Mechanical stretch aggravates the LPS-induced inflammatory reaction in intestinal smooth muscle cells and macrophages

Abstract

Introduction: The mechanical trauma is an iatrogenic consequence of abdominal surgery that results in a massive inflammation of the entire bowel wall, subsequently leading to postoperative ileus 1, 2. In addition to the trauma, bacterial translocation and LPS challenge are discussed as initiator of this inflammation. Although several cell types are involved, resident muscularis macrophages and smooth muscle cells play a central role. The aim of this study was to investigate the cellular and molecular reactions of both cell types after LPS-challenge or mechanical trauma. Methods: Rat intestinal (iSMC) and vascular (vSMC) smooth muscle cells and peritoneal macrophages (pMacs) were repetitively stretched for 6 hours in the presence or absence of 100 ng/ml LPS. Gene expression of iNOS, COX-2 and interleukin-6 was analyzed by quantitative PCR. In another experimental setting, intercellular inflammatory stimulation was analyzed after transfer of conditioned culture media. Therefore, media from stretched cells were transferred to unstretched cells — from SMC to macrophages or vice versaand MIP-1a, MIP-2, COX-2, IL-6 and iNOS expression was analyzed after 6 hours. Results: Mechanical stretch does not alter iNOS, COX-2 or IL-6 gene expression in iSMC compared to unstretched controls. However, LPS-challenge induced a significant gene expression of IL-6 (3-fold) and iNOS (292-fold) in iSMC. LPS and stretch together led to and further significant increase of IL-6 (5-fold) and iNOS (1250-fold). However, IL-6 expression was not altered in vSMC cultures by stretch or LPS stimulation, whereas COX-2 expression was increased by stretch (7.8-fold) and enhanced by stretch with LPS (15.4-fold). Stretch together with LPS stimulation did not increase the LPS induced iNOS expression (141-fold) in vSMC. In pMacs, gene expression was not altered by mechanical stretch but all genes were strongly increased after LPS induction. LPS induced proinflammatory gene expression was significantly enhanced in combination with mechanical stretch. The transfer of conditioned media from stretched iSMC induced a significant increase in MIP-2, iNOS and COX-2 expression in pMacs. On the other way, no effect on gene expression was observed in iSMC after transfer of media from stretched pMacs. Conclusion: A mechanical trauma does not induce a proinflammatory gene expression in macrophages and iSMC but it enhances LPS induced strong inflammatory reactions in both cell types. However, in response to a mechanical trauma, iSMC liberate unknown mediators that initiate a proinflammatory reaction in pMacs. Interestingly, vascular and intestinal smooth muscle cells differ in their molecular responses to stretch. Finally, LPS stimulation and a mechanical trauma together induce a massive inflammatory reaction in intestinal smooth cells and macrophages. The investigation of the molecular basics could help to identify targets responsible for the overwhelming inflammation in postoperative inflammations.