The Expression Of Interleukin-27 On Mast Cells In Human Chronic Periapical Diseases

Background and ObjectivePeriapical disease is a sequel to endodontic infection and manifests itself as the host defense response to microbial challenge emanating from the root canal system to the periapical tissues. Inflammatory cells, immune cells and cytokines(CKs)involve in the pathogenesis of periapical disease. Studies have shown that Interleukin-27(IL-27), as a new member of the IL-12 family, plays an important immunomodulatory role in cancer, autoimmune diseases and infectious diseases with two-way immunomodulatory effects. Although a large number of experimental and clinical studies have been done, but the mechanism of immune cells and CKs participating in onset and disease duration of periapical diseases and the expression and cellular localization of IL-27 have not yet been elucidated. Mast cells(MCs) are traditionally considered as the main effector cells of allergic inflammation. Recent studies have found that MCs have an important role in the innate and adaptive immune responses. The pathogenic mechanisms of CKs on MCs involved in periapical diseases are unknown. In the present study, the specimens of periapical diseases were stained with hematoxylin and eosin(HE) for histopathology, with toluidine blue and double immunofluorescence staining for identifying MCs, and with immunohistochemical staining for identifying IL-27 positive cells. Tryptase-IL-27 double positive MCs were identified by double immunofluorescence staining. Results from these studies will provide valuable insight into the role of immunopathogenesis of tryptase-IL-27 positive MCs in human chronic periapical diseases.MethodsSeventy five cases of specimen, including healthy control(n=25), periapical cyst(n=25) and periapical granuloma(n=25), were involved in the present study. Tissue material was fixed in 10 % buffered formalin for at least 48 h and then embedded in paraffin. Serial 5 μm thick sections were deposited onto Super Frost/Plus microscopeglasses. Routine staining of sections using HE was performed for histopathology.Toluidine blue staining was performed for identifying MCs. Immunohistochemical staining was performed for identifying IL-27 positive cells. IL-27 positive MCs were identified by double immunofluorescence staining. All statistical analyses were performed using SPSS software(version 13.0). Nonparametric tests were chosen for variables since the data were not distributed normally. Differences in the densities of MCs and degranulated MCs with toluidine blue staining, the density of IL-27 positive cells with immunohistochemical staining and the density of IL-27 positive MCs with double immunofluorescence staining of different groups were analyzed using Kruskal-Wallis H test. Two-group comparisons were assessed using Nemenyi test.The comparison between the density of MCs with toluidine blue staining and the density of tryptase-IL-27 double positive cells with double immunofluorescence staining in different groups was performed with paired sample Wilcoxon’s sign rank test. The comparison between the density of IL-27 positive cells with immunohistochemical staining and the density of tryptase-IL-27 double positive cells with double immunofluorescence staining in different groups was also performed with paired sample Wilcoxon’s sign rank test. A p value of <0.05 was considered statistically significant.Results1. Histological observation1) There was no inflammatory cell infiltration in normal periodontal ligament;2) We observed a large number of inflammatory cell infiltration in apical granuloma, mainly monocytes, lymphocytes and plasma cells showed focal infiltration, in which macrophage-like cells and neutrophils infiltrated scatteredly;3) The epithelial layer of radicular cyst showed intercellular edema and chronic inflammatory cells infiltration, the fibrous tissue layer was mainly comprised of collagen fiber and with no significant inflammatory cell infiltration.2. Toluidine blue staining results.1) Compared to healthy control, there were significantly higher densities of bothtotal and degranulated MCs in human periapical lesions(p<0.01);2) The number of MCs and degranulated MCs in periapical cyst were significantly higher than that of periapical granuloma(p<0.01).3. Immunohistochemical staining results.1) The density of IL-27 positive cells in periapical lesions was significantly higher than that of healthy control(p<0.01);2) The number of IL-27 positive cells in periapical cyst was significantly higher than that of periapical granuloma(p<0.01).4. Double immunofluorescence staining results.1) The density of tryptase-IL-27 double positive MCs in periapical lesions was significantly higher than that of healthy control(p<0.01);2) The number of tryptase-IL-27 double positive MCs in periapical cyst was significantly higher than that of periapical granuloma(p<0.01).5. The comparison of toluidine blue staining and double immunofluorescence staining.Compared with toluidine blue staining, the number of MCs with double immunofluorescence staining increased significantly(p<0.01).6. The comparison of immunohistochemical staining and double immunofluorescence staining.In each group, the number of IL-27 positive cells with immunohistochemical staining was significantly higher than that of tryptase-IL-27 double positive cells with double immunofluorescence staining(p<0.01).Conclusions1. The IL-27 positive MCs may play an important role in the pathogenesis of human chronic periapical diseases, particularly in the formation of fibrous tissue in periapical cyst.2. Double immunofluorescence staining is more sensitive than the traditional toluidine blue staining for identifying MCs.3. IL-27 not only expresses in MCs, but also in other cells.