Rheumatology. Semaphorin 3A: an immunoregulator in systemic sclerosis

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1 DOI /s Rheumatology INTERNATIONAL SHORT COMMUNICATION - PATHOLOGY REVIEWS Semaphorin 3A: an immunoregulator in systemic sclerosis Doron Rimar 1 Yuval Nov 2 Itzhak Rosner 1 Gleb Slobodin 1 Michael Rozenbaum 1 Katy Halasz 3 Tharwat Haj 3 Nizar Jiries 1 Lisa Kaly 1 Nina Boulman 1 Zahava Vadasz 3 Received: 17 January 2015 / Accepted: 9 April 2015 Springer-Verlag Berlin Heidelberg 2015 Abstarct Semaphorin 3A (sema3a) plays a regulatory role in immune responses, mainly affecting the activation of regulatory T cells. It has been found to correlate with disease activity in rheumatoid arthritis and systemic lupus erythematosus (SLE). To investigate the expression of sema3a in patients with systemic sclerosis (SSc) compared to healthy controls and SLE disease controls and to correlate it with clinical characteristics, 27 SSc patients, 42 SLE patients and 28 healthy controls were enrolled. Serum level of sema3a was measured by ELISA, and expression of sema3a on regulatory T cells was evaluated by FACS analysis. SSc patients were evaluated for demographics, clinical manifestations, routine laboratory results, nailfold videocapillaroscopy, pulmonary function tests, echocardiograms, modified Rodnan skin score, and disease activity and severity scores. Serum levels of semaphorin 3A were lower in SSc compared to healthy controls ± 5.7 versus ± 8.4 ng/ ml, p < and similar to SLE 15.7 ± 4.3 ng/ml. The expression of semaphorin 3A on regulatory T cells was also lower in SSc compared to healthy controls 61.7 ± 15.7 versus 88.7 ± 3. 7 % (p < ). Semaphorin 3A serum level inversely correlated with the duration of disease: r = 0.4, p = and with low C4 level r = 0.66, p = SCL- 70 antibody positivity was associated with a lower semaphorin 3A level (difference in mean of 3.44, p = 0.06). Sema3A expression is low in SSc serum and more specifically on regulatory T cells. This may help explain the reduced activation of regulatory T cells in SSc. Keywords Semaphorin 3A Systemic sclerosis Inflammation T regulatory cells Abbreviations Sema3A Semaphorin 3A SLE Systemic lupus erythematosus ACR American College of Rheumatology * Doron Rimar doronrimar@gmail.com Yuval Nov yuval@stat.haifa.ac.il Itzhak Rosner rosneri@tx.technion.ac.il Gleb Slobodin gleb.slobodin@gmail.com Michael Rozenbaum mrozenb@gmail.com Katy Halasz katy.halasz@b zion.org.il Tharwat Haj Tharwat.haj@b zion.org.il Nizar Jiries n_jiries@hotmail.com Lisa Kaly lisakaly@yahoo.fr Nina Boulman doctor.nina@yahoo.com Zahava Vadasz Zahava.vadas@b zion.org.il 1 Rheumatology Unit, Faculty of Medicine, Bnai Zion Medical Center, Technion, POB 4940, Haifa, Israel 2 Division of Statistics, Haifa University, Haifa, Israel 3 Division of Allergy and Clinical Immunology, Faculty of Medicine, Bnai Zion Medical Center, Technion, POB 4940, Haifa, Israel

2 SLEDAI Systemic lupus erythematosus disease activity index Ssc Systemic sclerosis VEGF Vascular endothelial growth factor Introduction Semaphorins are a large family of secreted and membranebound proteins originally discovered in the nervous system, involved in repulsive axon guidance during nervous system development [1]. Recent data have pointed to the involvement of semaphorins in the regulation of the immune system, thus denoted as immune semaphorins [2, 3]. Semaphorin 3A (sema3a), a secreted member of this family, is now recognized as a potent immuno-regulator during all immune response stages, from early initiation to the late phase of inflammatory processes [4]. Sema3A expression has been found to be increased on differentiating macrophages and activated T cells, suggesting that it has a role in modulating inflammatory conditions [5]. Sema3A expression on T regulatory cells has been recognized as a suppressive marker, contributing to the regulatory properties of these cells [6]. In 2010, Catalano et al. were the first to report on the defective expression of sema3a in CD4+ T cells derived from patients with rheumatoid arthritis (RA). The altered expression on T cells was shown to correlate with the progression of RA [7]. Recently, we described the presence of low serum semaphorin 3A levels in systemic lupus erythematosus (SLE) patients, correlating with SLEDAI scores, reflecting disease activity [8]. Becoming a frontier player in the regulation of immune responses and the maintenance of self-tolerance, sema3a should be expected to be involved in the pathogenesis of many autoimmune diseases. Materials and methods Patient populations Twenty-seven consecutive patients with SSc who fulfilled the American College of Rheumatology (ACR) classification criteria for SSc were recruited from the Bnai Zion Medical Center rheumatology clinics and ambulatory care facility after obtaining informed consent. Twenty-eight ageand gender-matched individuals from the hospital staff were recruited as healthy controls. A serum level of sema3a from SLE patients was also evaluated as disease control. Demographic characteristics and clinical manifestations of SSc patients were assessed and recorded in advance of blood sampling including: age, gender, BMI, smoking history, disease duration defined as years from diagnosis of non-raynaud s phenomena, SSc subtype (limited cutaneous or diffuse cutaneous SSc according to LeRoy criteria), evaluation of skin involvement by modified Rodnan skin score (mrss), digital ulcer currently or in the past, critical ischemia in the past, fissures, telangiectasia, calcinosis, myositis, gastritis or diarrhea, diastolic dysfunction per echocardiography, lung fibrosis by auscultation and plain roentgenography or high-resolution CT (if pulmonary fibrosis was suspected by auscultation or chest X-ray or reduced DLCO <60 %) and pulmonary function tests (FVC and DLCO). Laboratory results were evaluated: blood count, creatinine, urea, autoantibodies (anti-centromere, anti-scl-70, ANCA), C3, C4, ESR, CRP and spot urine test or 24-h urine collection for protein. Each patient underwent nailfold videocapillaroscopy (NVC) with findings classified as early, active or late [12]. Disease severity and activity were determined by the Medsger disease severity scale and Valentini activity index [13]. Finally, current and previous medical treatments were recorded. Exclusion criteria were as follows: pregnancy, age below 18, other coexistent connective tissue disease including systemic lupus erythematosus and mixed connective tissue disease and other known fibrotic states including cirrhotic liver disease. The study was approved by the local research ethics board, and all patients gave their informed consent. Semaphorin 3A serum level The measurement of sema3a serum level was conducted using a commercial ELISA kit (MBS-MyBiosource, San Diego, California, USA) according to the manufacturer s instructions. The serum samples were stored at 20 C until ELISA evaluation. The expression of semaphorin 3A on CD25 high CD4+ T cells The expression of sema3a on CD4+CD25+ high T cells from healthy controls and SSc patients was assessed by staining mononuclear cells with monoclonal antibodies, human anti-cd4 PE and CD25 PC5 (Immunotech, Beckman Coulter, Marsellie, France), and human anti-sema3a AlexaFluor 488 (R&D, Minneapolis, MN, USA), and evaluated in flow cytometry software (FC500 and CXP software, Beckman Coulter, Brea, CA, USA). Statistical analysis Continuous data were described by means and SD and categorical variables as frequencies and percentages (Table 1). Comparisons between the four patient groups were made using one-way ANOVA followed by Tukey s post hoc test. We further evaluated relationships between semaphorin 3A level and disease-related covariates. For numerical

3 covariates, the relationship was studied by a correlation test and is reported via Pearson s r. For binary covariates, the relationship was studied by a two-sample, two-tailed t test and is reported via Δ, the difference in means of the log-transformed data (and the difference in means of the raw data in parentheses). p value of <0.05 was considered significant. Statistical analysis was performed using the R Foundation for Statistical Computing Results Patient population Twenty-seven SSc patients, 42 SLE patients as a disease control group and 28 healthy controls were enrolled in this study. There were no significant differences between the groups with respect to their age, gender, BMI and smoking habits (Table 1). Table 1 summarizes the demographic, clinical and immunological data as well as treatments of the patient populations. Sixty-three percent of the SSc patients (17 patients) had limited cutaneous disease, and the other 37 % (10 patients) had diffuse cutaneous disease, with lung involvement in eight of them. Semaphorin 3A serum levels As seen in Fig. 1a, the serum levels of semaphorin 3A were lower in SSc compared to healthy controls 14.4 ± 5.7 versus 27.1 ± 8.4 ng/ml, p < and similar to SLE 15.7 ± 4.3 ng/ml. Semaphorin 3A expression on T regulatory cells The expression of semaphorin 3A on regulatory T cells was also lower in SSc compared to healthy controls 61.7 ± 15.7 versus 88.7 ± % (p < ), Fig. 1b. The level of sema3a expression on T reg cells in the SLE patient group was similar to that in the control group. There was no significant difference between patients with limited compared to diffuse SSc with respect to serum semaphorin 3A levels, 14.3 ± 3.9 versus 14.4 ± 6.6 ng/ml, nor on regulatory T cells 64.7 ± 8 versus 64.1 ± 15 % (data not shown in the table). Clinical correlation On univariate analysis, semaphorin 3A serum level in SSc correlated inversely with the time since diagnosis r = 0.4, p = (Fig. 2) and with low C4 level r = 0.66, p = Although no difference was found between limited and diffuse disease with regard to semaphorin 3A level, anti-scl-70 antibody positivity was associated with a lower semaphorin 3A level in serum (difference in mean of 3.44, p = 0.06). Discussion In this study, we have demonstrated for the first time that the serum level and the expression of semaphorin 3A on regulatory T cells in patients with SSc are reduced. This lower expression is correlated with low levels of C4 and anti-scl-70 antibody and inversely correlated with disease duration. This lower expression was not found to correlate with disease activity nor severity and thus may be related only to the pathogenesis of this disease. Semaphorin 3A has been found to be reduced in other autoimmune diseases such as rheumatoid arthritis and SLE. Thus, it has been speculated to be regulator of autoimmunity, such that its reduced activity may tip the balance toward autoimmunity. The finding of reduced expression of semaphorin 3A on T regulatory cells in SSc is in line with former studies, which suggested either increased or decreased number of these cells but denoted inefficient T regulatory activity in autoimmune diseases such as SSc [9 11]. Another study demonstrated increased number of T regulatory cells in patients with SSc, correlating with activity and severity of the disease. However, this expansion of T reg cells was not accompanied by increased levels of TGF-β or IL-10 production, namely consistent with impaired regulatory function [12]. Thus, the finding of reduced sema3a expression on the T regulatory cells in SSc patients may be associated with impaired regulatory function. Vasculopathy is one of the hallmarks of systemic sclerosis. Along with ischemia and hypoxia, SSc is characterized by compensatory but dysfunctional neoangiogenesis. These phenomena can be readily visualized on nailfold videocapillaroscopy, which demonstrates enlarged giant capillaries and microhemorrhages in the active stage, a sign of immature and unstable microvessels formed during an uncontrolled angiogenic response and, on the other hand, deletion of capillaries along with bizarre-shaped capillaries in the late stage, reflecting ischemia and ineffective neoangiogenesis. Despite the overall decrease in the angiogenic response to hypoxic stimuli, several proangiogenic mediators have been found to be up-regulated in the skin and serum of SSc patients [14]. This proangiogenic response is mediated by several important factors including: VEGF, platelet-derived growth factor (PDGF), transforming growth factor-β 1 (TGF-β 1 ) and others. VEGF is one of the major regulators of angiogenesis [15]. Several studies have shown that VEGF expression is markedly increased in different cell types both in the epidermis and in the dermis of patients with SSc [16, 17]. VEGF exerts its biological functions by binding to the tyrosine kinase receptors VEGFR-1 (flt-1) and VEGFR-2 (flk-1/kdr), which are both up-regulated on dermal endothelial cells in SSc-affected skin [16, 17], and by the neuropilin-1, which is also a sema3a receptor. Several

4 Table 1 Demographics and clinical characteristics of the study population Variable SSc N = 27 SLE N = 42 Healthy controls N = 28 Age (years), mean ± SD 48.4 ± ± ± 3.9 NS Sex: female 24 (88 %) 38 (90 %) 27 (96 %) NS BMI (kg/m 2 ), mean ± SD 24 ± ± ± 4.3 NS Smoker current 2 (7.4 %) 4 (5 %) 4 (14 %) NS Smoker past 7 (26 %) Years since diagnosis of non-raynaud s phenomena 5.9 ± 4.6 Lung fibrosis 8 (30 %) Diffuse cutaneous SSc 10 (37 %) Limited cutaneous SSc 17 (63 %) DLCO (% predicted) 69 ± 29 FVC (% predicted) 85.9 ± 20.8 SPAP (mmhg) 29.8 ± 8.9 mrss 14.6 ± 11.3 Active digital ulcer in past 3 months 15 (56 %) Critical ischemia in the past 7 (26 %) Capillaroscopy pattern Early 5 (19 %) Active 7 (26 %) Late 14 (52 %) Diastolic or systolic dysfunction 6 (22 %) Diarrhea 6 (22 %) Calcinosis 17 (63 %) Myositis 4 (15 %) SRC 0 (0 %) ACA 11 (40 %) SCL 70 7 (26 %) ANA 27 (100 %) Proteinuria 3 (11 %) Activity score 2.5 ± 1.7 Severity score 5.5 ± 3.2 Iloprost 17 (63 %) Bosentan 16 (59 %) Sildenafil 2 (7 %) ACE inhibitors 5 (19 %) CCB 7 (26 %) Methotrexate 16 (59 %) Azathioprine 6 (22 %) Cyclophosphamide (ever) 8 (30 %) Cyclosporin 8 (30 %) Rituximab 2 (7 %) Mycophenolate mofetil 1 (4 %) IV immunoglobulins 4 (15 %) p value SSc systemic sclerosis, SLE systemic lupus erythematosus, PMF primary myelofibrosis, BMI body mass index, CRP C-reactive protein, DLCO diffusing capacity of the lung for carbon monoxide, FVC forced vital capacity, SPAP systolic pulmonary artery pressure, mrss modified Rodnan skin score, SRC scleroderma renal crisis, ACA anti-centromere antibody, Scl70 scleroderma 70 antibody, ANA antinuclear antibody, ACE angiotensin converting enzyme, CCB calcium channel blocker, NS nonsignificant

5 the formation of a chaotic capillary network with irregularly shaped, enlarged capillaries, similar to the altered capillary morphology commonly seen in SSc [20, 21]. Hence, an uncontrolled, chronic overexpression of VEGF in SSc may result in the improper neoangiogenesis that typically characterizes this disease. Semaphorin 3A is also known for its anti-angiogenic activity. It inhibits VEGF biological function via competitive inhibition through neuropilin-1 binding. Thus, the low level of sema3a in SSc patients may result in increased biological activity of VEGF contributing to increased but ineffective neoangiogenesis. Further, future studies may indicate more accurately the contribution of sema3a to the pathogenesis of SSc. Conclusion Fig. 1 SSc, systemic sclerosis; SLE, systemic lupus erythematosus. a Semaphorin 3A serum concentration in systemic sclerosis patients is lower than healthy controls (14.38 ± 5.7 vs ± 8.4 ng/ml, p < ) but similar to systemic lupus erythematosus patients (mean ± SD). b The expression of semaphorin 3A on regulatory T cells in systemic sclerosis is lower than healthy controls (mean ± SD) This preliminary small study suggests that sema3a expression is low in SSc serum and more specifically on regulatory T cells. This may help explain the reduced activation of regulatory T cells in SSc and contributes to our understanding of the pathogenesis of the disease. Semaphorin 3A serum level inversely correlated with the duration of disease. Further large-scale studies are needed in order to validate these findings and to focus on the therapeutic and follow-up potential of sema3a in Ssc. Conflict of interest The authors declare that they have no competing interests. References Fig. 2 Linear regression between serum concentration of semaphorin 3A reveals inverse correlation with time since diagnosis of systemic sclerosis (r = 0.4, p = 0.036) studies have demonstrated that serum levels of VEGF are also significantly increased in SSc patients during different disease stages of the disease [18, 19]. It was demonstrated in a mouse model that while a short-period overexpression of VEGF induced the formation of new mature and functional capillaries, prolonged exposure to VEGF resulted in 1. Kolodkin AL (1996) Semaphorins: mediators of repulsive growth cone guidance. Trends Cell Biol 6: Vadasz Z, Toubi E (2014) Semaphorins: their dual role in regulating immune-mediated diseases. Clin Rev Allergy Immunol 47(1): Vadasz Z, Attias D, Kessel A, Toubi E (2010) Neuropilins and semaphorins from angiogenesis to autoimmunity. Autoimmun Rev 9: Suzuki K, Kumanogoh A, Kikutani H (2008) Semaphorins and their receptors in immune cell interactions. Nat Immunol 9: Ji JD, Park-Min KH, Ivashkiv LB (2009) Expression and function of semaphorin 3A and its receptors in human monocytederived macrophages. Hum Immunol 70: Lepelleteir Y, Moura IC, Hadj-Slimane R, Renand A, Fiorentino S, Baude C et al (2006) Immunosuppressive role of semaphorin 3A on T cell proliferation is mediated by inhibition of actin cytoskeleton reorganization. Eur J Immunol 36: Takagawa S, Nakamura F, Kumagai K, Nagashima Y, Goshima Y, Saito T (2013) Decreased semaphorin3a expression correlates with disease activity and histological features of rheumatoid arthritis. BMC Musculoskelet Disord 23: Vadasz Z, Haj T, Halasz K, Rosner I, Slobodin G, Attias D et al (2012) Semaphorin 3A is a marker for disease activity and a

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