The chronic autoimmune disorder Sjögren's syndrome (SS) affects exocrine organs, particularly salivary and lachrymal glands (1). Destruction and functional derangement of these glands lead to oral and ocular dryness, in addition to which patients may experience arthralgias and chronic fatigue. B cells and autoantibodies play an important role in the disease, and disturbances in the peripheral and glandular B cell population in patients with SS have been demonstrated (2, 3), specifically in terms of clonal expansion (4), changed V- and J-gene segment usage (5), increased plasma cell occurrence in the glands (6), and hypergammaglobulinemia. Autoreactive specificities commonly detected within this antibody pool are the anti-Ro/SSA and anti-La/SSB antibodies, directed against intracellular ribonucleoprotein targets (7).
The glandular lymphocytic infiltration is a progressive feature (8), which, when extensive, may replace large portions of the organs. The glandular infiltrates in some patients closely resemble organized lymphoid tissue with germinal centers (GCs) (9–13). Signs of ectopic lymphoid tissue and lymphoid neogenesis have also been observed in affected tissue in other autoimmune conditions, including synovia in rheumatoid arthritis (14, 15), the thymus in myasthenia gravis (16), and the thyroid in Hashimoto thyroiditis (17). In addition, structures resembling GCs can be observed in chronic inflammation without apparent autoimmune origin, as during infection with Helicobacter pylori of the gastric mucosa (18) and chronic inflammatory diseases of the liver (19).
Lymphoid neogenesis with formation of ectopic lymphoid follicles in chronic inflammatory disease is a complex process regulated by expression of an array of cytokines, chemokines, and adhesion molecules (for review, see refs. 20 and 21). Several studies have characterized such ectopic lymphoid tissue and shown that it has morphologic features of secondary lymphoid organs with the presence of activated postcapillary high endothelial venules (HEVs), GCs in the interface of B and T cell areas, and expression of adhesion molecules and chemokines mediating homing of naive cells (22, 23).
The signals governing normal lymphoid organogenesis during development seem to be involved also in formation of ectopic lymphoid tissue at sites of chronic inflammation (for review, see ref. 24). In particular, the B cell–attracting chemokine CXCL13, required for normal polarization of GCs, has been implicated as a key regulator of lymphoid neogenesis. A number of studies have shown this molecule to be expressed in the glandular infiltrates of patients with SS (11–13). Other chemokines implicated in lymphoid neogenesis are the homing chemokines CCL21 and CXCL12. CCL21 is highly expressed in HEVs and in the T cell area in secondary lymphoid organs and is a chemoattractant for T cells and dendritic cells, while CXCL12 is a potent chemoattractant for both naive and memory T cells and is essential for the earliest stages of B cell lymphopoiesis (for review, see ref. 25). Further, both CCL21 and CXCL12 induce integrin-mediated adhesion of naive cells (26), and CCL21 was recently reported to be ectopically induced in association with chronic inflammatory liver disease (19).
Adhesion molecules are of critical importance in cell trafficking to the inflamed organ. In an animal model of SS, both the adhesion pathways of integrins lymphocyte function–associated antigen 1 (LFA-1; α1β2) and very late activation antigen 4 (VLA-4; α4β1) and their ligands intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) have been shown to be important for lymphocyte migration from blood to inflamed glands (27).
Little is yet known about the functional activity of the ectopic lymphoid tissue seen in autoimmune diseases such as SS. Also, the frequency with which SS patients develop such structures has not been determined. To investigate these issues, salivary gland biopsy samples from patients with focal sialadenitis indicating SS were screened for occurrence of GC-like structures, and 17% of these were found to be positive. High levels of autoantibody production and apoptosis were detected in patients with GCs, indicating a higher level of activity and organization in the glands of these patients. In contrast to previous findings, however, only moderate differences in chemokine and adhesion molecule expression were observed between patients with and those without ectopic GCs. Our data suggest that ectopic GC formation and lymphoid neogenesis take place in the salivary glands of patients with SS. This active participation in the chronic inflammatory process might sustain the autoimmune response and contribute to tissue destruction and loss of function in the exocrine organs of these patients.
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- PATIENTS AND METHODS
Destruction of salivary glands in patients with SS is related to chronic inflammation, manifested by focal lymphocytic infiltration. By using a series of phenotypic markers, we identified an architecture and activity in the salivary glands of SS patients normally found in organized secondary lymphoid tissue. The observations of large aggregates of B and T cells and dense populations of proliferating cells in close proximity to FDC networks indicate that GCs are formed in patients with SS. High expression of adhesion molecules and chemokines, involved in attracting and organizing lymphocytes in secondary lymphoid organs, as well as apoptosis and local production of autoantibodies against the Ro/SSA and La/SSB proteins were observed in the salivary glands of SS patients. Taken together, we demonstrate that highly organized structures are formed and activity normally associated with secondary lymphoid organs takes place in the target organ of patients with SS.
GC-like structures could be identified in 17% of the 165 investigated patients. No significant difference in their occurrence was observed between primary and secondary SS. For diagnostic purposes, at least 5 minor salivary glands are recommended. This amount is considered sufficient to provide a representative sample of material for the generalized exocrinopathy, lending support to a fairly proper assessment of GC development in our study. To our knowledge, this is the first report to describe the scanning of a larger sample of material obtained from patients evaluated for SS, although it is possible that the occurrence of GC-like structures might be different in the major glands. The frequency we found in minor salivary glands is nevertheless comparable with the incidence in synovial tissue of patients with rheumatoid arthritis, where GCs were detected in 23% of the biopsy samples (14).
In the minor salivary glands, we observed GCs only in manifest infiltrates, and in most cases proliferating areas were seen in several glands from the same patient. This indicates that a mass of cells is required to provide the microenvironment where GCs may develop, but that when it is initiated, the GC formation is a general phenomenon occurring at multiple sites. Autoantibody-producing cells were detected at significantly higher frequency and in higher numbers in patients with GCs compared with patients lacking such structures, indicating a functional activity in the organized lymphocytic infiltrates. A correlation was also detected between the presence of local antibody-producing cells in the salivary glands and higher anti-Ro and anti-La levels in sera as we previously described (2, 35), suggesting a more active disease status in these patients.
Reports of clonal B cell proliferation and hypermutation within the salivary glands of patients with SS (9), together with direct and indirect signs of local production of autoantibodies (13, 36, 37), support the interpretation of functional activity in the GCs. Differences in the microenvironment and structure, as observed in chronically inflamed tonsils compared with traditional GCs (38), possibly in combination with easy access to autoantigens, might contribute to a disturbance in the selection process allowing autoreactive clones to develop more frequently. Even though local production of autoantibodies in the salivary glands is correlated with higher antibody levels, it should be noted that the autoantibodies are not confined to patients with ectopic GCs, indicating the possibility of anti-Ro/anti-La autoantibody production taking place at additional sites. In this respect, some autoantibody-producing cells were found to recirculate (37), but production elsewhere, including bone marrow and spleen, remains to be investigated.
Hypergammaglobulinemia and persisting levels of autoantigen-specific IgM (39) have been demonstrated in SS patients, and one might speculate that at least some of the plasma cells encountered within the inflamed glands belong to the pool of long-lived plasma cells, for which inflamed tissue has recently been shown to provide survival niches equivalent to those in the bone marrow (40). A regulation of plasma cell survival has been suggested to be mediated by competition for survival niches in the bone marrow; de novo formation of survival niches in chronically inflamed tissue might unbalance this regulation and promote survival of autoreactive B cell clones, thus further sustaining the disease process.
Moderate levels of apoptotic cells were detected both in patients with and in those without GCs. The apoptotic cells were encountered at the margins of large infiltrates, as well as in interstitial space, partly coinciding with the localization of autoantibody-producing cells, in concordance with previously reported findings (2, 13, 34). The low numbers of apoptotic cells observed could be a result of B cells escaping apoptosis and proper selection in the ectopic GCs. Indeed, attenuated apoptosis of B cell–activating factor–expressing cells has recently been demonstrated in SS (41). The low apoptosis rate might also partly be explained by the fact that apoptosis is a relatively short event, spanning ∼3 hours, possibly making detection difficult with the TUNEL method used.
The investigated chemokines CXCL13, CCL21, and CXCL12 were expressed by acinar and ductal epithelial cells in all SS patients studied. Notably, no correlation was observed between level of chemokine expression and presence of GCs or size of lymphocytic infiltrates, implying a constant level of chemokine expression maintained during the entire inflammatory process in the investigated patients. This partly contradicts the interpretation of previous findings, where expression of CXCL13 was suggested to be closely associated with ectopic GC development (11). In another study (14), tissue containing ectopic GC reactions was reported to have a 10–20-fold increase in transcription levels of CXCL13 and CCL21, although it should be noted that these chemokines were also detected in tissues lacking such structures. In conformity with findings of others, our results suggest that the epithelial cells are active participants in the inflammatory process in the glands of SS patients, both in producing chemokines and cytokines (11, 13, 42–44) and in expression of major histocompatibility complex class II (45) and costimulatory molecules (46).
The adhesion molecules LFA-1 and VLA-4 were expressed in all salivary glands investigated. Patients with GCs showed a lower expression of LFA-1 in the central region of large infiltrates, possibly indicating a decrease in LFA-1–mediated adhesion interactions when infiltrates are being organized. LFA-1 has a central role in lymphocyte trafficking and interaction, as implied by the findings of in vitro experiments where antibodies against LFA-1 have been reported to inhibit adhesion of both naive and activated T cells (for review, see ref. 47). Patients lacking LFA-1 have abnormalities in a wide spectrum of adherence-dependent functions of granulocytes, monocytes, and lymphoid cells (48).
ICAM-1 was highly expressed on infiltrating mononuclear cells in all biopsy samples investigated. Similar to LFA-1, expression of ICAM-1 was lower in the central region of large infiltrates. ICAM-1 was also detected on fibroblast-like cells, as reported earlier in SS patients (49). In contrast to the findings of Kapsogeorgou and colleagues (49), we could not detect ICAM-1 on the epithelium, except for a discrete staining in the basement membrane region where acini or ducts appeared in close proximity to infiltrates. VCAM-1 was expressed on endothelial and fibroblast-like cells. A reticular staining pattern for VCAM-1 on mononuclear cells was observed in large infiltrates, possibly representing FDCs (10). In concordance with the findings of others, we found increased expression of adhesion molecules in the glands of patients with SS, suggesting activation and active recruitment of inflammatory cells (49–51). The ratios of both ICAM-1– and VCAM-1–expressing cells were increased in focal infiltrates in SS patients with GCs, indicating a higher degree of activation and lymphocyte trafficking in the salivary glands of these patients, although differences were not statistically significant.
An association of GC development with the increased risk of B cell lymphomas seen in patients with SS has been suggested (52), and formation of proliferating GCs has been proposed to contribute to the malignant transformation and development of mucosa-associated lymphoid tissue–type lymphoma (53). Patients with SS and chronic H pylori infection are at an elevated risk for developing lymphomas, possibly related to prolonged lymphocytic activation in the target organs of these patients. Disturbances of the B cell maturation have been demonstrated in patients with SS (3, 54), part of which might be explained by overstimulation and neglected selection in ectopic GCs in the affected glands. To include assessment of GCs in the clinical routine evaluation of diagnostic minor salivary gland biopsy samples might therefore prove beneficial in predicting lymphoma development, suggesting a need for prospective studies confirming the association. Also, biopsy sample size will be important in obtaining representative material for such a study.
In conclusion, our results suggest that formation of ectopic lymphoid microstructures in nonlymphoid organs participates in the pathogenesis of organ-specific autoimmunity, here illustrated by the chronic inflammatory disease SS. Whether this is a primary or secondary event in the initiation of the inflammatory and autoimmune disease process remains to be investigated further; our observations nonetheless underscore a key role of the target organ in cell recruitment and disease progression. The involvement of salivary glands as a site of ectopic GC formation and of the selection of high-affinity autoantibodies mediating this autoimmune state suggests novel targets for future immunomodulatory therapeutic strategies.