We have previously shown that the absence of Fas/Fas ligand significantly reduced tissue damage and intestinal epithelial cell (IEC) apoptosis in an in vivo model of T cell-mediated enteropathy. This enteropathy was more severe in IL-10-deficient mice, and this was associated with increased serum levels of IFN-gamma and TNF-alpha and an increase in Fas expression on IECs. In this study, we investigated the potential of IL-10 to directly influence Fas expression and Fas-induced IEC apoptosis. Mouse intestinal epithelial cell lines MODE-K and IEC4.1 were cultured with IFN-gamma, TNF-alpha, or anti-Fas monoclonal antibody (mAb) in the presence or absence of IL-10. Fas expression and apoptosis were determined by FACScan analysis of phycoerythrin-anti-Fas mAb staining and annexin V staining, respectively. Treatment with a combination of IFN-gamma and TNF-alpha induced significant apoptosis. Anti-Fas mAb alone did not induce much apoptosis unless cells were pretreated with IFN-gamma and TNF-alpha. These IECs constitutively expressed low levels of Fas, which significantly increased by preincubation of the cells with IFN-gamma and TNF-alpha. Treatment with cytokine or cytokine plus anti-Fas mAb increased apoptosis, which correlated with a decreased Fas-associated death domain IL-1-converting enzyme-like inhibitory protein (FLIP) level, increased caspase-8 activity, and subsequently increased caspase-3 activity. IL-10 diminished both cytokine- and anti-Fas mAb-induced apoptosis, and this was correlated with decreased cytokine-induced Fas expression, increased FLIP, and decreased caspase-8 and caspase-3 activity. In conclusion, IL-10 modulated cytokine induction of Fas expression on IEC cell lines and regulated IEC susceptibility to TNF-alpha, IFN-gamma, and Fas-mediated apoptosis. These findings suggest that IL-10 directly modulates IEC responses to T cell-mediated apoptotic signals.
In the present study, we show therapeutic amelioration of established ovalbumin (OVA)-induced allergic airway disease following house dust mite (HDM) peptide therapy. Mice were sensitized and challenged with OVA and HDM protein extract (Dermatophagoides species) to induce dual allergen sensitization and allergic airway disease. Treatment of allergic mice with peptides derived from the major allergen Der p 1 suppressed OVA-induced airway hyperresponsiveness, tissue eosinophilia, and goblet cell hyperplasia upon rechallenge with allergen. Peptide treatment also suppressed OVA-specific T-cell proliferation. Resolution of airway pathophysiology was associated with a reduction in recruitment, proliferation, and effector function of T(H)2 cells and decreased interleukin (IL)-17⁺ T cells. Furthermore, peptide immunotherapy induced the regulatory cytokine IL-10 and increased the proportion of Fox p3⁺ cells among those expressing IL-10. Tolerance to OVA was not associated with increased IL-35. In conclusion, our results provide in vivo evidence for the creation of a tolerogenic environment following HDM peptide immunotherapy, leading to the therapeutic amelioration of established OVA-induced allergic airway disease.
These observations suggest that immune tolerance induced by peptide immunotherapy can be used experimentally to treat an allergic response to another allergen and that the molecular mechanisms underlying induction of tolerance to a treatment-specific allergen and a bystander allergen might be different.
The role of invariant NKT (iNKT) cells in reactive arthritis is unknown. We explored the functional role of NKT cells in reactive arthritis using an established murine model of Chlamydia trachomatis-induced arthritis (CtIA). CtIA in wild-type and CD1d knockout (KO) mice was induced by intra-articular injection of C. trachomatis. The effect of alpha-galactosylceramide (alpha-GalCer) activation of iNKT cells was investigated by intra-peritoneal administration of alpha-GalCer. Histopathological and phenotypic changes, chlamydial clearance and cytokine and chemokine production in synovial tissue of the knee joint were investigated after onset of the arthritis. The severity of CtIA was significantly increased in CD1d KO mice, which was associated with decrease in bactericidal cytokine IFN-gamma, regulatory cytokines IL-4 and IL-10 and increase in pro-inflammatory chemokines macrophage inflammatory protein-2 (MIP-2) and IFN-gamma-inducible protein-10 (IP-10). Local clearance of the pathogen from the joint was also decreased. Prior treatment of mice with alpha-GalCer, a potent activator of iNKT cells, significantly reduced the severity of CtIA in mice. The amelioration of CtIA was associated with decrease in chlamydial load and induction of cytokines IFN-gamma, IL-4 and IL-10 and significant suppression of MIP-2 and IP-10. Treatment of established CtIA with alpha-GalCer also demonstrated modulation of CtIA and decrease in chlamydial load. These results suggest that iNKT cells are protective against CtIA and alpha-GalCer-activated iNKT cells have an immunoregulatory role not only in preventing the induction of reactive arthritis but also in modulating established disease.
Intraduodenal priming of mice with reovirus serotype 1/strain Lang (reovirus 1/L) stimulates gut lymphocytes and generates precursor and effector CTLs. Our earlier studies demonstrated that germinal center and T cell Ag (GCT) is a marker which identifies reovirus 1/L-specific precursor CTL and effector CTL in Peyer’s patches (PP) of reovirus 1/L-inoculated mice. In this study, we characterized the expression of the activation markers, GCT and CD11c, on reovirus 1/L-stimulated gut lymphocytes and the effector mechanisms involved in reovirus 1/L-specific cytotoxicity. We found that intraduodenal reovirus 1/L inoculation of mice induced the expression of both GCT and CD11c on PP lymphocytes (PPL), intraepithelial lymphocytes (IEL), and lamina propria lymphocytes (LPL), and these activated cells expressed Fas ligand (FasL). The majority of the GCT+CD11c+ IEL and LPL expressed a phenotype, TCRαβ+Thy-1+CD8+ similar to that expressed on reovirus 1/L-stimulated PPL. However, splenic lymphocytes expressed GCT but not CD11c after stimulation with reovirus 1/L. Perforin, Fas-FasL, and TRAIL pathways were found to be involved in PPL, IEL, and LPL cytotoxic activity against reovirus 1/L-infected targets. In PPL, perforin and Fas-FasL pathways were more effective than TRAIL. In IEL, all three cytotoxic mechanisms were equally as effective. However, LPL prefer Fas-FasL and TRAIL over perforin. Further, we demonstrated the preferential migration of GCT+ PPL to the intraepithelial compartment and the lamina propria. These results suggest that GCT and CD11c can be used as activation markers for gut lymphocytes and CD11c can also be used to differentiate between activated gut and systemic lymphocytes.
Intestinal intra-epithelial lymphocytes (IELs) form a highly specialized lymphoid compartment. IELs consist primarily of T cells that are dispersed as single cells within the epithelial cell layer that surrounds the intestinal lumen. These lymphocytes along with lamina propria lymphocytes are considered to play an important role in the regulation of immune responses. IELs are heterogeneous with regard to phenotype, and they contain sub-populations with diverse functions. In our most recent study, we found that intra-duodenal inoculation of mice with reovirus serotype 1/strain Lang (reovirus 1/L) induced expression of both germinal center and T cell antigen and CD11c on IELs suggesting these cells to be the recently stimulated cells in gut mucosal tissue. We also demonstrated that IELs from these mice when cultured in vitro in the presence of reovirus 1/L-pulsed antigen-presenting cells generated reovirus 1/L-specific MHC-restricted CTL whose function was mediated utilizing perforin, Fas-FasL and TRAIL mechanisms. This present study provides a comprehensive analysis of the diverse subsets of IELs, which function with other mucosal cells to provide a strong, protective immunity in a highly regulated fashion inside the microenvironment of the intestinal epithelium. We demonstrated that the IEL population contains both thymus-dependent (TD) and thymus-independent (TI) lymphocytes in mice and that a complex phenotype is present when sub-populations are analyzed for TCR, Thy-1, CD4, CD8 and B220 expression in a comprehensive manner. In reovirus 1/L-inoculated mice, we found a decrease in the TI population and an increase in the TD population characterized by significant alterations in various sub-populations. This increase was largely due to an increase in CD4(+), CD8(+) and CD4/CD8 double-positive sub-populations of TD IELs. Intracellular cytokine analysis demonstrated induction of IFN-gamma and an increase in effector/cytotoxic CD8 and CD4 cells after reovirus 1/L infection. These results suggest that TD IELs may play an important role in the clearance of reovirus 1/L infection from gut.
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