Inducible CD40 expression mediates NF B activation and cytokine secretion in human colonic fibroblasts

Abstract

Background: CD40 has been shown to be a functional activation antigen on a variety of cell types involved in immune responses. As intestinal fibroblasts and myofibroblasts may play a role during mucosal inflammation, we investigated the functional consequences of CD40 induction in primary cultures of human colonic fibroblasts. Methods: Primary colonic lamina propria fibroblasts (PCLF) were isolated from endoscopic biopsies and surgical specimens. Cultures were used between passages 3 and 9. CD40 surface display was determined by FACS analysis and mRNA expression by reverse transcription-polymerase chain reaction. Secretion of cytokines was determined by ELISA. Nuclear factor kB (NFkB) activation was shown by electrophoretic mobility shift assay (EMSA). Results: After priming with interferon c (IFN-c) (200 U/ml) for 72 hours, five of eight tested PCLF cultures showed induction of CD40 surface display (up to 10-fold). Induction of CD40 mRNA expression was demonstrated by semiquantitative polymerase chain reaction. In the responder-PCLF cultures, IFN-c alone caused a 1.5-5-fold increase in interleukin (IL)-8 secretion. Addition of 1 ng/ml CD40L was sufficient to achieve a further increase in IL-8, IL-6, or monocyte chemotactic protein 1 (MCP-1) secretion (2.5-18-fold of controls). Incubation with CD40L alone without priming with IFN-c had no effect. The proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN 100 mM) reduced IFN-c/CD40L mediated cytokine induction, suggesting participation of NFkB, which was directly demonstrated by EMSA. CD4+ T cells induced MCP-1 secretion by PCLF, which was prevented by addition of an excess of CD40-IgG fusion protein. CD40 expression on PCLF could also be demonstrated in vivo by immunohistochemistry. Conclusion: The CD40-CD40L pathway augments mucosal inflammatory responses via mucosal PCLF. CD40-CD40L mediated T cell/PCLF interactions could play an important role during intestinal mucosal inflammation.

TNF-α Blockade Down-Regulates the CD40/CD40L Pathway in the Mucosal Microcirculation: A Novel Anti-Inflammatory Mechanism of Infliximab in Crohn’s Disease

“…These and other observations make the CD40/CD40L pathway a rational target for therapeutic intervention because disrupting CD40-CD40L interactions, either with blocking Abs or antisense oligonucleotides, could represent a novel approach to turn off intestinal inflammation. Supporting this possibility are reports showing that shutting down the CD40/CD40L system is very effective in dampening inflammation in in vitro cellular systems (7)(8)(9) as well as in vivo in animal models of experimental colitis (16 -19).…”

The CD40-CD40L Pathway Contributes to the Proinflammatory Function of Intestinal Epithelial Cells in Inflammatory Bowel Disease

“…12 Subsequently, CD40 was identified on various cell types such as professional APCs, endothelial cells, epithelial cells of different organs, and fibroblasts. 13,14 Its ligand, CD40L, is a 39-kDa surface glycoprotein and a member of the TNF gene superfamily. CD40L is mainly expressed on activated CD4 ϩ T-helper cells and platelets but could also be found on cytotoxic CD8 ϩ T cells.…”

A Novel Strategy to Identify mRNA 3′ UTR and mRNA Level Difference in Crohn’s Disease

“…FAF1 could suppress CD40-induced NF κB activation by interacting with the CD40 receptor [26]. And previous studies have demonstrated that CD40 expression level was high in intestinal mucosa and dendritic cells in CD patients [27]. What's more, IFN-γ/CD40L induced cytokine secretion plays an important role in intestinal mucosal inflammation [28].…”