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* = Presenting author

P068. Prolonged survival of neutrophils in Crohn's disease

R. Somasundaram1, C.J. van der Woude2, M.P. Peppelenbosch3, G. Fuhler4

1Erasmus Medical Center, MDL laboratory – Gastroenterology, L462, Rotterdam, Netherlands; 2Erasmus Medical Center, Department of Gastroenterology & Hepatology, Rotterdam, Netherlands; 3Erasmus Medical Center, Department of Gastroenterology and Hepatology, Rotterdam, Netherlands; 4Erasmus Medical Center, Gastroenterology and Hepatology, Rotterdam, Netherlands

Background: Although the factors responsible for initiating and maintaining Crohn's disease (CD) remain obscure, it has become clear that innate immunity plays an important role in CD pathology. Patients with CD display reduced innate cell function, whereas increased numbers of neutrophils (polymorphonuclear cells, PMN) are found in chronic lesions. After clearing bacterial infections, PMN at the site of inflammation should proceed to undergo programmed cell death (apoptosis). Enhanced survival of PMN could exacerbate the inflammatory response, and may contribute to the chronic inflammation observed in CD. Here, we investigate the survival and apoptosis of PMN to provide better insights into the poorly studied field of PMN biology in CD.

Methods: Freshly isolated PMN from 10 CD patients and 10 healthy controls (HC) were included in the study. Spontaneous early PMN apoptosis was quantified by flowcytometry of Annexin V positive cells at timepoints 0 h, 6 h and 15 h. Late apoptosis was studied by western blot analysis of cleaved caspase 8 and caspase 3. In addition, apoptosis was induced by incubation of PMN with Fas antibody CH11 for 6 hours, whereas PMN were rescued from spontaneous apoptosis by culturing them for 15 hours in the presence of GM-CSF or IL8. Survival signalling in PMN was assessed by determining GM-CSF-induced priming of protein kinase B (PKB/Akt) phosphorylation.

Results: No differences in spontaneous early apoptosis between CD and HC PMN were observed by Annexin V staining. CH11-induced early apoptosis, as well as GM-CSF or IL‑8-induced rescue thereof, was similar between HC and CD patients. Interestingly, the levels of cleaved caspase 8 and 3 were significantly decreased in CD PMN cultured with GM-CSF, suggesting an enhanced rescue of these cells from apoptosis. This effect was specific for GM-CSF, as neither Fas-induced caspase cleavage, nor IL‑8-induced rescue thereof, were altered in CD patients. Enhanced PKB activation in GM-CSF primed, but not IL8 stimulated PMN from CD patients was observed.

Conclusions: Although initiation of apoptosis in CD PMN is normal as determined by Annexin V staining, an enhanced survival signal provided by GM-CSF-induced PKB activation may lead to a decreased cleaving of caspases, thereby rescuing PMN from the apoptotic pathway. In toto, our results suggest an enhanced GM-CSF-mediated PMN survival, which may play a role in the chronic intestinal inflammation in CD.