Dragan Gazivoda

Proinflammatory and immunoregulatory mechanisms in periapical lesions

Proinflammatory and immunoregulatory cytokines are important for the pathogenesis of periapical lesions. However, little is known about how their functions are balanced and controlled at different phases of lesion development. The aim of this study was to examine the relationship between the production of Th1, Th2, Th17 and T regulatory cell (T reg) cytokines by human periapical lesion mononuclear cells (PL-MNC) in culture and their correlation with cellular composition and clinical presentation of the lesions. We show that symptomatic lesions are characterized by the infiltration of neutrophils, high production of IL-17, positive correlation between IL-17 and IFN-gamma, but not between IL-17 and IL-23 production. Most IL-17(+) cells coexpressed IFN-gamma. Asymptomatic lesions were phenotypically heterogeneous. The lesions with the predominance of T cells over B cells/plasma cells expressed higher levels of IFN-gamma which correlated with higher production of IL-12 and the frequency of macrophages. In contrast, in most B-type lesions higher levels of IL-5 and TGF-beta were observed, as well as positive correlation between the production of TGF-beta and IL-10. The addition of Th cytokines in PL-MNC cultures confirmed that Th1, Th2 and Th17 cytokines are mutually antagonistic, except that IL-17, unexpectedly, augmented the production of IFN-gamma. IL-10 and TGF-beta inhibited the production of both Th1 and Th17 cytokines. Dendritic cells (DCs) from periapical lesions, composed of immature (CD83(-)), and mature (CD83(+)) myeloid type DCs and plasmacytoid (BDCA2(+)) DCs produced higher levels of IL-12 and IL-23 but lower levels of IL-10 and TNF-alpha than monocyte (Mo) -derived DCs. IL-23 stimulated the production of IL-17 by PL-MNC, whereas the secretion of IFN-gamma was enhanced by both IL-12 and IL-23. Cumulatively, these results suggest that: (1) Th1 immune response is most probably important for all stages of periapical lesion development; (2) Th2 and immunoregulatory cytokines are more significant for advanced types of lesions with the predominance of B cells/plasma cells; (3) Th17 immune response seems to play a dominant role in exacerbating inflammation.

Production of proinflammatory and immunoregulatory cytokines by inflammatory cells from periapical lesions in culture

BACKGROUND The role of cytokines in pathogenesis of periapical lesions is not well understood. The aim of this study was to study the correlation between proinflammatory and immunoregulatory cytokines in periapical lesions and their relationship with cellular composition and clinical presentation. METHODS Inflammatory cells were isolated from 67 human periapical lesions and cultivated for 24 h. The levels of proinflammatory cytokines: interleukin-1 beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor alpha (TNF-alpha) and immunoregulatory cytokines: transforming growth factor-beta (TGF-beta) and IL-10 were determined in culture supernatants using a fluorescent bead immunoassay or ELISA. The phenotype of cells was analysed by immunocytochemistry. RESULTS Inflammatory cells from symptomatic lesions which contained higher proportion of granulocytes, secreted higher levels of IL-1, IL-6 and IL-8 compared with asymptomatic lesions. Large-size lesions contained lower percentages of mononuclear phagocytes, higher percentages of CD8(+) T cells and produced higher levels of TNF-alpha, IL-6 and IL-10 compared with small-size lesions. There were negative correlations between the concentrations of TGF-beta and proinflammatory cytokines. TGF-beta, added to cultures, downregulated the levels of proinflammatory cytokines more strongly than IL-10, independently of clinical presentation of the lesions. By contrast, exogenous IL-10 was mainly immunosuppressive in cultures of asymptomatic lesions. CONCLUSION Symptomatic lesions are characterized by higher production of proinflammatory cytokines. Immunoregulatory cytokines are more important for suppression of inflammation in asymptomatic lesions and in this context the effect of TGF-beta is more potent and different from IL-10.

Regulatory T-cells in Periapical Lesions

CD4+CD25hiFoxp3+ regulatory T-cells (Tregs) are of crucial importance in regulating the immune response, including the control of any defense against infection. Their presence in periapical lesions has not been demonstrated, as yet. We hypothesized that Tregs infiltrate periapical lesions, where they inhibit T-cell proliferation. The aim of this study was to characterize Tregs in periapical lesions by confocal microscopy, flow cytometry, and functional assays. We showed that CD4+CD25hiFoxp3+ cells in periapical lesions expressed IL-10 and TGF-β. Their frequency was significantly higher than in peripheral blood and correlated with the levels of TGF-β and IL-10 in culture supernatants of periapical lesion mononuclear cells. Tregs inhibited the proliferation of responder T-cells in vitro, at least in part, by stimulating the production of IL-10. These findings suggest that CD4+CD25hiFoxp3+ cells in periapical lesions may play regulatory roles in controlling local immune/inflammatory processes.

Production of IL‐10 and IL‐12 by antigen‐presenting cells in periapical lesions

BACKGROUND Interferon-γ (IFN-γ) plays an important role in the pathogenesis of periapical lesions. Its expression is up-regulated by interleukin (IL)-12) and down-regulated by IL-10. The aim of this work was to study the cellular source of these cytokines and their mutual interactions in human periapical lesions. METHODS Mononuclear cells, macrophages and dendritic cells were isolated from periapical lesions using plastic adherence and osmotic gradients. Cytokines were measured in culture supernatants by a microbeads fluorescence assay. Phenotypic characteristics of cells were studied by immunocytochemistry, whereas allostimulatory activity of antigen-presenting cells was tested using a mixed leukocyte reaction. RESULTS We observed the positive correlations between the levels of IL-12 and IFN-γ as well as IL-12 and IL-10 in cultures of mononuclear cells. As IL-10 and IL-12 are produced by dendritic cells and activated macrophages, we examined their contribution to the production of these cytokines. Macrophages, CD14(+) adherent cells, produced high levels of IL-10 and very low levels of IL-12. In contrast, non-adherent, strongly HLA-DR(+) dendritic cells, potent stimulators of the alloreactive T-cell response, produced low levels of IL-10 and moderate levels of IL-12. Dendritic cells stimulated the production of IFN-γ by allogeneic CD4(+) T cells. In contrast, the level of IFN-γ was significantly decreased and the production of IL-10 was enhanced by addition of macrophages to the culture system. CONCLUSION Our results suggest that a fine balance between the production of IL-10 and IL-12 by different antigen-presenting cells, through IFN-γ, may control the course of chronic inflammation in periapical lesions.

Immunomodulatory Activity of IL-27 in Human Periapical Lesions

IL-27, a cytokine with pro-inflammatory and anti-inflammatory properties, is a new member of the IL-6/IL-12 family, whose function in periapical lesions is unknown. We hypothesized that the production of IL-27 and its effect depend upon the type of immune/inflammatory response and clinical presentation of periapical lesions. We tested this hypothesis by studying the expression and function of IL-27 in human periapical lesions, both in situ and in culture. Immunohistochemistry demonstrated the strongest expression of IL-27 by endothelial cells and mononuclear phagocytes. Its production by periapical lesion mononuclear cells (PL-MNC), especially in symptomatic lesions, was significantly higher compared with that in peripheral blood MNC and correlated with the frequency of CD14+ and CD3+ cells. Exogenous IL-27 stimulated Th1 and down-regulated Th17 cytokine production by PL-MNC from symptomatic lesions, but down-regulated Th1 and Th2 responses in asymptomatic lesions. These findings suggest that IL-27 is an immunomodulatory cytokine in periapical lesions, with complex biological effects.