Endocrine Abstracts

Introduction

Leukocyte recruitment is a key feature in (I/R)-induced tissue injury. We have developed and validated a novel method to study molecular mechanisms of leukocyte-endothelium interactions in the colonic microcirculation in vivo by use of inverted intravital fluorescence microscopy.

Methods & Results

In male C57/BL6 mice the superior mesenteric artery was occluded 30 min and leukocyte rolling and adhesion were analyzed in colonic venules after 120 min of reperfusion. It was found that 30 min of ischemia and 120 min of reperfusion induced the strongest and most reproducible leukocyte response, i.e. leukocyte rolling and adhesion was 29 ± 3 cells/min and 75 ± 25 cells/mm, respectively.

The mechanisms of leukocyte rolling was inhibiting by investigated using anti L-, E- and P-selectin antibodies. We found that pretreatment with P-selectin antibody significantly reduced leukocyte rolling and adhesion by 88% and 85%, respectively, whereas antibodies directed against L- and E-selectin had no effect.

Next we evaluated the role of CXC chemokines and found that injection of antibodies directed against MIP-2 and KC markedly reduced both leukocyte rolling and adhesion. Furthermore RT-PCR and ELISA demonstrated a marked increase in mRNA and protein levels of MIP-2 and KC in the colonic tissue after I/R.

Conclusions

This study presents a new method to study I/R-induced leukocyte responses in the colon and provide evidence demonstrating that leukocyte rolling is mediated by P-selectin. Moreover we show an important role for CXC chemokines (MIP-2 and KC) in mediating the leukocyte response in the colon. Taken together, this data may provide important targets to control pathological inflammation in I/R-induced tissue injury in the colon.