Intracellular signaling pathways in Peripheral Blood Mononuclear Cells in patients with inflammatory bowel disease.

Crohn*s disease is a chronic inflammatory disease of the gastrointestinal
mucosa that affects approximately 1:1000 persons in the Western world. The
molecular etiology of Crohn*s disease remains unclear but involves an
overactivity of the adaptive immune response with accumulation of IL-12
producing dendritic cells and CD4+ T cells which are geared towards a Th1
phenotype. One theory is that this overactivity may be the indirect result of a
defective mucosal innate immune response. According to this theory mucosal
recruitment of neutrophils in response to damage or infection is impaired,
resulting an immune response which is overly dependent on other phagocytic cell
types such as macrophages and dendritic cells resulting in a chronic
granulomatous disease.

It has recently been shown that patients with Crohn*s disease indeed fail to
induce production of an important chemokine (IL-8) in response to mucosal
damage and infection compared to healthy control patients or patients with
ulcerative colitis, an other chronic inflammatory intestinal disease. . This
phenotype appears systemic as it was found in the intestinal mucosa, skin and
Peripheral Blood Mononuclear Cells (PBMCs). Induction of the IL-8 gene involves
activation of mitogen activated protein kinases (MAPKs) and nuclear
translocation of the NF-kappaB transcription factor complex and we hypothesize
that the function of one of these signaling pathways may be compromised in
patients with Crohn*s disease.

The current project aims to confirm this defective production of IL-8 and
explore the possible underlying defect by examining the functionality of the
major signal transduction pathways involved in IL-8 production.

We will study the activation of four major signaling pathways in the innate
immune response in PBMCs isolated form the blood of the patients included in
this study. After isolation PBMCs will be stimulated with TNFalfa or complement
factor 5a (C5a). The induction of phosphorylation of relevant kinases will be
measured on western blot at two time points relative to unstimulated PBMCs
using antibodies specific for the phosphorylated (activated) state of the
kinases. We will examine phosphorylation of three different mitogen activated
protein kinase (MAPK) pathways and of the NF-kappaB pathway.

The burden is limited to periferal sampling of 100 ml blood. This induces no
signficant risk.