Characteristics of germinal center-like structures in patients with Sj ogren s syndrome

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1 International Journal of Rheumatic Diseases 2016 ORIGINAL ARTICLE Characteristics of germinal center-like structures in patients with Sj ogren s syndrome Jing HE,* Yuebo JIN,* Xia ZHANG, Yunshan ZHOU, Ru LI, Yijun DAI, Xiaolin SUN, Jingzhong ZHAO, Jianping GUO and Zhanguo LI Department of Rheumatology and Immunology, Peking University People s Hospital & Beijing Key Laboratory for Rheumatism Mechanism and Immune Diagnosis, Beijing, China Abstract Aim: To analyze the relationship between ectopic germinal centers (GCs) in the salivary glands and the clinical/ laboratory characteristics of patients with Sj ogren s syndrome (SS). Methods: Retrospectively, 126 patients with primary SS (pss) and 16 patients with secondary SS (sss) were analyzed. Minor salivary gland biopsies were evaluated for the presence of GC-like morphology by hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining for CD21. Clinical and serological data were obtained from medical records. Results: GC-like structures were observed in 36/126 (28.6%) pss patients and 4/16 (25.0%) sss patients. The mean inflammatory focus score of the gland was significantly higher in samples than in ones in both pss and sss patients (P = and 0.024, respectively). In pss, significantly elevated titers of rheumatoid factor (RF)-IgM (P = 0.023) and antinuclear antibodies (ANA) (P = 0.036), increased levels of IgA (P = 0.012) and IgG (P = 0.017) were encountered in patients. The group also presented higher prevalence of anti-ssa antibodies, lower levels of white blood cells, higher levels of erythrocyte sedimentation rate and c-globulin, although not statistically significant. In sss patients with ectopic GC formation, ANA titers were remarkably elevated. The anticyclic citrullinated peptide (anti-ccp)-igg titers and the prevalence of antikeratin antibody (AKA)-IgG, antiperinuclear factor (APF)-IgG were also increased, yet not significantly. GCs were found to be associated with antibody and immunoglobulin production. Conclusion: This study indicates that SS patients with ectopic GCs have distinct features. Ectopic GC structures were particularly noted in patients with higher focus scores, and might play an essential role in sustaining antibody production as well as B cell activation. Key words: autoantibodies, germinal center, Sj ogren s syndrome. INTRODUCTION Sj ogren s syndrome (SS) is an autoimmune disease characterized by progressive lymphocytic infiltration Correspondence: Dr Zhanguo Li, Department of Rheumatology and Immunology, Peking University People s Hospital & Beijing Key Laboratory for Rheumatism Mechanism and Immune Diagnosis (BZ0135), Beijing, No. 11 Xizhimen South Street, Xicheng District, Beijing , China. zgli99@aliyun.com *These authors contributed equally to this study. and destruction of exocrine (lacrimal and salivary) glands, which leads to a marked reduction in the secretion of tears and saliva. 1 Previously, studies have classified SS as primary (pss) or secondary (sss) depending on its coexistence with other connective tissue diseases (CTDs). Secondary SS is associated with diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). 2,3 The clinical manifestations of this disease include not only organ-specific disorders (autoimmune exocrinopathy) but also systemic processes such as impairments in hematologic, pulmonary, 2016 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd

2 J. He et al. vascular, renal, musculoskeletal, puerperal and nervous systems. 1,4 The pathogenesis of SS is unclear. Studies have suggested that the autoimmune abnormalities in SS are the result of a combination of immunologic, genetic and environmental factors. 1,4 B-cell activation is a feature of SS, and many autoantibodies such as rheumatoid factors (RFs), antinuclear antibodies (ANAs), antibodies to SS antigen A or B (SSA or SSB), anti-m3 receptor and anti-a-fodrin antibodies have reportedly been detected in both pss and sss. 1,2,5,6 Histopathologically, the disease manifests as infiltration of focal mononuclear cells in the salivary and lacrimal glands, which is the golden standard for its diagnosis. The infiltration is progressive and mainly consists of B lymphocytes, T lymphocytes and macrophages. 7 Germinal centers (GCs) were first described by Walther Flemming in Immunization experiments have demonstrated that GCs arise in lymphoid tissues following antigenic stimulation, and provide a milieu for the proliferation of B cells, somatic hypermutation of immunoglobulin variable-region gene segments and class-switch recombination of immunoglobulin. Recent studies carring out salivary gland biopsy have shown that approximately % of patients with SS demonstrated ectopic GC-like structures. 9 Although several articles have reported the presence of GC-like structures in SS patients, 10 there is considerable heterogeneity in these findings. Therefore, in this study, we aimed to thoroughly explore the role of GC-like structures in SS, and determine whether ectopic GCs are associated with autoantibody production and disease activity. MATERIALS AND METHODS Patients and tissue samples We retrospectively analyzed the data of 126 pss patients and 16 RA patients with sss who had been admitted to Peking University People s Hospital between January 2010 and December 2010 and undergone salivary gland biopsy. All SS patients fulfilled the revised US-EURO classification criteria for SS (2002) 2 and all RA patients fulfilled the American College of Rheumatology (ACR) classification criteria for RA. 11 No corticosteroid treatment or immunosuppressive drugs were given before tissue and blood samples were obtained. All the subjects provided their written informed consents before the samples were collected. These patients minor salivary gland (MSG) tissues were sectioned, embedded in paraffin and stained with hematoxylin and eosin (H&E) for histological evaluation. This study was approved by the Medical Ethics Committee of Peking University People s Hospital and was performed according to the Declaration of Helsinki. Immunohistochemistry For immunohistochemical (IHC) analysis, paraffinembedded salivary gland tissues were sectioned into 4 6 mm sections with a microtome (Leica Instruments GmbH, Nussloch, Germany) onto microscope slides (Sail Brand Co., Ltd., Jinan, China). Following deparaffinization, rehydration and heat-induced epitope retrieval (HIER), the sections were incubated with primary antibodies against CD21 (1 : 50 dilution; monoclonal mouse anti-human IgG1 kappa), 10 and then peroxidase-conjugated goat anti-mouse antibodies, counterstained with hematoxylin, dehydrated and mounted with a non-aqueous mounting medium (Eukitt, O. Kindler GmbH & Co, Germany) prior to analysis. Unless otherwise indicated, all reagents were purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China. All tissue sections were analyzed in duplicate. 10 Evaluation of staining The sections were evaluated under a light microscope (Leica DMLB, Leica Microsystems Wetzlar, Wetzlar, Germany) with a objective. Upon observation of H&E staining, focus score was defined as the number of inflammatory cell aggregates containing at least 50 mononuclear cells per 4 mm 2. Meanwhile, the sections were morphologically screened for the presence of ectopic GC-like structures, that is, a well-circumscribed chronic inflammatory cell infiltrate consisting of at least 50 mononuclear cells, presenting with a lymphoid-like organization (containing a densely packed dark zone and a light zone) within normal MSG tissue. 10,12 In the analysis of immunohistochemical staining for CD21, ectopic GC-like features were further confirmed according to CD21-defined follicular dendritic cell (FDC) networks. Samples negative for CD21 and samples exhibiting only a few scattered CD21-positive cells were considered, and the remainder were GCpositive. All sections were randomly and independently assessed by two expert observers, blinded to clinical and immunological data. Clinical and laboratory evaluation Clinical features were obtained from medical records of all patients, including age, gender, disease duration, parotid swelling, lower limb purpura, Raynaud s phenomenon, pulmonary interstitial fibrosis (PIF), arthritis 2 International Journal of Rheumatic Diseases 2016

3 Germinal center-like structures in SS and renal tubular acidosis. Laboratory data such as the white blood cell (WBC), hemoglobin, platelet (PLT), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), c-globulin, IgA, IgG, IgM, complement factors C3 and C4 were also detected. All the patients underwent extensive immunological analyses for the detection of antibodies, including rheumatoid factor (RF)-IgM (rate nephelometry), anti- SSA, anti-ssb (double immunodiffusion) and ANA (indirect immunofluorescence) for both pss and sss patients, and anticyclic citrullinated peptide (anti-ccp)- IgG (enzyme-linked immunosorbent assay), antikeratin antibody (AKA)-IgG and antiperinuclear factor (APF)- IgG (indirect immunofluorescence) for sss patients. All the detections were performed by standard commercial kits according to the manufacturer s instructions. European League Against Rheumatism SS Disease Activity Score (ESSDAI) and Disease Activity Index of 28 joints (DAS28) were assessed for pss and sss patients, respectively, to indicate disease activity. Statistical analysis All statistical analyses were performed by SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The Mann Whitney test was used to study the differences between groups and Spearman s correlation was used to analyze the relationships between variables. Categorical data were analyzed using the chi-square test. Multiple logistic regressions were performed with autoantibody production and immunoglobulins as the dependent variable. P-values < 0.05 were considered statistically significant. RESULTS GCs in pss and sss We reviewed the data of 138 patients with SS. Table 1 presents the general data of the 126 patients with pss and 16 RA patients with sss in this study. Focal lymphocyte infiltration with morphological features of ectopic GC-like structures appeared in 36/126 (28.6%) and 4/16 (25.0%) MSG biopsies in patients with pss and sss respectively, which were screened by H&E staining (Fig. 1a,c) and further confirmed by CD21 IHC staining (Fig. 1b,d). The remaining sections exhibited conventional focal infiltrates with either only scattered CD21-positive cells (Fig. 1e,f) or no IHC staining for CD21 (Fig. 1g,h). All pss patients were divided into two groups based on the presence of CD21-positive GC-like structures. and GCnegative groups showed no significant difference in terms of age, female proportion and disease duration in both pss and sss patients. GC-like structures associated with clinical and laboratory aberrations in patients with pss In patients with pss, as compared to the group, the mean focus score was significantly higher in the group (P = 0.007), and elevated titers of RF-IgM (P = 0.023), ANA (P = 0.036) as well as increased levels of IgA (P = 0.012) and IgG (P = 0.017) were notable in pss patients with ectopic GC formation. As shown, the group also presented higher prevalence of anti-ssa antibodies, as well as lower levels of WBC, higher levels of ESR and c-globulin, although the differences were not statistically significant (Table 2). Relationship of GC-like structures and scores and serological findings in RA patients with sss In RA patients with sss, a remarkably higher focus score (P = 0.024) and ANA titers (P = 0.047) were also seen in the group in comparison with the GCnegative group. However, similar DAS28 and ESSDAI scores were demonstrated in these groups. Furthermore, the anti-ccp-igg titers and the prevalence of AKA-IgG and APF-IgG were increased in sss patients with ectopic GC formation, yet not significantly (Table 3). Table 1 General data of 126 patients with primary Sj ogren s syndrome (pss) and 16 RA patients with secondary Sj ogren s syndrome (sss) Patient characteristics Ectopic GC-like morphology in pss Ectopic GC-like morphology in sss (n = 36, 28.6%) (n = 90, 71.4%) P-value (n = 4, 25.0%) (n = 12, 75.0%) P-value Age, years, mean SD Female proportion, n (%) 36 (100.0) 88 (97.8) (100.0) 11 (91.7) Duration, months, mean SD GC, germinal center. International Journal of Rheumatic Diseases

4 J. He et al. (a) (b) (c) (d) (e) (f) (g) (h) Figure 1 Germinal centers (GCs) were identified by hematoxylin and eosin (H&E) and anti-cd21 immunohistochemisty (IHC) stain biopsies. The figure illustrates focal infiltrates with GC-like morphology in H&E biopsies from primary Sj ogren s syndrome and secondary Sj ogren s symdrome patients (a,c). Accordingly, in IHC sections, GClike inflammatory structures were confirmed by GC-marker CD21 (b,d), which is present on follicular dendritic cells. While in other sections, anti-cd21 IHC stained biopsies showed no GC-like inflammatory structures, but conventional focal infiltrates with only scattered CD21 positive cells (e,f) or no IHC staining for CD21 (g,h). Ectopic GC may be interrelated with the production of autoantibodies and immunoglobulins In this study, we made use of general data, clinical and laboratory information in correlation with ectopic GC results to explore the association between GC and autoantibody and immunoglobulin production. As shown in Table 4, ectopic GC was found to be significantly associated with RF-IgM and IgG secretion, and it may be interrelated with autoantibody and immunoglobulin production. DISCUSSION Compared to the normal population, patients with primary SS manifested aberrant B-cell activity, exocrine 4 International Journal of Rheumatic Diseases 2016

5 Germinal center-like structures in SS Table 2 Comparison of clinical and laboratory findings in 126 pss patients with GC-like structures () and those without GC-like structures () Patient characteristics Ectopic GC-like morphology P-value (n = 36, 28.6%) (n = 90, 71.4%) Clinical findings Parotid swelling, n (%) 8 (22.2) 17 (18.9) Purpura: lower limbs, n (%) 8 (22.2) 21 (23.3) Raynaud s phenomenon, n (%) 6 (16.7) 16 (17.8) PIF, n (%) 12 (33.3) 22 (24.4) Arthritis, n (%) 14 (38.9) 40 (44.4) Renal tubular acidosis, n (%) 4 (11.1) 10 (11.1) ESSDAI score, mean SD Focus score, mean SD ** Autoantibodies RF-IgM (IU/mL), median SD * Anti-SSA positive, n (%) 24 (66.7) 48 (53.3) Anti-SSB positive, n (%) 12 (33.3) 24 (26.7) ANA titer, median SD * Other laboratory data WBC (10 9 /L), median SD Hemoglobin (g/l), median SD PLT (10 9 /L), median SD ESR (mm/h), median SD CRP (mg/l), median SD c-globulin (%), median SD IgA (g/l), median SD * IgG (g/l), median SD * IgM (g/l), median SD C3 (g/l), median SD C4 (g/l), median SD GC, germinal center; PIF, pulmonary interstitial fibrosis; ESSDAI, European League Against Rheumatism SS Disease Activity Index; RF, rheumatoid factor; SSA, Sj ogren s syndrome antigen A; SSB, Sjögren s syndrome antigen B; ANA, antinuclear antibodies; WBC, white blood cell; PLT, platelet; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein. *P < 0.05; **P < gland infiltration, production of various antibodies and hyper-immunoglobulin response, and were reported to have a 16- to 44-fold higher risk of developing lymphoma. 13,14 The high serum immunoglobulin levels and high risk of malignant transformation both seem to be associated with abnormal ectopic GC formation in autoimmune diseases. 15 GCs are specialized microenvironments within secondary lymphoid tissues in which B cells undergo extensive rounds of proliferation, somatic hypermutation and antigen affinity-driven selection, 15,16 especially in antibody-related autoimmune diseases such as SS, SLE and RA. GC is a unique formation that occurs during the immune response to many types of antigenic stimuli, and they are formed with the help of antigenspecific B and T cells that were previously activated in the early stages of the response. Despite this general understanding of GCs, studies on human diseases have not been well worked out. Nonetheless, a growing body of clinical, laboratory and pathological evidence has sparked interest in the possibility of GCs participating the pathogenesis of SS. 17 Studies on the formation of GCs in ectopic sites in SS have reported inconsistent results. In this study, we addressed the significance of ectopic GC-like structures and their relationship with clinical and laboratory data in SS patients. Some in vivo studies on experimental animals have demonstrated that, after immunization, the affinity of the antibodies in serum increased dramatically with time. It is difficult to isolate the specific antigen and identify the duration of antigenic exposure; however, the affinity maturation phenomenon is indicated by antibody production in humans. In this study, patients with GC formation showed elevated titers of International Journal of Rheumatic Diseases

6 J. He et al. Patient characteristics Ectopic GC-like morphology P-value (n = 4, 25.0%) (n = 12, 75.0%) Scores DAS28, mean SD ESSDAI score, mean SD Focus score, mean SD * Autoantibodies RF-IgM (IU/mL), median SD Anti-CCP-IgG (RU/mL), median SD AKA-IgG positive, n (%) 3 (75.0) 6 (50.0) APF-IgG positive, n (%) 4 (100.0) 8 (66.7) Anti-SSA positive, n (%) 2 (50.0) 4 (33.3) Anti-SSB positive, n (%) 1 (25.0) 2 (16.7) ANA titer, median SD * Other serological data ESR (mm/h), median SD CRP (mg/l), median SD Table 3 Comparison of scores and serological findings in 16 RA-sSS patients with GC-like structures () and those without GC-like structures () GC, germinal center; RA, rheumatoid arthritis; sss, secondary Sj ogren s syndrome; DAS28, Disease Activity Score of 28 joints; ESSDAI, European League Against Rheumatism SS Disease Activity Index; RF, rheumatoid factor; Anti-CCP, anticyclic citrullinated peptide AKA, antikeratin antibody; APF, antiperinuclear factor; SSA, Sj ogren s syndrome antigen A; SSB, Sj ogren s syndrome antigen B; ANA, antinuclear antibodies; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein. *P < Table 4 Multiple logistic regression analysis of autoantibody and IgG production associated with GCs OR 95% CI P-value RF-IgM * Anti-SSA Anti-SSB IgG * GC, germinal center; OR, odds ratio; CI, confidence interval; RF, rheumatoid factor; SSA, Sj ogren s syndrome antigen A; SSB, Sj ogren s syndrome antigen B. *P < autoantibodies such as RF, IgG and IgA. GCs were mostly found in seriously infiltrated lymphocytes. Thus, ectopic GCs may play an essential role in sustaining antibody production. GC has been shown to modulate B-cell maturation and differentiation into autoreactive B cells, 15 which led to autoantibody production. Our study also indicated that GC formation might be interrelated with RF-IgM production and IgG secretion. However, it remains to be understood whether the immune responses in the salivary glands are restricted to the antigens in the local environment, or if antigen trapping and enrichment could also become relevant for antigens that are not primarily involved in the disease process. Many papers have reported that GC B cells express enzyme activationinduced deaminase (AID), which deaminates cytidine residues in the V(D)J and switches on regions of the Ig gene, leading to somatic hypermutation and classswitch recombination. 18 For GCs, one key approach to unravel their function is B-cell differentiation and ontogeny. In secondary lymphoid organs, emigrated B lymphocytes give rise to type I and type II transitional B cells, which differentiate into marginal zone or follicle B cells, depending on the affinity of the BCRs B-cell receptors to their antigens. Once the B cells enter the follicles, the BT2 cells initiate ectopic GCs. 19 These processes have long been described as quite similar to those in lymphoid organs. 17 These ectopic GCs do not exclude autoreactive B cells. 17 Considering the involvement of GCs in autoimmune diseases, the expression of the transcription factor and AID support the functionality of ectopic GCs. AID is required for immunoglobulin switch and somatic hypermutations in follicular DC networks and interfollicular large B cells. Thus, GC was viewed as one of the most important entities in the development of autoimmune diseases. 6 International Journal of Rheumatic Diseases 2016

7 Germinal center-like structures in SS However, the present study is only an elementary study on ectopic GCs in autoimmune diseases. Other factors such as inflammatory mediators, specific helper T cells, Toll-like receptors and adhesion molecules may also play a role in the formation of GC. Many aspects of the autoimmune disease model and antigen-induced immunized animal model have proved the regulatory function of GCs, 20 while clinical confirmation of these features has been difficult as a result of the difficulty involved in establishing GCs in vitro. The aim of the present study is to summarize the clinical findings and validate the association of GC in SS. While this study primarily focuses on human disease, in vitro experiments are required to reproduce GCs in vivo. We hypothesized ectopic GCs located in regions such as the labial gland as seen in SS patients are prone to antigenic stimulation. GCs in the labial gland are sequentially colonized by cells specific for different antigens, which is known as the process of epitope spreading. Then, positively selected GC cells are exported from the GC as plasmablasts, which are precursors of the plasma cells that will secrete antibodies into the serum, or as memory B cells, which upon re-exposure to antigen will rapidly differentiate into plasma cells or re-enter the GC reaction for further diversification. Although the data of our study only demonstrated the limited role of ectopic GCs in SS, our findings suggest SS patients with ectopic GCs have distinct features, and ectopic GCs are necessary for the production of antibodies and aberrant immunoglobulin, which is the principal factor involved in the pathogenesis of SS. ACKNOWLEDGEMENTS JH and YJ contributed equally to this study. JH and XZ performed most of the experiments. JH and YJ drafted the manuscript. ZL conceived the study and participated in its design and coordination, and interpreted the data. All authors read and approved the final manuscript. This work was supported by National Key Technology Support Program, No. 2014BAI07B01, National Natural Science Foundation of China, No , Beijing Municipal Science & Technology Commission, No. Z REFERENCES 1 Fox RI (2005) Sjogren s syndrome. Lancet 366, Vitali C, Bombardieri S, Jonsson R et al. (2002) Classification criteria for Sjogren s syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 61, Ramos-Casals M, Brito-Zeron P, Font J (2007) The overlap of Sjogren s syndrome with other systemic autoimmune diseases. Semin Arthritis Rheum 36, Fox RI, Stern M, Michelson P (2000) Update in Sjogren syndrome. Curr Opin Rheumatol 12, He J, Guo JP, Ding Y et al. (2011) Diagnostic significance of measuring antibodies to cyclic type 3 muscarinic acetylcholine receptor peptides in primary Sjogren s syndrome. Rheumatology (Oxford) 50, He J, Chen QL, Li ZG (2006) Antibodies to alpha-fodrin derived peptide in Sjogren s syndrome. Ann Rheum Dis 65, Jonsson R, Kroneld U, Backman K, Magnusson B, Tarkowski A (1993) Progression of sialadenitis in Sjogren s syndrome. Br J Rheumatol 32, Nieuwenhuis P, Opstelten D (1984) Functional anatomy of germinal centers. Am J Anat 170, Risselada AP, Looije MF, Kruize AA, Bijlsma JW, van Roon JA (2013) The role of ectopic germinal centers in the immunopathology of primary Sjogren s syndrome: a systematic review. Semin Arthritis Rheum 42, Jonsson MV, Skarstein K, Jonsson R, Brun JG (2007) Serological implications of germinal center-like structures in primary Sjogren s syndrome. J Rheumatol 34, Arnett FC, Edworthy SM, Bloch DA et al. (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31, Carubbi F, Alunno A, Cipriani P et al. (2014) Is minor salivary gland biopsy more than a diagnostic tool in primary Sjogrens syndrome? Association between clinical, histopathological, and molecular features: a retrospective study. Semin Arthritis Rheum 44, Youinou P, Saraux A, Pers JO (2012) B-lymphocytes govern the pathogenesis of Sjogren s syndrome. Curr Pharm Biotechnol 13, Dong L, Chen Y, Masaki Y, Okazaki T, Umehara H (2013) Possible Mechanisms of Lymphoma Development in Sjogren s Syndrome. Curr Immunol Rev 9, MacLennan IC (1994) Germinal centers. Annu Rev Immunol 12, Honjo T, Kinoshita K, Muramatsu M (2002) Molecular mechanism of class switch recombination: linkage with somatic hypermutation. Annu Rev Immunol 20, Le Pottier L, Devauchelle V, Fautrel A et al. (2009) Ectopic germinal centers are rare in Sjogren s syndrome salivary glands and do not exclude autoreactive B cells. J Immunol 182, Pavri R, Nussenzweig MC (2011) AID targeting in antibody diversity. Adv Immunol 110, Schwickert TA, Alabyev B, Manser T, Nussenzweig MC (2009) Germinal center reutilization by newly activated B cells. J Exp Med 206, Victora GD, Nussenzweig MC (2012) Germinal centers. Annu Rev Immunol 30, International Journal of Rheumatic Diseases