ITAN20100083A1 - PHARMACEUTICAL USE OF SEMAFORINE - Google Patents

Description

â € œPharmaceutical use of Semaforineâ €,

TEXT OF THE DESCRIPTION

The present invention relates to traffic lights, and in particular it concerns the pharmaceutical use of these molecules, as well as the pharmaceutical use of plasmids containing the gene instruction whose expression involves the production of traffic lights.

The semaphorin family includes soluble and membrane-bound proteins that act in the nervous system, where they are involved in establishing a correct neuronal network. A growing body of data shows that this family also exerts its action outside the nervous system, playing a fundamental role in cardiac and skeletal development, epithelial morphogenesis, angiogenesis and tumor progression. Furthermore, some studies suggest that these molecules are important actors during an immune response, as they regulate the interactions between T cells and antigen presenting cells during a primary immune response. Several members of the traffic lights act as amplifiers of the immune response, while others, in particular the class 3 secreted traffic lights, are capable of carrying out a negative control on immune functions.

In the course of the research that led to the present invention, semaforin-3A (hereinafter referred to as Sema3A), whose amino acid sequence is attached, has been identified as a potent immunosuppressive factor. Sema3A inhibits T cell functions by promoting growth arrest and / or blocking the secretion of proinflammatory cytokines. The downstream pathways through which Sema3A exerts its action on immune cells remain to be elucidated, but it is likely that they involve a receptor complex consisting of one of two neuropilins (NPs) and one of four plexin-A proteins. NPs act as primary binding sites of the ligand, while plexins-A act as signal transduction components.

It has recently been shown that the Sema3A-dependent pathway is involved in the complex system of interactions between epithelial cells and thymocytes within the thymus. More recently, Sema3A was shown to enhance the in vitro apoptosis of T cells. This proapoptotic effect of Sema3A depended on the activation state of T cells, and was mediated by the engagement of death receptors, particularly the Fas system. (CD95 / APO-1).

Taken together, these observations suggest that Sema3A may play an important role in the generation and maintenance of central and peripheral immune tolerance.

Rheumatoid arthritis (RA) is a chronic, destructive and autoimmune disease of the joints characterized by high levels of proinflammatory cytokine production. Specifically, within an inflamed RA synovial membrane, proinflammatory cytokine levels exceed those of anti-inflammatory mediators. Furthermore, an effective therapeutic localization of cytokines in RA, in particular of TNF-Î ± and IL-6, has demonstrated their fundamental importance in pathogenesis. In contrast to other semaphorins, such as Sema3C, no expression of Sema3A was detected in the RA synovium, and lymphoblastoid cells from RA patients were shown to be resistant to Fas-mediated cell death.

On the basis of the experimental findings described above, an aim of the present invention is to provide a pharmaceutical cure for autoimmune diseases, which takes into account the properties and characteristics of traffic lights.

A further object of the present invention is to provide a drug for the treatment of rheumatoid arthritis, osteoarthritis and joint inflammatory states in general.

The object of the present invention is therefore the use of traffic lights for the preparation of a drug; another object of the present invention is the use of plasmids encoding gene information for the production of traffic lights for the preparation of a drug.

In particular, the present invention relates to the use of semaphorin 3A, or the plasmid encoding the gene information for the production of semaphorin 3A for the preparation of a drug. The drug in question is a drug capable of fighting rheumatoid arthritis, osteoarthritis and similar pathologies.

Further advantages and characteristics of the present invention will become evident from the following description of some executive examples, and with reference to the attached drawings, in which:

Figures 1A and 1B are two diagrams representing respectively the production of cytokines in cells following a treatment with Sema3A or Sema3C at the different concentrations indicated, and stimulated with plate-bound antibodies specific for CD3 and CD28 over the course of 24 hours;

Figure 2 is a diagram relating to the production of cytokines by PBMC following a treatment with Sema3A or Sema3C (both at 360 ng / ml) and exposure to LPS 1 ng / ml for 24 hours;

Figures 3A to 3C are diagrams illustrating the production of cytokines in PBMC cells of individuals affected by RA;

Figures 4A and 4B are diagrams representing respectively the expression of CD25 from CD4 + T cells derived from five different donors and the expression on the cell surface of CD25 for one of five experiments;

Figures 5A and 5B are diagrams representing respectively the mean expression ± e.s.m. (n = 3) of IL-17, and a representative intracytoplasmic staining of IL-17 from CD4 + T cells after 7 days of treatment;

Figures 6A to 6C represent the mRNA expression levels (multiplicative factor of induction with respect to T cells without exogenous cytokines) of RORC2, T-bet and GATA3, shown after stimulating CD4 T cells;

Figure 7 is a diagram illustrating the apoptosis assessment for human CD4 + T cells stimulated with antibodies;

Figure 8 is a diagram illustrating the apoptosis assessment for human CD4 + T cells stimulated with antibodies;

Figure 9 is a diagram illustrating cytokine expression in antibody-stimulated CD4 + T cells;

Figures 10A and 10B are diagrams representing the response of guinea pigs to a preventive treatment according to the present invention;

Figures 11A and 11B are diagrams representing the response of guinea pigs to a curative treatment according to the present invention;

Figures 12A and 12B are representative sections of joint tissue stained with H&E of mice treated in prevention according to the present invention;

Figure 13 is a diagram representing the histopathological scores of inflammation (a), tissue formation (b) and bone and cartilage erosion (c) in mice treated for prevention, evaluated against a control;

Figures 14A to 14C are diagrams which represent the evaluation of the titer of the anti-CII IgG isotypes by ELISA in the sera of mice treated according to the present invention;

Figures 15A to 15C are diagrams representing the evaluation of cytokine levels by ELISA in CD4 + T cells of mice treated according to the present invention;

Figures 16A and 16B are diagrams representing the hypodiploid DNA content after PI staining in mice treated with Sema3A or treated with the control plasmid;

Figure 17 is a diagram illustrating the cell death of lymph node cells which have been treated in accordance with the present invention; And

Figures 18A and 18B are diagrams which respectively represent the evaluation of paw swelling parameters and arthritis score in mice treated according to the present invention.

Materials and methods

Cell culture and apoptosis evaluation.

CD4 + T cells were purified from PBMC (peripheral blood mononuclear cells), total spleen or lymph node cells, by incubation with an anti-CD4 mAb (GK1.5), followed by a positive selection of CD4 + cells with specific isolation kits. for T cells (all from Miltenyi Biotec). We synthesized recombinant human Sema3A and Sema3C proteins as described in Banu, N., J. Teichman, M. Dunlap-Brown, G. Villegas, and A. Tufro.

2006. Semaphorin 3C regulates endothelial celi function by increasing integrin activity. PHASEB J. 20: 2150-2152.

Subsequently, apoptosis was assessed by flow cytometry, using fluorescein isothiocyanate (FITC; Roche) conjugated with annexin V in combination with PI (propidium iodide, Sigma), and we used light microscopy to confirm the presence of variations. morphological characteristics of apoptosis.

Induction and evaluation of the CIA.

Collagen-induced arthritis (CIA) in DBA / l mice was induced and classified. Briefly, DBA / l (Charles River Laboratories) mice received intradermal immunization with bovine CII 100 µg / mouse (Chondrex) emulsified in CFA containing heat-killed Mycobacterium tuberculosis H37Ra, 250 µg / mouse (BD). Twenty-one days after immunization, the mice were recalled by subcutaneous injection, at the base of the tail, with bovine CII, 100 µg / mouse. For the treatment experiments, only mice with clinical arthritis (mean visual arthritis score of 4) were randomized and treated. The number of mice used in these studies was the minimum required to obtain statistical significance. Mice were classified for arthritis using the following visual classification system: grade 0, no swelling or erythema; grade 1, mild swelling and erythema, or inflammation of the fingers; grade 2, moderate swelling and erythema, confined distal to the median region of the paw; grade 3, more pronounced swelling and erythema with ankle extension; Grade 4, severe swelling, erythema and joint stiffness of the ankle, paw and fingers. Each limb was graded with a score from 0 to 4, with a maximum possible score of 16 for each individual mouse.

The thickness of the paw was determined by measuring, with calibers from 0 to 10 mm, the thickness of the hind leg affected with greater severity. Mice were treated with each i.p.plasmid, starting the day before CIA induction for prevention experiments, and following arthritis development and randomization for treatment experiments.

Treatment protocols

Mice (22-25 g) were treated with the Sema3A plasmid (e.g. pAGNT-Sema3A plasmid; 1 pg / animal; equivalent to 40 pg / kg, in a final volume of 0.2 ml PB S) i.p. once daily, up to day 50. Control mice received a psDNA3 at the same time points. The selected dose of DNA complies with preliminary animal experiments, where it was observed that the expression of the Sema3A protein in the PBMC, spleen and liver was upregulated by the administration of a plasmid encoding Sema3A (data not shown). The expression level of the Sema3A protein was assayed by Western blot in newly isolated splenic cells from mice treated with Sema3A or with the control plasmid. Mice were sacrificed 24 hours after the last treatment, and cellular used were separated in 10% SDS-PAGE, transferred to membrane, and probed with anti-Sema3A Ab (antibodies). For in vivo administration of zVAD-fmk, twenty-four hours after intrapleural injection of the Sema3A plasmid, mice received an i.p. of zVAD-fmk (z-Val-Ala-DL-Asp-fluoromethylketone; Bachem) 5 mg / kg, at a final concentration of 1 mg / ml in saline, or appropriate control amounts, the DMSO vehicle, as described previously (23). Two additional doses of zVAD-fmk were given i.p. 10 and 25 days later, and all mice were sacrificed after 50 days.

Histopathological studies.

The hind limbs were fixed and decalcified in Cal-Ex II (Fischer Scientific) for 3 days prior to paraffin embedding. Sections were stained with H&E and evaluated by a researcher, blinded to the treatment status, in terms of synovitis, tissue formation and bone and / or cartilage destruction, based on a previously described classification system: grade 0, normal; grade 1, mild inflammation, mild hyperplasia of the synovial lining layer, mild cartilage destruction without bone erosion; grades 2-4, increasing grades of inflammatory cell infiltrates, hyperplasia of the synovial lining, tissue formation, and destruction of cartilage and bone.

ELISA.

After exposure to the compound, cells were stimulated with LPS (Escherichia coli, 01 1 1: B4, Sigma) at 1 pg / ml and 5 pg / ml, respectively, for 24 hours, and the supernatants were analyzed for IL-23 (eBiosciences), TNF-a (BD Bioscience) and IL-6 (BD Bioscience) by ELISA. We analyzed immune cells with antibodies against CD3 and CD28 for 24 hours in terms of IFN-γ and IL-2 or IFN-γ production by ELISA (BD Bioscience). The expression of CD25, NP- 1 and NP-2 on the CD4 subset of PBMCs was measured by flow cytometry (BD Pharmingen).

Differentiation of type 17 helper T cells. CD4 + T cells (7.5 x 104 per well) were stimulated with antibodies against CD3 and CD28 in the presence of TGF-β 5 ng / ml (from R&D Systems) and IL-21 25 ng / mL (by Celi Sciences), for a period of 7 days in the absence of IL-2, as described above (24). Intracytoplasmic staining was performed using standard methodologies and anti-IL-17-APC (R&D Systems) and anti-IFN-γ-ΡΠ• or anti-IL-10-PE (BD Biosciences) antibodies. Cells were stimulated with phorbol 12-myristate 13-acetate 5 ng / ml and ionomycin 200 ng / ml, in the presence of brefeldin A (Sigma), before intracellular staining.

Real-time PCR.

We included, without further selection, ten RA patients, 10 OA patients undergoing knee joint replacement surgery, and 5 control subjects without inflammatory conditions and undergoing arthroscopic knee surgery. Synovial tissue samples were obtained immediately after opening the knee joint capsule, or during arthroscopic surgery (in controls). Total RNA was isolated, and real-time RT-PCR analysis was performed on a Chromo4 sequence detector (Bio-Rad, Milan, Italy). All primers and probes were obtained from Applied Biosystems and used according to standard methodologies. Details on thermal cycling sequences and conditions are available on request. The data was acquired and analyzed with the Chromo4 sequence detector software.

Determination of anti-collagen Ab.

CII-specific IgG (Chondrex) was measured using an ELISA kit and following the manufacturer's protocol. Levels of IgG isotypes of anti-collagen type II Ab in sera from arthritic DBA / l mice were assessed using the assay kit for anti-collagen type II Ab, substituting total TAb IgG conjugated to HRP with IgG1, IgG2a, Isotype-specific goat anti-mouse IgG2b and IgG3 conjugated to HRP (Jackson ImmunoResearch Laboratories).

Statistical analysis.

To perform comparisons between treatment groups, we conducted unpaired t-test (Mann-Whitney), paired t-test, and one- and two-way ANOVA (where appropriate). For the ANOVA we used a Bonferroni post hoc analysis to compare the treatment groups. We conducted all statistical analyzes with GraphPad Prism software (version 4.01). Data are expressed as mean ± e. s.m. (standard error of the mean), considering P values less than 0.05 to be significant.

First, the different expression of Sema3A and Sema3C was confirmed in synovial tissue samples obtained from 10 patients with RA, 10 patients with osteoarthritis (OA), and 5 patients without inflammatory conditions. Most of the OA and RA synoviums expressed Sema3C, while all synovial tissue specimens derived from OA and RA patients did not show Sema3A mRNA expression. It is interesting how Sema3A and Sema3C are expressed differently in inflammatory joints, as these two traffic lights can exert different effects on autoimmune disorders.

To evaluate the impact of these semaphorins on immune cells, the effects of Sema3A and Sema3C on cytokine production in stimulated PBMCs were compared. When the PBMCs treated with Sema3A were stimulated through antibodies to CD3 and CD28, the release of IFN-γ was inhibited by up to 60%, and the production of IL-2 was inhibited by 50%, at all concentrations. tried (180-480 ng / mL) (Figure 1A). Conversely, Sema3C did not substantially affect cytokine release (Figure 1B). We also compared cytokine production in endotoxin-stimulated PBMCs. In contrast to Sema3C, Sema3A reduced IL-23 production by 70%, and tumor necrosis factor a (TNF-a) and IL-6 by approximately 50% (Figure 2). These results indicate that Sema3A selectively regulates the production of inflammatory cytokines in PBMCs stimulated with LPS or with anti-CD3 and CD28 monoclonal antibodies.

To better understand the effect of Sema3A and Sema3C on immune cells, it was necessary to determine whether their natural receptors, NP-1 and NP-2, were expressed in PBMCs derived from healthy donors and RA patients. PBMCs derived from RA patients exhibited higher NP-1 expression in single-positive CD4 + cells, compared to PBMCs derived from healthy donors. Furthermore, NP-1 expression on CD4 + T cells increased during stimulation with antibodies to CD3 and CD28. In contrast, no difference in NP-2 expression on CD4 + T cells was observed between PBMCs derived from healthy donors and from RA patients. Stimulation conditions did not affect NP-2 expression, further supporting the view that the Sema3A NP-1 receptor participates in T cell activation.

Subsequently, the assessment was made with respect to whether Sema3A regulates the production of cytokines in stimulated and derived PBMCs from individuals with active RA. Similar to the findings described above (see Fig. 2), Sema3A blocked the production of IL-23 up to 80%, and the secretion of IL-6 and TNF-Î ± up to 50% (Figures 3A-3C) . Taken together, these results may indicate that, in RA patients, Sema3A expression controls the final production of proinflammatory cytokines.

To better understand the role of Sema3A on T cell response, we examined the effect of Sema3A on early T cell activation and on the differentiation of T helper type 17 (TH17). Initial T cell activation, as measured by CD25 expression, was affected by Sema3A (Figures 4A, 4B). In addition, Sema3A treatment partially reduced TH17 cell differentiation, as measured by IL-17 expression, after 7 days of culturing T cells in the presence of IL-21 and transforming growth factor β (TGF-β ) (Figures 5A, 5B). Furthermore, treatment with Sema3A was confirmed to inhibit the mRNA expression of the transcription factor RORC2 (Figures 6A-6C), a known master gene of the differentiation of human IL-17 secreting T cells (24). T-bet (also known as TBX21) is the master regulator of TH1 cells that secrete IFN-γ, while GATA3 induces TH2. Notably, it was found that treatment with Sema3A also reduced T-bet mRNA levels and increased GATA3 expression in IL-21-stimulated CD4 + T cells (Figures 6A-6C). In human cells treated with Sema3C, no differences were observed in the mRNA levels of RORC2, T-bet and GATA3 compared to untreated cells (data not shown). These data suggest that Sema3A affects both initial T cell activation and differentiation into inflammatory effector cells in the presence of polarizing cytokines.

Having shown in vitro that T cell apoptosis was markedly influenced by Sema3A, the possibility was evaluated that the effects described above could be related to the apoptotic properties of Sema3A. At 20 hours, Sema3A accelerated apoptosis in the presence of anti-CD3 and CD28 monoclonal antibodies, as well as in the presence of polarizing cytokines (Figure 7), without directly inducing apoptosis per se. It was therefore found that the proapoptotic effect of Sema3A was caspase-dependent since preincubating PBMCs with zVAD-fmk, a broad-spectrum caspase inhibitor, avoided Sema3A-enhanced apoptosis (Figure 8). Furthermore, we found that Sema3A-induced inhibition of IL-2 and IL-17 was attenuated by zVAD-fmk (Figure 9), offering a potential mechanism explaining the inhibitory activity of Sema3A on immune cells.

To determine the impact of Sema3A on inflammatory responses in vivo, it was verified, using a plasmid coding for Sema3A, whether the overproduction of Sema3A had the ability to prevent and treat autoimmune arthritis in the CIA model. For CIA prevention studies, an i.p. of Sema3A expressing plasmid (1 pg) 1 day before CIA induction, and then once every 5 days, until day 50. Paw swelling (Figure 10A) and clinical score (Figure 10B) increased significantly time-dependent, in mice immunized with type II collagen and injected with the control plasmid without insert (pcDNA3). On the other hand, mice treated with the Sema3A coding plasmid showed less paw swelling and lower clinical scores (Figures 10A and 10B). At the end of the experiment (day 50), treatment with Sema3A markedly reduced the incidence of CIA. There was no evidence of toxicity or weight loss in the mice that received the DNA. To determine the expression levels of the Sema3A protein, splenic cells were isolated from mice 24 hours after the last DNA treatment (day 48). A western blotting was then conducted. The Sema3A protein was effectively overexpressed by injection with the plasmid while, in spleen Usates derived from arthritic mice that had received pcDNA3, only a slight expression of Sema3 A was evident.

For the CIA treatment studies, mice with overt clinical arthritis (mean score of 4; day 20-22 after primary immunization) were randomized and injected with either a Sema3A encoding plasmid or a control plasmid. Similar to prevention studies, Sema3A inhibited the progression of overt arthritis, as assessed by both paw swelling and clinical score (Figures 12A and 12B).

A histopathological analysis was conducted on the hind legs collected from the CIA affected mice. Representative images of H&E stained joint tissue sections from mice injected with the Sema3A plasmid and the control plasmid in CIA prevention studies are shown (Figures 12A, 12B). Histopathological evaluation, conducted by a researcher blinded to the treatment group, showed that Sema3A caused a sharp reduction in synovitis, cloth and erosion scores in CIA prevention experiments (Figure 13). A similar result was observed in the treatment of full-blown CIA (data not shown).

To determine whether Sema3A could affect humoral responses for CII during the treatment period, an analysis of anti-CII IgG levels was conducted on sera from Sema3A-treated and control mice. In normal mice, the levels of anti-collagen type II Ab were below the detection limits in the assay (data not shown), while in arthritic mice injected with the control plasmid, the concentrations of anti-collagen type II they were very high (Figure 14A). Type II anti-collagen Abs were mainly IgG1 and IgG2a (Figures 14B and 14C). Total concentrations of type II anti-collagen Ab and serum levels of the IgG2a subclass of type II anti-collagen Ab were lower in mice treated with Sema3A (Figures 14A and 14B). In particular, at the end of the treatment, a slight but significant increase in the absolute levels of anti-CII IgGl was observed in the sera of the DBA / l mice injected with Sema3A compared to the control mice (Figure 14C).

To clarify whether the class shift to IgGl was related to variations in the cytokine secretion pattern, the production of IFN-γ, IL-17 and IL-10 in CD4 + T cells was measured 7 days after the second immunization of the mice. with CII. Sema3A treatment lowered IFN-γ and IL-17 expression (Figures 15A and 15B). Furthermore, it was found that continuous treatment with Sema3A in vivo significantly increased IL-10 production (Figure 15C). As observed in human T cell differentiation assays, treatment with Sema3A was also confirmed to enhance the expression of transcription factor GATA3 mRNA in spleen CD4 + T cells obtained at day 45 (data not shown). These results suggested that repeated treatment with Sema3A in chronic inflammation reduces anti-CII IgG levels and induces a deviation from a type 1 to type 2 mediated immune response.

Starting from Sema3A-induced caspase-dependent apoptosis of activated T cells in vitro (see Figures 7 to 9), it was determined whether the best CIA resolution by Sema3A was mediated by a similar induction of caspase apoptosis. dependent in vivo. The mice involved in the gene therapy protocol were sacrificed at the end of the treatment, the draining lymph nodes were excised, and the cells were cultured for 24 hours in the presence or absence of CII for the detection of apoptotic cells. Lymph node cells from all Sema3A-treated mice showed increased susceptibility to antigen-induced apoptosis, as shown by a FACS analysis of hypodiploid DNA content (Figures 16A, 16B). In contrast, mice treated with pCDNA3 showed background levels in terms of hypodiploid DNA content (Figures 16A, 16B). In the absence of CII, cell death levels were between 2 and 5% in both experimental groups (data not shown). Furthermore, the increase in lymph node cell apoptosis observed in association with Sema3A treatment was markedly inhibited by zVAD-fmk treatment (Figure 17). Finally, systemic administration of zVAD-fmk prevented Sema3A-induced resolution of paw swelling (Figure 18A) and clinical in vivo score (Figure 18B). Taken together, the results presented here suggest a correlation between the molecular properties reported for Sema-3A in vitro and its therapeutic potential in vivo.

Traffic lights play a central role in guiding the axon, and emerging evidence points to different functions of the numerous traffic lights (including Sema3A) in the immune system. For example, Sema4D and Sema4A play crucial roles in mutual stimulation between T cells and APCs (26). Furthermore, Sema3A inhibits human monocyte migration in response to cytokine stimulation, while also reducing the proliferation of leukemic T cells following TCR stimulation.

The overexpression of Sema3A was sufficient to prevent CIA and to achieve a drastic arrest of pathological progression, as assessed by the clinical, histopathological and immunological manifestations of the arthritis. The present results extend the findings demonstrating that Sema3A plays a distinct role in the regulation of apoptosis of human T cells and monocyte-derived macrophages, showing that the proapoptotic activity of Sema3A is essential in the regulation of inflammatory responses in vivo.

One of the areas studied most frequently in the etiology of autoimmune diseases is the role of dysregulated apoptosis. The first clue that a loss of apoptosis may affect the onset of autoimmune disorders comes from animal models carrying spontaneous mutations in the Fas or Fas ligand genes. Later, evidence that resistance to apoptosis could promote autoimmune disorders was derived from the observation that the dominant Fas gene mutation was related to Canale-Smith syndrome, a lymphoproliferative disease.

Here it is clarified how susceptibility to apoptosis was increased in lymph node cells derived from mice injected with Sema3A DNA. Furthermore, administration of zVAD-fmk prevented Sema3A-induced resolution of paw swelling and clinical score in arthritic mice. Murine CIA is induced in genetically susceptible DBA / l mice, and resembles RA in both cellular and humoral mechanisms involved in the pathogenic process. The blood-derived cells that migrate into the joints, together with the activated synovial cells, produce inflammatory and type 1 cytokines, as well as degradative enzymes that progressively lead to the destruction of cartilage and bone. Therefore, the apoptosis promoted by Sema3A could provide an ideal mechanism that uses an endogenous protein of natural origin capable of terminating the attack of autoimmune T cells, preventing the expansion of dominant self-aggressive clones and guaranteeing a minimum of harmful damage at a distance ( bystander) to the local parenchyma.

Treatment with Sema3A produced an overall reduction in anti-CII Ab levels, shifting the balance towards a type 2 mediated immune response, a new function of traffic lights that remains to be examined further. However, it could be hypothesized that the ongoing autoimmune response, mainly controlled by proinflammatory Thl cells, may be inhibited by the apoptosis of memory and activated T cells. Once the inflammatory action of type 1 cytokines is removed, a previously repressed Th2 response may come to light. This observation may provide a potential association between Sema3A-induced apoptosis and immune deviation. Consequently, only Thl effector cells are said to be susceptible to TCR- and Fas-mediated apoptosis, leading to selective Th2 survival. Furthermore, the results we obtained on the Thl / Th2 shift through Sema3A treatment are quantitatively more robust than those previously reported for Sema-related hepatocyte growth factor (HGF).

It is worth noting that Sema3A reduces IgG2a levels and increases IgGl levels. IgG2a is an antibody that fixes the complement, contributing to tissue damage by triggering the production of anaphylatoxins that cause the extravasation and infiltration of neutrophils which, in turn, secrete pathogenic mediators of inflammation. Therefore, Sema3A can also have a therapeutic impact in multiple sclerosis and myasthenia gravis, where antibodies and complement have been involved as well as RA, as regards the pathology of the disease.

There has already been evidence of a higher prevalence of Sema3C in the synovial tissue of RA patients. This nerve repellent factor of sympathetic nerve fibers could be an element responsible for reduced sympathetic innervation in an RA tissue. We found a negative correlation in RA-associated expression between Sema3C and Sema3A in human subjects. Furthermore, Sema3A, but not Sema3C, blocked the production of cytokines in PBMC stimulated with both CD3 / CD28 and LPS. The results obtained here demonstrate that class III secreted semaphorins have distinct roles in the regulation of cell function, showing that cytokine release is selectively dependent on Sema3A. The molecular basis of these different models is currently unknown. Recent data suggest that NP-1, the cell surface receptor for Sema3A, mediates contacts between dendritic cells and T cells through homotypic interactions, and that this receptor is essential for the initiation of primary immune responses. Unlike NP-2, NP-1 was found to be strongly expressed on activated CD4 + T cells, suggesting that Sema3A can control cytokine release via NP-1. This is in agreement with previous evidence where apoptosis of Sema3A-induced T cells were reduced by a neutralizing anti-NP-1 antibody. Nonetheless, the intracellular domain of NP-1 is short and does not appear to be able to mediate functional responses against Sema3A. Plexin-Al is a Sema3A co-receptor in the nervous system, and plays a signaling role in immune responses. Further studies are needed to understand whether plexin-Al may play a role in the immunoregulatory activity of Sema3A.

Claims (10)

CLAIMS 1. Use of a protein belonging to the class of semaphorins as a drug. 2. Use of a protein belonging to the class of semaphorins as a drug, in which said protein is semaphorin 3A. 3. Use of a protein belonging to the class of semaphorins as a drug for the treatment of autimmune diseases. 4. Use of a protein belonging to the class of semaphorins as a drug for the treatment of joint inflammatory states. 5. Use of a protein belonging to the class of semaphorins according to claim 4, in which said protein is semaphorin 3A and the inflammatory states are rheumatoid arthritis or osteoarthritis. 6. Use of a plasmid encoding the expression of a protein belonging to the class of semaphorins as a drug. 7. Use of a plasmid encoding the expression of a protein belonging to the class of semaphorins as a drug, in which said protein is semaphorin 3 A. 8. Use of a plasmid encoding the expression of a protein belonging to the class of semaphorins as a drug for the treatment of autimmune diseases. 9. Use of a plasmid encoding the expression of a protein belonging to the class of semaphorins as a drug for the treatment of joint inflammatory states. 10. Use of a plasmid encoding the expression of a protein belonging to the class of semaphorins according to claim 8, in which said protein is semaphorin 3A and the inflammatory states are rheumatoid arthritis or Î "osteoarthritis.