The findings of this study are: (1) the expression of all Th subset-related molecules in whole LSGs from SS patients was higher than those in the controls. (2) The expression of Th2 and Tfh-related molecules was associated closely with strong lymphocytic accumulation in whole LSGs from SS patients. (3) In the selectively extracted lesions of LSGs, expression of Th1- and Th17-related molecules in infiltrating lymphocytes without ectopic GC was higher than those with ectopic GC. In contrast, expression of Th2 and Tfh-related molecules in infiltrating lymphocytes with ectopic GC was higher than in those without ectopic GC.
Analyses over time of the mRNA expression of cytokines and transcription factors in whole LSGs performed neither a prospective cohort study nor multiple biopsies. From a practical viewpoint, it is difficult to evaluate the initiation and progression of SS. The acquisition of lymphoid features by inflammatory foci in the LSGs of SS is associated critically with enlargement of the inflammatory foci and with secondary lymphoid follicles [29
]. Therefore, in order to evaluate accurately the propagation of the disease process, in the same SS patients a novel strategy was thus used to compare the expression of cytokines and transcription factors in the infiltrating lymphocytes with/without ectopic GC in the LSGs specimens by using LCM.
In SS-susceptible mice, the elimination of Th1 cells ameliorated all pathological and clinical signs of the disease [30
]. Our previous study suggested a model of the pathogenesis of SS [17
]. The mutual stimulation of Th1 cells and the target organ via the production of various cytokines plays a key role in the induction and/or maintenance of the disease and results in the eventual destruction of the target organ. Recently, CD4+
Th17 cells have been shown to be tissue-seeking and involved intimately in the initiation of SS [31
]. The results of the present study concerning lymphocyte subsets and cytokine production in the LSGs are consistent with this model. Youinou et al
] reported that Th17 cells orchestrate autoreactive GCs. Our results were consistent with this report. However, in the selectively extracted lesions of LSGs, expression of Th17-related molecules in infiltrating lymphocytes without the ectopic GC was higher than in those with the ectopic GC. Interestingly, Th17/Th1 cells reportedly co-express IFN-γ with IL-17 [34
], and such a subset has been identified in the gut in Crohn's disease [35
]. Both Th1 and Th17 cells together were involved in the pathogenesis of SS [36
], and there was an early induction of a CD4+
Th1/Th17 pathway leading to systemic release of IL-17 in mice [31
]. Our observations suggest that both Th1 and Th17 cells around the ductal epithelial cells might be of critical importance in the initiation of SS.
Mitsiasis et al
] have reported that the balance between Th1 and Th2 shifted in favour of the former in LSG with a high infiltration score. Our results are consistent with these results. In our present data, mRNA expression of Th1 cytokines in the LSG with both weak and strong lymphocytic infiltration from SS patients was also significantly higher than that from controls. Therefore, these results suggest that Th1 cytokines play a key role in the induction and maintenance of the disease. Conversely, we reported that the levels of mRNA for both Th1 and Th2 cytokines and chemokines in LSGs with strong lymphocytic infiltration from patients with SS were significantly higher than in controls [26
]. As described above, Theander et al
] reported that the ectopic GC-like structures, including ectopic GC in the LSGs, was involved in the development of malignant lymphoma in primary SS. We thus speculate that additional Th2 cells play a role in the lympho-aggressiveness of the disease. This paper focused on infiltrating lymphocytes, particularly Th subsets, around the ductal epithelial cells and ectopic GCs in LSGs, using a LCM technique to obtain tissue samples exclusively from specific regions of interest. We have positive results indicating that expression of Th2 cytokines in infiltrating lymphocytes with the ectopic GC was higher than that around the ductal epithelial cells. From the results obtained in this study, we speculated that Th2 cells might be involved in the progression of the disease, especially in the growth and activation of ectopic GC formation. Furthermore, several studies on autoimmune diseases in mice and humans have indicated a pathogenic role for Th1 cells and a possible protective role for Th2 cells [38
]. Our results, showing that expression of Th1-related molecules in infiltrating lymphocytes with the ectopic GC was lower than without GC, and that of Th2-related molecules with GC was higher than without GC, are consistent with this report. Previous studies have reported that Th2 effector cells was important role for GC formation [40
], and that Th2 cytokines induced infiltrating B lymphocytes to produce autoreactive antibodies [41
]. Furthermore, the salivary gland environment in SS, in association with tissue trophic viruses such as the Epstein–Barr virus [42
], cytomegalovirus [43
] and retrovirus [44
], might increase the risk of pseudolymphoma and hypergammaglobulinaemia promotion, and might hasten the progression to B cell malignant lymphoma. Considering the possible role of Th2 cells in the induction of B cell abnormalities, these cells might have a harmful (rather than a protective) effect on SS. Conversely, several studies have reported that expression of the chemokine receptor CXCR5 allows Tfh to home to the B cell follicle [45
], IL-21 was increased in serum from SS patients [46
] and high levels of IL-21 receptor were present at the surface of most B cells [7
]. Furthermore, mice that lack the receptors for both IL-4 and IL-21 have greatly reduced IgG responses, indicating that IL-21 co-operates with IL-4 to regulate humoral immune responses [47
]. In the LSG lesions, Tfh cells in infiltrating lymphocytes in/around the ectopic GC was significantly higher than that around the ductal epithelial cells ( and ). Our results strongly support that Th2 and Tfh cells are involved in the progression of the disease process as a lympho-aggressive disorder, particularly growth and activation of the ectopic GC formation.
Kolkowski et al
. revealed that salivary glands in SS consistently express IL-10 and TGF-β[17
]. Our results obtained in the present study are in accord with this report. Other studies have reported that the remarkable reduction of Treg
in LSGs and peripheral blood might be involved in the pathogenesis of salivary gland destruction in SS [49
]. Contrary to this study, Gottenberg et al
. reported that Treg
cell numbers were increased in the peripheral blood of patients with SS [50
]. Therefore, it is unclear whether or not Tregs
are involved in the pathogenesis of SS. Recently, the immunoregulatory role of CD4+
T cells might be different at each stage of the disease process in rheumatoid arthritis [51
]. However, our results indicated that FoxP3 mRNA expression showed no relationship to the degree of lymphocytic infiltration in whole LSGs from SS patients, between weak and strong lymphocytic infiltration (). The lower levels of Treg
cells in/around the ectopic GC might not result in ectopic GC formation ( and ). Furthermore, recent studies, in which SS patients were divided into three groups (mild, intermediate and severe lymphocytic infiltration in the LSGs), reported that FoxP3+
cells at the LSGs of severe SS stage was reduced in comparison with that of intermediate stage SS [3
]. Our previous study, which concerned the frequency of reduced Treg
in the severe stage of LSGs from SS patients, is consistent with this report [26
]. Furthermore, increased levels of Treg
cells in the whole LSGs from SS patients might suggest that negative feedback is more active in the LSGs from SS patients than in healthy subjects (). Therefore, these results suggest that Tregs
might be not involved in the initiation, growth and activation of ectopic GC formation of SS. However, the immunoregulatory role of Treg
might be different at each stage of disease status in SS [52
]. Conversely, we have also investigated whether Mikulicz's disease, which apparently differs from SS, was a unique IgG4-related disease characterized by Th2 and regulatory immune reactions [26
Furthermore, we also examined the mRNA expression of Th1-, Th2-, Th17- and Treg-related molecules in peripheral blood mononuclear cells (PBMCs) from SS patients and controls. The mRNA expression of IFN-inducible protein 10 (IP-10) and CXCR3 in PBMCs from SS patients was slightly higher than that from controls, but that of the other molecules was not. In addition, the mRNA expression of all these molecules in the PBMCs showed no significant relationship to the degree of lymphocytic infiltration in the LSGs from the SS patients. These results indicate that few T cells involved in the pathogenesis of SS circulate in the periphery (manuscript in preparation).
In conclusion, we provide new evidence concerning the selective localization of Th subsets in LSGs from SS patients. In addition, it is still necessary to elucidate the mechanisms underlying Th2 and Tfh cell induction to provide functional evidence on the direct role of Th2 and Tfh cell progression, which might lead eventually to the creation of therapeutic strategies for inhibiting the disease progression. A more thorough understanding of the complex mechanisms of the disease, especially those involved in these Th subsets, might lead to pharmacological strategies to disrupt the cytokine network as a further means of inhibiting the initiation and/or progression of SS as a lympho-aggressive disorder.
Although our present study focused on Th subsets, B cells also play a key role in the formation of ectopic GC, and we are thus trying to evaluate the progression of SS more thoroughly (manuscript in preparation).