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Serum sCTLA-4 levels and clinical manifestations in ankylosing spondylitis patients.

Introduction

Ankylosing spondylitis (AS) is a type of inflammatory rheumatic disorder and a prototype of spondylarthritis, characterized by axial skeleton and sacroiliac joint involvement. In the beginning of the disease, CD[4.sup.+] and CD[8.sup.+] T cells, macrophages, and elevated amounts of tumor necrosis factor alpha (TNF-[alpha]) were detected in the sacroiliac joint. Two signals were needed by T cell for activation. In addition to the first antigenic signal presented via the major histocompatibility complex, a costimulatory signal is reguired. The costimulatory signal is produced by cooperation between CD28 attached to T cell and B7 molecules on antigen-presenting cells (1-3). The cytotoxic T lymphocyte-associated molecule-4 (CTLA-4, also termed CD152)on activated T cells connects to CD86 and CD80, and some of its inhibitory effects may be modulated by SH2-containing phosphatase (3, 4).

CTLA-4 attached to the cell surface is increased by activation of T-cell and a CD28-ligand association. CTLA-4 negatively regulates T-cell functions (5, 6). Because of CTLA-4-ligand interactions, early T-cell activation, regulatory T cells, and cell-cycle progression are suppressed, and IL-2 production, cyclin production, and T-cell receptor signal transduction are reduced (7-10).

Recent studies have shown increased levels of soluble CTLA-4 (sCTLA-4) in thyroiditis, myasthenia gravis, and systemic lupus erythematosus (SLE) patients (11-13). Toussirot et al. (1) reported a statistically significant increase in serum CTLA-4 levels in patients with spondyloarthritis. Furthermore, there was correlation between the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and the levels of C-reactive peptide (CRP) and sCTLA-4 (1).

We aimed to determine whether levels of serum sCTLA-4 were indicative of clinical severity in AS patients, and to compare levels of serum sCTLA-4 between groups of participants.

Methods

Thirty-eight participants diagnosed with AS, 28 patients diagnosed with rheumatoid arthritis (RA), and 27 disease-free controls were involved in this study. Written informed consent was collected from each participant. The Ethics Committee and Institutional Review Board approved the study. The patient and control groups'demographic and laboratory data such as sex, age, medical history, physical examination results, CRP and erythrocyte sedimentation rate (ESR) were collected. The BASDAI was used as a measure of disease activity. To measure the serum sCTLA-4 levels, we examined peripheral blood by using an enzyme-linked immunosorbent assay (ELISA), applying the CTLA-4 (Human) ELISA commercial kit (Abnova, Taipei City, Taiwan). Operations were performed with a fully automated ELISA Triturus device.

The Statistical Package for Social Sciences for Windows software, version 19 (SPSS Inc.; Chicago, IL, USA), was used for statistical analysis. The Kolmogorov-Smirnov test was used for guantitati ve data with a norma I distri bution. The i ndependent t-test and Mann-Whitney U test were applied to make comparisons between the independent groups. A one-way analysis of variance and the Kruskal-Wallis test were applied to make comparisons between multiple groups. The Bonferroni-corrected Mann-Whitney U test and Fisher's least significant difference test were applied for post-hoc analysis. Pearson's chi-sguare and Fisher's exact tests were applied for the comparison of categorical data The categorical data were expressed as percentages and numbers of patients (n). The data were examined at the confidence level of 95%, and p-values <0.05 were accepted as significant.

Results

The demographic characteristics of participant groups are shown in Table 1. Up to guarter percent (n=10) of all AS patients received indomethacin, while the remaining patients were treated with naproxen, diclofenac, and meloxicam. Only a few patients were treated with sulfasalazine (n=5) and methotrexate (n=2). Participants who were receiving or had consumed anti-TNF-a agents were removed from the study due to possible confounding factor effects. The levels of serum sCTLA-4 in the AS group were higher than those in the disease-free control and RA groups (p<0.001). There was no significant difference between healthy control and RA patients (p>0.05). The serum sCTLA-4 levels and p values of the different groups are shown in Table 2.

The correlations between age (r=-0.382, p=0.001), disease duration (r=0.06, p=0.949), BASDAI (r=0.115, p=0.370), CRP (r=0.136, p=0.067), ESR (r=-0.114, p=0.129) and serum sCTLA-4 levels in AS patients are shown in Table-3. CRP and ESR levels were correlated with BASDAI scores.

Discussion

Although the etiology of AS is unknown, several studies suggest T-cell autoreactivity, genetic and molecular associations, and infections contribute to its progression (14). Defects in coinhibitory molecules may cause autoimmune disorders. The sCTLA-4 molecule and various other costimulatory signal molecules may have a role in the pathogenesis of AS and correlate with clinical findings (15). This hypothesis was initially supported by the demonstration of lymphocyte hyperactivation in CTLA-4-deficient mice. It was reported that CTLA-4-deficient mice died by one month of age because of lymphocytic system disorder and multi-organ lymphocytic infiltration (16, 17).

Previous studies indicate that levels of sCT-LA-4 are high in most autoimmune originated disorders. The sCTLA-4 is considered as double-edged molecule which means ability of both activation and inhibition effect on T-cell. Toussirot et al. (1) reported correlations between levels of serum sCTLA-4, intracellular CTLA-4, CTLA-4 mRNA, lymphocytes, and clinical characteristics of the disease in 165 participants with spondyloarthropathy (SpA) taking immunosuppressive drugs. The findings were compared with participants with 71 RA and 88 disease-free controls. Higher sCTLA-4 levels were detected in patients with AS compared with RA and disease-free controls, and the differences were statistically significant. There was a positive correlation between BASDAI and sCTLA-4. So, serum sCTLA-4 will be used as a biomarker to measure activity of the disease in AS. Although high levels of sCTLA-4 are detected in patients with autoimmune disease. The plasticity of sCTLA-4 may cause different outcomes in clinical severity of the disease (18).

We have detected higher levels of sCTLA-4 in AS patients than in RA and control groups (p=0.001), but no correlation between clinical severity of disease and sCTLA-4 levels (r=0.115, p=0.115).

Serum sCTLA-4 levels of the AS group were higher than those in the healthy control and RA groups (p<0.001). There was no significant difference between the healthy control and RA groups (p>0.05). The mean duration of the disease was 19 months in our study, which is less compared with Toussirot's study (9.2 years) (1). Different results may be due to CTLA-4 polymorphism or genetic variations. CTLA-4 gene allele polymorphism and cytokine genes that control inflammation can be variable in patients with AS (2). The differences may be due to the use of a different ELISA kit and eguipment (Abnova, Taipei City, Taiwan) or different immunosuppressive therapies. Also, all of the AS patients received non-steroidal anti-inflammatory drugs and 7 of them had an immunosuppressive therapy (sulfasalazine or methotrexate). In our study, the patients were receiving one or more disease-modifying anti-rheumatic drugs. The patients using anti-TNF therapy were excluded. This use of drugs by patients with AS is because of their suppressive effects on disease activity; however, the effects of drugs on sCTLA-4 levels are unknown.

There was not a statistically significant difference between the serum sCTLA-4 levels in the 28 patients with RA compared to the healthy group (p>0.05). A significant positive correlation has been reported between Disease Activity Score (DAS-28) and sCTLA-4 levels in RA patients (19). Furthermore, immunosuppressive drugs such as leflunomide may reduce the levels of serum sCTLA-4 and other costimulatory molecules. In our study, low sCTLA-4 levels in patients with RA could be associated with genetic variation or immunosuppressive therapy. 24 of the RA patients used methotrexate, 4 of the RA patients received leflunomide, and 26 of them received combined immunosuppressive therapy (methotrexate+sulfasalazine, methotrexate+hydroxychloroguine, or methotrexate+sulfasalazine+hydroxychloroguine).

The small numbers of patients and controls are major limitations of the study. We investigated the serum sCTLA-4 levels in the pathogenesis of AS and established a significant difference in the levels of serum sCTLA-4 between patients with AS, RA, and a disease-free control group. Earlier diagnosis is substantial for patients with AS and laboratory tests with high specificity for disease detection are needed. In our study, although higher sCTLA-4 levels were found in AS patients than in the control groups, there was no correlation between clinical severity of disease and serum sCTLA-4 levels. An increase in CTLA-4 levels may be involved in the pathogenesis of AS. The lack of correlation between CTLA-4 levels and disease activity may exhibit the complex mechanism of the disease, and the existence of other possible inflammatory molecules related to disease activity that we couldn't examine. Further studies are reguired to evaluate the potential role of serum sCTLA-4 in terms of early diagnosis of AS and its impact on the severity of the disease.

Ethics Committee Approval: Ethics committee approval was received for this study from the Ethics Committee of Adrian Menderes University.

Informed Consent: Written informed consent was obtained from patients who participated in this study.

Peer-review: Externally peer-reviewed.

Author Contributions: Concept - S. P. C., T. S.; Design - S.P.C., T.S., G.S.; Supervision -T.S., S.P.C.; Resources - S.P.C., T.S.; Materials - N.A., S.P.C.; Data Collection and/or Processing - S.RC, G.S.; Analysis and/or Interpretation - S.P.C., N.A., G.S.; Literature Search - S.P.C. T.S., G.S.; Writing Manuscript - S.P.C., T.S., G.S.; Critical Review - T.S., S.P.C., G.S.

Conflict of Interest: The authors have no conflict of interest to declare.

Financial Disclosure: This study was founded by Ad-nan Menderes University Department of Scientific Research Projects (TPF 13011).

References

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(2.) Lee WY, Chang YH, Lo MK, Chang CP. Yang SC, Yang TR et aI. Polymorphisms of cytotoxic T lymphocyte-associated antigen-4 and cytokine genes in Taiwanese patients with ankylosing spondylitis. Tissue Antigens 2010; 75: 119-26. [CrossRef]

(3.) Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7 system of T cell costimulation. Annu Rev Immunol 1996; 14:233-58. [CrossRef]

(4.) Thompson CB, Allison JP. The Emerging Role of CTLA-4 as an Immune Attenuator. Immunity 1997; 7:445-50. [CrossRef]

(5.) WalunasTL, Lenschow DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM, et al. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1994; 1:405-13. [CrossRef]

(6.) Walunas TL, Bakker CY, Bluestone JA. CTLA-4 ligation blocks CD28-dependent T cell activation. J Exp Med 1996; 183:2541-50. [CrossRef]

(7.) Luhder F, Chambers C, Allison JP. Benoist C, Mathis D. Pinpointing whenTcell costimulatory receptor CTLA-4 must be engaged to dampen diabetogenic T cells. Proc Natl Acad Sci 2000; 97:12204-9. [CrossRef]

(8.) Chikuma S, Imboden JB, Bluestone JA. Negative Regulation of T Cell Receptor-Lipid Raft Interaction by Cytotoxic T Lymphocyte-associated Antigen 4. J Exp Med 2003; 197:129-35. [CrossRef]

(9.) Tang Q, Boden EK, Henriksen KJ, Bour-Jordan H, Bi M, Bluestone JA. Distinct roles of CTLA-4 and TGF-(3 in CD4+CD25+ regulatory T cell function. Eur J Immunol 2004; 34: 2996-3005. [CrossRef]

(10.) Tai X, Cowan M, Feigenbaum L, Singer A. CD28 costimulation of developing thymocytes induces Foxp3 expression and regulatory T cell differentiation independently of interleukin 2. Nat Immunol 2005; 6:152-62. [CrossRef]

(11.) Oaks MK, Hallett KM. Cutting Edge: A Soluble Form of CTLA-4 in Patients with Autoimmune Thyroid Disease. J Immunol 2000; 164:5015-8. [CrossRef]

(12.) Wang XB, Kakoulidou M, Giscombe R, Qiu Q, Huang D, Pirskanen R, et al. Abnormal expression of CTLA-4 by T cells from patients with myasthenia gravis: effect of an AT-rich gene seguence. J Neuroimmunol 2002; 130:224-32. [CrossRef]

(13.) Liu MF, Wang CR, Chen PC, Fung LL Increased Expression of Soluble Cytotoxic T-Lymphocyte-Associated Antigen-4 Molecule in Patients with Systemic Lupus Erythematosus. Scand J Immunol 2003; 57:568-72. [CrossRef]

(14.) Ramos M, De Castro JAL. HLA-B27 and the pathogenesis of spondyloarthritis. Tissue Antigens 2002; 60:191-205. [CrossRef]

(15.) Cao J, Zou L, Luo R Chen R Zhang L Increased production of circulating soluble co-stimulatory molecules CTLA-4, CD28 and CD80 in patients with rheumatoid arthritis. Int Immunopharmacol 2012; 14:585-92. [CrossRef]

(16.) Tivol EA, Borriello F, Schweitzer AN, Lynch WR Bluestone JA, Sharpe AH. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 1995;3:541-7. [CrossRef]

(17.) Waterhouse R Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP., et al. Lymphoproliferative Disorders with Early Lethality in Mice Deficient in Ctla-4. Science 1995; 270: 985-8. [CrossRef]

(18.) Pawlak E, Kochanowska IE, Frydecka I, Kietbihski M, Potoczek S, Bilihska M. The soluble CTLA-4 receptor: a new marker in autoimmune diseases. Arch Immunol Ther Exp (Warsz). 2005; 53:336-41.

(19.) Sato S, Fujimoto M, Hasegawa M, Komura K, Yanaba K, Hayakawa I, et al. Serum soluble CTLA-4 levels are increased in diffuse cutaneous systemic sclerosis. Rheumatology 2004; 43: 1261-6. [CrossRef]

Sultan Pinar Cetintepe (1) Taskin Senturk (2), Gokhan Sargin (2), Neriman Aydin (3)

(1) Department of Occupational Medicine, Hacettepe University School of Medicine, Ankara, Turkey

(2) Department of Rheumatology, Adnan Menderes University School of Medicine, Aydin, Turkey

(3) Department of Clinical Microbiology, Adnan Menderes University School of Medicine, Aydin, Turkey

Address for Correspondence: Gokhan Sargin, Department of Rheumatology, Adnan Menderes University School of Medicine, Aydin, Turkey

E-mail: gokhan_sargin@hotmail.com

Submitted: 30 June 2017

Accepted: 1 October 2017

Available Online Date: 22 January 2018

DOI:10.5152/eurjrheum.2018.17114
Table 1. Demographic characteristics of the patients and control group

              Ankylosing spondylitis  Rheumatoid arthritis
              (n=38)                  (n=28)

Male (n/%)    24 (63.2%)                 14 (36.8%)
Female (n/%)  23 (82.1%)                  2 (7.4%)
Age (years)   39.37 [+ or -] 12.11    58,32 [+ or -] 13,88

              Control group (n=27)

Male (n/%)     5 (17.9%)
Female (n/%)  25 (92.6%)
Age (years)   54.89 [+ or -] 12.94

Table 2. Serum sCTLA-4 levels in patients with AS and RA and in the
control group

                  Ankylosing spondylitis  Rheumatoid arthritis

sCTLA-4  Median   0.22                    0.04
         IQR (*)  0.09                    0.01
         p(AS-RA)<0.001; p(AS-Control)<0.001; p (RA-Control)>0.05

         Control group  p

sCTLA-4  0.04           <0.001
         0.03
         p(AS-RA)<0.001; p(AS-Control)<0.001; p (RA-Control)>0.05

Kruskal-Wallis test (post-hoc test: Bonferonni-corrected Mann-Whitney U
Test, [alpha] = 0.017) sCTLA-4: soluble Cytotoxic T-Lymphocyte
Associated Antigen-4; AS: ankylosing spondylitis; RA: rheumatoid
arthritis; IQR: interquartile rang

Table 3. Demographic, biochemical, and clinical correlations of AS
patients

                     ESR    CRP     CTLA-4  Disease Duration

Age               r  0.203  -0.012  -0.382  0.160
                  p  0.005   0.866   0.001  0.065
ESR               r          0.310  -0.114  0.017
                  p          0.001   0.129  0.846
CRP               r                  0.136  0.128
                  p                  0.067  0.138
CTLA-4            r                         0.006
                  p                         0.949
Disease Duration  r
                  p

                  BASDAI ([dagger])

Age               0.001
                  1.000
ESR               0.309
                  0.012
CRP               0.730
                  0.001
CTLA-4            0.115
                  0.370
Disease Duration  0.192
                  0.123

ESR: erythrocyte sedimentation rate; CRP: C-reactive peptide; sCTLA-4:
soluble cy totoxic T-lymphocyte associated antigen-4; BASDAI: bath
ankylosing spondylitis disease activity index; AS: ankylosing
spondylitis
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Title Annotation:Original Article
Author:Cetintepe, Sultan Pinar; Senturk, Taskin; Sargin, Gokhan; Aydin, Neriman
Publication:European Journal of Rheumatology
Date:Jun 1, 2018
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