Molecular proinflammatory mechanism in the intestinal muscularis following intestinal manipulation

Background

We have previously demonstrated that manipulation of the rat intestine leads to a local inflammatory response within the intestinal muscularis followed by a decrease in gastrointestinal motility. Furthermore, locally produced mediators play a major role in systemic inflammatory syndromes. The aim of this study was to delineate the initial steps of the local inflammatory cascade within the gut wall in respect to the known prototypic inflammatory cytokine interleukin-6.

Methods

ACI rats, IL-6^-/- and IL-6^+/+ mice underwent a standardized intestinal manipulation (IM) and were sacrificed at various time points postoperatively. One group of rats received repeated i.v. doses of blocking antibodies against the adhesion molecules (1A29 + WT.3) pre- and postoperatively. The small bowel was separated in mucosal and muscularis layers and specimens were used for RNA and protein extraction. STAT protein was quantified using EMSA. IL-6 protein was measured by ELISA in tissue culture and by immunohistochemistry in muscularis whole-mounts.

Results

Muscularis extracts demonstrated an early upregulation (12.3 fold at 3 hours) in IL-6 mRNA by RT-PCR. This was in contrast to mucosal extracts that did not show significant changes. Adhesion molecule blockade resulted in a significant decrease in infiltrating cells, but did not change mRNA expression. IL-6 immunohistochemistry stained resident muscularis macrophages, smooth muscle cells and infiltrated leukocytes. Muscularis tissue culture after IM demonstrated a significant increase in IL-6 protein compared to untreated control cultures (420 vs 230 pg/100 mg tissue). Postoperatively STAT proteins showed a significant increase in activation (32 fold at 30 min) with a prototypic IL-6 supershift profile (Stat3α). IL-6^-/- mice demonstrated a significantly lower STAT activation following IM compared to IL-6^+/+ mice.

Conclusion

These results demonstrate for the first time that operative trauma leads to an early and signifcant production of IL-6 within the intestinal muscularis. IL-6 is mainly produced by resident cells within the muscularis and has functional activity. Therefore, the intestinal muscularis plays a role in the postoperative production of the proinflammatory mediator IL-6.