Low prevalence of ectopic germinal centre formation in patients with HTLV-I-associated Sjogren's syndrome

Sir, We have proposed that HTLV-I infection can be a possible environmental factor for SS based on high prevalence of an anti-HTLV-I antibody in primary Sjögren's syndrome (pSS) in Nagasaki [1–3], and recently confirmed that labial salivary glands (LSGs) of the HTLV-I-seropositive SS patients are less damaged in radiography [4]. Amft et al. [5] revealed the prominent expression of B cell-attracting chemokine 1 (BCA-1/CXCL13) on endothelial cells and lymphocytic aggregates in the ectopic germinal centre (GC) of LSGs in SS, speculating that ectopic GC is associated with the autoantibodies production as well as the salivary destruction [5]. We focused on the presence of ectopic GC formation in situ as well as the expression of CXCL12/CXCL13. Sixty-four pSS patients were registered and classification of pSS was determined by the revised criteria proposed by the American-European Consensus group [6]. LSGs from a control subject who complained of sicca but did not meet the pSS criteria have been obtained. The presence of anti-HTLV-I antibody was determined by ELISA (Eitest-ATL kit; Eisai, Tokyo, Japan) or particle agglutination assay (Serodia-ATL Kit; Fujirebio, Tokyo, Japan). Informed consent for the usage of samples obtained by the biopsy was obtained from all the participating patients as of the commencement of the study, and the study was conducted with the approval of the Human Ethical Committee of our institution. Immunohistochemistry was performed by the labelled streptavidin–biotin method (Histofine Staining Kit; Nichirei, Tokyo, Japan) using mouse anti-CXCL12 monoclonal antibody or goat anti-CXCL13 polyclonal antibody (R&D Systems, Minneapolis, MN, USA) [2] with microwave epitope retrieval for the detection of CXCL13. Negative control sections was treated with normal mouse IgG or normal goat serum. Mann–Whitney U-test, the chi-square test or Fisher's exact probability test was used for the statistical analysis. A P-value < 0.05 was statistically significant. The gender and age were similar in 32 HTLV-I-seronegative (male/female 3/29; age 56.9 ± 14.9 years) and 32 HTLV-I-seropositive (male/female 4/28; age 58.5 ± 12.6 years) pSS patients, as well as in 9 HTLV-I-associated myelopathy (HAM)–pSS patients (male/female 1/8; age 61.6 ± 8.9 years). Sicca symptoms were observed in 83.3–100% of patients among the groups. Differences in anti-SS-A/SS-B antibodies (Mesacup SS-A/Ro test and SS-B/La Test; Medical & Biological Laboratories, Nagoya, Japan) and IgG concentrations at the time of biopsy were not statistically significant irrespective of HTLV-I infection. Strikingly, ectopic GC was low in HTLV-I-seropositive pSS (1/32, 3.1%) as compared with HTLV-I-seronegative pSS (6/32, 18.8%) (P = 0.045), and 0% in HAM–pSS. Expression of CXCL13 was observed in 0–10% of mononuclear cells (MNCs) of HTLV-I-seronegative pSS without ectopic GC patients or HTLV-I-seropositive pSS patients (Fig. 1). In HTLV-I-seronegative pSS (n = 6) with ectopic GC patients, the expression of CXCL13 was found dominantly in the light zone of ectopic GC. All of the cases showed >50% of MNCs in the light zone of ectopic GC expressing CXCL13. One pSS in HTLV-I carrier showed a relatively small size of ectopic GC whose CXCL13 expression pattern was similar. However, interestingly, the MNCs of HAM–pSS patients demonstrated no expression of CXCL13. In contrast to CXCL13, CXCL12 was commonly expressed on ductal epithelial cells of all the pSS patients irrespective of anti-HTLV-I antibody. In a normal subject, no expression of CXCL13 was observed with positive expression of CXCL12 similar with pSS. Expression of CXCL13 and CXCL12 in HTLV-I-seronegative and HTLV-I-seropositive patients with SS. Immunohistochemical analysis of CXCL13 (BCA-1) in minor LSGs was demonstrated (A–D). After pre-treatment with microwave epitope retrieval, goat anti-CXCL13 polyclonal antibody was used to detect the expression of CXCL13. Expression of CXCL12 (SDF-1) was examined using mouse anti-CXCL12 monoclonal antibody (E–H). (A and E) Expression of CXCL13 and CXCL12 in minor LSG from an HTLV-I-seronegative SS patient with ectopic GC. (B and F) Expression of CXCL13 and CXCL12 in minor LSG from an HTLV-I-seronegative SS patient without GC. (C and G) Expression of CXCL13 and CXCL12 in minor LSG from an HTLV-I-seropositive SS patient without GC. These are representative of five patients with HTLV-I-seronegative SS with ectopic GC, HTLV-I-seronegative SS patients without ectopic GC and HTLV-I-seropositive SS patients without GC, respectively. (D and H) Expression of CXCL13 and CXCL12 in minor LSG from an HAM–SS patient. (I and J) Expression of CXCL13 and CXCL12 in minor LSG section from a normal subject, respectively. (K and L) Expression of CXCL13 in the light zone and CXCL12 in mantle zone of human tonsil tissue as a positive control, respectively. Haematoxylin and methyl green were used for counterstaining of CXCL13 and CXCL12, respectively (original magnification ×100). The lymphoid aggregates of LSGs are responsible for autoantibody production that locally occurs in ectopic GC [5,7,8]. Radiographic destruction of the ductal structure in HTLV-I-seropositive pSS occurs to a lesser extent than in HTLV-I-seronegative pSS, which is a unique characteristic of the former [4]. The chemokines have been found to regulate ectopic GC formation of SS [5]. Xanthou et al. [9] also demonstrated the significance of lymphoid chemokines for lymphoid structure formation in SS, while others have demonstrated an association of CXCL13 expression and ectopic GC formation in SS [7, 8]. Barone et al. [8] found a B cell-dominant expression pattern, whereas the selected expression in acinar and ductal epithelial cells was observed by Salomonsson et al. [7], although the exact roles of these results remain unclear. Our data suggest an important interaction of CXCL13 and ectopic GC in sialadenitis in SS. The tendency towards low levels of radiographic damage in patients with HAM–pSS suggests that salivary-specific cytotoxicity is modified by HTLV-I infection. Due to even expression of CXCL12 irrespective of HTLV-I infection, HTLV-I presumably affects the CXCL13 expression of infected CD4+ T cells. Via inflammatory mediators modulated by HTLV-I tax protein, dysfunction of MNC-lineage cells due to HTLV-I infection is supposed to play an important role. Disclosure statement: The authors have declared no conflicts of interest.