Gliadin increases iNOS gene expression in interferon-γ-stimulated RAW 264.7 cells through a mechanism involving NF-κB

Abstract

Nitric oxide (NO) plays an important role in the pathogenesis of the histological changes seen in coeliac disease. We have investigated the effect of peptic-tryptic digest of gliadin (Pt-G) and gliadin (G) on inducible nitric oxide synthase (iNOS) protein expression in RAW 264.7 macrophages stimulated with interferon-gamma (IFN-gamma). Pt-G and G enhanced in a concentration and time-dependent manner NO production by IFN-gamma-stimulated RAW 264.7 cells. The increase of iNOS protein expression was correlated with NF-kappa B/DNA binding activity and occurred at transcriptional level. Pyrrolidine dithiocarbamate and N-alpha-para-tosyl-L-lysine chloromethyl ketone, two known inhibitors of NF-kappa B activation, decreased significantly NO production and iNOS protein expression as well as NF-kappa B/DNA binding activity. Our results show that the effect of Pt-G and G on enhancement of iNOS protein expression in IFN-gamma-treated RAW 264.7 cells is mainly mediated through NF-kappa B and suggest that blockage of NF-kappa B activation reduces enhancing effect of gluten on NO production in inflamed mucosa of coeliac patients.

Ascorbate-dependent decrease of the mucosal immune inflammatory response to gliadin in coeliac disease patients

“…Additionally, gliadin increased the binding activity of NF-B/DNA, the degradation of IB␣ and the nuclear translocation of p50 and p65 subunits. 25 With these findings we have confirmed that by using a non-toxic (20 mM) supplementation of ascorbate to biopsy culture challenged with gliadin, the gliadin-induced production of nitrites is inhibited. The effects of gliadin are probably mediated by iNOS, and ascorbate acts blocking its pathway.…”

“…The effects of gliadin are probably mediated by iNOS, and ascorbate acts blocking its pathway. 25,31 The expression levels of the cytokines IFN␥, TNF␣, IFN␣, and IL-6 are decreased to below those levels observed in basal cultures (Fig. 2).…”

“…Both effects were reduced by NF-B activation inhibitors, thereby indicating that gliadin should modulate iNOS gene expression through NFKB activation. 25 Given the role of the NF-B pathway in the pathogenesis of CD, we wondered whether ascorbate has an effect on the inhibition of the early/innate immune response triggered by gluten and, therefore, can be used as a supplementary therapeutical strategy to GFD on CD patients. To address this question we have cultured biopsies from treated CD patients stimulated in vitro with gliadin, with and without supplementation of ascorbate.…”

Gliadin Stimulation of Murine Macrophage Inflammatory Gene Expression and Intestinal Permeability Are MyD88-Dependent: Role of the Innate Immune Response in Celiac Disease

“…Previous studies have suggested the possibility that gliadin, the immunologically active component of wheat gluten associated with CD (2), has the capacity to activate cytokine production in monocytes and macrophages (43)(44)(45). To confirm and extend these findings, the expression of a panel of proinflammatory genes known to be up-regulated by LPS was measured in murine peritoneal macrophages treated with concentrations of PT-gliadin or peptides derived from gliadin (p31-43 or 33-mer) that have been reported previously to be either immunomodulatory or cytotoxic (43)(44)(45).…”

Antioxidant status of the celiac mucosa: implications for disease pathogenesis

“…The results of various investigations suggest that gliadin disturbs the pro-oxidant-antioxidant balance in small intestinal mucosa of affected persons through overproduction of ROS (Boda et al, 1999;Dugas et al, 2003). Several in vitro studies have also reported redox imbalance and increased levels of free radicals after exposure of cells to gliadin (Dolfini et al, 2002;Maiuri et al, 2003;Rivabene, 1999;Tucková et al, 2002).…”