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), arthritis, diabetes, thyroiditis, hemolytic anemia, atrophic orchitis, goodpasture's disease, autoimmune retinopathy, autoimmune thrombocytopenia, myasthenia gravis, primary biliary cirrhosis, aggressive chronic hepatitis, ulcerative colitis, dermatitis, chronic glomerulonephritis, sjogren's syndrome, reiter's syndrome, myositis, systemic sclerosis, or polyarthritis.

3. Use according to claim 1 or 2, wherein the long pentraxin PTX3 is naturally occurring PTX 3.

4. Use according to claim 3, wherein the long pentraxin PTX3 is human pentraxin.

5. Use according to claim 1 or 2, wherein the long pentraxin PTX3 is synthetically obtained PTX 3.

6. The use of claim 1, wherein the autoimmune disease is systemic lupus erythematosus.