Evaluation of Autoantibodies in patients with Systemic Sclerosis

*Corresponding Author: Mahdi Mahmoudi, Ph.D., Rheumatology Research Center (RRC), Shariati Hospital, Tehran University of Medical Sciences (TUMS), Tehran, Iran. PO-Box: 1411713137, E-mail: mahmoudim@tums.ac.ir, Telefax: +98-218-822-0067. And Farhad Gharibdoost, Rheumatology Research Center (RRC), Shariati Hospital, Tehran University of Medical Sciences (TUMS), Tehran, Iran. PO-Box: 1411713137, E-mail: gharibdoost@sina.tums.ac.ir, Telefax: +98-218-8220067. Received: 31 October 2019; Accepted: 11 November 2019 1 Original Article Open Access Vol. 5, No. 1, Jan 2020, Webpage: http://rheumres.org Email: editor@rheumres.org ISSN:2476-5856 doi: 10.22631/rr.2020.69997.1083 ©2020, Iranian Rheumatology Association


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Rheumatology Research., Vol. 5, No. 1, Jan. 2020 The subgrouping of systemic sclerosis patients based on their serum ANA in the early stages of the disease may be practical for assessing the risk and nature of organ involvement and estimating survival in these patients [6]. Each subgroup is associated with a different type of autoantibody [7].
The presence of ACA is most often associated with a variant of SSc, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) [8], and has a better prognosis than ATApositive SSc patients [7].
The presence of both anti-RNAP I and III and a higher prevalence of renal crisis but not pulmonary fibrosis are seen with dcSSc [15,16]. Thus, the survival rate in patients with anti-RNAP is better than in patients with ATA [4,17].
It has been documented that purified human ATAs impede relaxation of super helical DNA [18], anti-RNAP I /III autoantibodies inhibit RNA transcription [19], and ACAs disrupt mitosis [20,21]. However, how these autoantibodies bind to the intracellular antigen resulting in cellular damage is not clear [5]. Henault et al. demonstrated that by binding to the surface of dermal fibroblast cell lines, topo I provides a binding site for anti-topo I and stimulates adhesion and activation of monocytes in vitro [22].
The current study assessed the prevalence of ANA, ATA, ACA, and anti-RNAP III among Iranian systemic sclerosis patients and investigated their prevalence separately in patients with dcSSc and lcSSc.

Materials and Methods___________________ Patients
The study population comprised 481 systemic sclerosis patients from the rheumatology clinic of Shariati Hospital, Tehran, Iran, and the Iran Rheumatism Center (IRC) from 2013 to 2016. All of the cases fulfilled the American College of Rheumatology (ACR) 2013 criteria [23], and clinical data was available for all cases. The patients were categorized into dcSSc and lcSSc according to the 2013 classification criteria for SSc [23]. Written informed consent was obtained from all participants, and ethical approval for this study was obtained from Tehran University of Medical Sciences (TUMS).
Whole blood samples from each patient were collected in test tubes, and the sera were separated by centrifugation. Aliquots of sera were stored at -70 °C until used.

Indirect immunofluorescence (IIF) method for detection of ANA
The ANAs were quantitatively detected using the indirect immunofluorescence method via mosaic HEp-20-10 cells and primate liver cells (EUROIMMUNE, Medizinische Labordiagnostika AG, Germany). Sera were diluted with phosphate buffered saline (PBS) at a ratio of 1/160 and 1/640. Twenty-four microliters of each serum sample was added to each well; then the chambers were capped and incubated for 30 minutes at room temperature (RT). Each slide was washed quickly two times with PBS. The slides were located again in the humidifier chamber, subjected to appropriate diluted goat anti-human IgG fluorescein isothiocyanate (FITC) conjugated antibody, and further incubated for 30 minutes. Subsequently, the slides were rinsed thrice with PBS for 5 minutes each. Completely blotted slides were then overlaid with a coverslip and positioned on the slide tray. The slides were stored at 4 °C until visualized using a Zeiss fluorescence microscope.

Enzyme-linked immunosorbent assay (ELISA) for detection of ATA, ACA, and anti-RNAP III
The ATA and ACA of IgG class in sera were detected using an ELISA kit (Euroimmune, Medizinische Labordiagnostika AG, Germany), and the anti-RNAP III was also determined qualitatively by an ELISA kit (Cusbio, China) according to the manufacturer's protocols.

Statistical Analysis
Statistical analyses were performed using statistical package SPSS version 22 (SPSS Inc., Chicago IL, USA), and p value < 0.05 was considered to be statistically significant. The Benjamini and Hochberg method was used to control for false discovery rate in multiple comparisons [24].

The level of non-specific and specific systemic sclerosis-relevant autoantibodies
The frequency rates of autoantibodies detected in these patients are shown in Table 2. Among all patients, 434 (90.2%) were ANA positive and the remaining 47 (9.7%) were ANA negative (Table 3). Overall, ANA was detected in 158 (87.3%) and 276 (92.0%) of lcSSc and dcSSc patients, respectively, which was not significantly different (p Value = 0.092). Anti-RNAP III, anti RNA polymerase III antibody; ACA, anti-centromere antibody; ATA, anti-topoisomerase I antibody Table 3. The prevalence of the autoantibodies in different populations.
The presence of ACA was found in 16 (8.8%) and 13 (4.3%) individuals with lcSSc and dcSSc, respectively, which lost its significant correlation after adjustment with the Benjamini and Hochberg method (p value = 0.073).
The presence of ATA was detected in 98 (54.1%) lcSSc patients and 250 (83.3%) dcSSc patients. ATA was the most common autoantibody in both subtypes, and it was significantly higher in dcSSc (adjusted p value < 0.001).

Discussion_____________________________
Systemic sclerosis is a highly heterogeneous disorder with connective tissue involvement and an autoimmune nature, clinically characterized by the triad of endothelial dysfunction, inflammation and autoimmunity, and tissue fibrosis [25]. The importance of the study of autoantibodies lies in the fact that some of them are involved in the disease pathogenesis. Therefore, laboratory examinations to detect systemic sclerosis-relevant autoantibodies (e.g., ANA, ATA, anti-RNAP III, and ACA) provide an effective tool for the diagnosis of systemic sclerosis and the classification of disease subsets [26]. It has been estimated that over 95% of patients with systemic sclerosis have a positive ANA test, and over 85% have one or more serum autoantibodies [27]. DNA topoisomerase I (topo I), centromere proteins, RNA polymerases I, II, and III are antigens defined as intracellular targets for ANAs [28].
Systemic sclerosis is more common in women than men; however, the course of the disease may be more progressive in men [29]. Similar to other populations, i.e. U.S and French [30], Mexican [31], German Network [16], EULAR 1 Scleroderma Trials And Research (EUSTAR) group [13], and the mid-western region of Brazil [32], patients in the current study were mostly female.
ACA is directed against centromere proteins, and it has been detected in 20-25% of SSc populations that are strongly correlated with lcSSc [27]. The frequency of ACA in different studies varies from 4-52.2% [16,[30][31][32][33][34], although in a study of Japanese patients by Kuwana et al., 93% were ACA positive [6]. In the current study, the frequency of ACA, which is more associated with lcSSc [9,10], was low and 6.09% were ACA positive.
Anti-RNAP III has 98% to 100% specificity for SSc and occurs in 16%-20% of patients, mainly in those with the dcSSc subtype [35]. Several researchers have reported 1.4-9.9% anti-RNAP III positivity in SSc patients [30,31,34]. Consistent with these studies, the current results demonstrated that 5.19% of SSc patient were anti-RNAP III positive. However, two studies showed U.S and Brazil SSc patients were 25% and 15.2% anti-RNAP III positive, respectively [30,32]. 1 The European League Against Rheumatism Taken together, the current data showed that anti-RNAP III and ATA were higher in dcSSc patients, whereas ACA was higher in lcSSc patients. In comparison with other studies, the frequency of ATA was significantly high in Iranian patients, but ACA frequency is low. With regards to the correlation of these autoantibodies with disease prognosis, it seems that more Iranian SSc patients have a poor prognosis. However, a study has reported that survival rates in Iranian SSc patients are 93% and 83% at 5 and 10 years from diagnosis, respectively [36], which is not different from other countries [37][38][39][40][41].
There were several discrepancies in frequency rates of autoantibodies among different studies. These conflicting results may be attributable in part to differences in sample size, ethnic background, and geographic factors, differences in the ACR criteria, the system assay used to detect autoantibodies, and diverse statistical tests.

Conclusion____________________________
A systemic sclerosis diagnosis is mainly clinical; nevertheless, quantification and identification of autoantibodies can play important roles in diagnosis. The present study illustrated differences in the autoantibodies associated with SSc disease. In accordance with the literature, this study confirms that the clinical subtype of SSc may correlate positively with the presence of specific autoantibodies. Further research is required in order to investigate the exact role of autoantibodies in SSc patients and the cause of their dissimilar frequencies among different cohorts.