1 The purpose of this study was to examine the impact of CXC chemokines, i.e. macrophage in¯ammatory protein-2 (MIP-2) and KC, on leukocyte-endothelium interactions in detail and to evaluate the role of P-selectin by use of intravital microscopy in the mouse cremaster muscle. 2 Administration of MIP-2 and KC provoked a dose (5 ± 500 ng)-and time (0 ± 4 h)-dependent increase in leukocyte rolling, adhesion and tissue recruitment. Neutrophils comprised more than 92% of the leukocyte response. Pretreatment with an antibody directed against P-selectin (RB40.34) signi®cantly inhibited MIP-2-and KC-induced leukocyte rolling by more than 96%. This marked decrease in rolling abolished ®rm adhesion and extravascular accumulation of neutrophils (489% reduction), suggesting that CXC chemokines induce P-selectin-dependent rolling, which in turn apparently is a precondition for the subsequent stationary adhesion and extravasation of neutrophils. 3 Moreover, the extravascular recruitment of leukocytes was evaluated in whole-mounts of the cremaster muscle without preceding intravital microscopy. Using this approach, it was again observed that MIP-2-and KC-induced neutrophil accumulation was completely dependent on P-selectin function. In contrast to the CXC chemokines, administration of the classical chemoattractant formyl-methionyl leucyl phenylalanine (fMLP) did not provoke extravascular tissue accumulation of neutrophils. 4 We could not detect gene expression of CXCR2 in murine endothelial cells, whereas neutrophils were positive, indicating that the stimulatory eect of CXC chemokines on leukocyte-endothelium interactions is not a direct eect on the endothelium but rather an indirect eect via activation of an intermediary tissue cell. However, challenge with MIP-2 and KC did not increase the number of degranulated mast cells. 5 In conclusion, our data demonstrate that CXC chemokines induce all steps in the extravasation process of leukocytes, including rolling, adhesion and transmigration in vivo. Moreover, these results show that P-selectin plays a critical role in MIP-2 and KC provoked neutrophil recruitment as a critical mediator of initial leukocyte rolling. Additionally, our study suggest that a restricted action of MIP-2 and KC on neutrophils is far too simplistic to explain the complex mechanisms of action of CXC chemokines on neutrophil in®ltration in vivo.

We present a novel approach to study anastomotic healing using radiological examination. While bursting pressure appears to be suitable for measuring early anastomotic healing in the colon, we demonstrate that breaking strength is not sufficiently sensitive to be used in examination of early healing. Moreover, our data suggest that the acute inflammatory response and associated neutrophil recruitment in the anastomosis does not negatively affect healing in the rat colon.

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Leukocyte-endothelium interactions are dependent on a coordinated expression and function of specific adhesion molecules. The objective of the present study was to examine the role of selectin function and leukocyte rolling in tumor necrosis factor-alpha (TNF-alpha)-induced leukocyte adhesion and extravasation in venules in vivo. For this purpose, we used intravital microscopy in the mouse cremaster muscle stimulated for 2-3 h with TNF-alpha intrascrotally. Pretreatment with fucoidan, which inhibits P- and L-selectin, and a P-selectin monoclonal antibody (RB40.34) abolished TNF-alpha-stimulated leukocyte rolling. This great reduction in rolling caused a marked attenuation of firm adhesion and extravascular accumulation of leukocytes. When fucoidan and RB40.34 were administrated after stimulation with TNF-alpha, it was found that leukocyte rolling was greatly reduced whereas the number of firmly adherent leukocytes was completely unchanged, suggesting that the inhibitory effect of blocking P-selectin function on firm leukocyte adhesion and recruitment was due to the reduction in leukocyte rolling along the endothelium. Moreover, pretreatment with a monoclonal antibody against intercellular adhesion molecule-1 (ICAM-1) and a platelet-activating factor (PAF)-receptor antagonist had no effect of TNF-alpha-induced leukocyte rolling and adhesion, indicating that molecules other than ICAM-1 and PAF mediate firm adhesion and recruitment of leukocytes in TNF-alpha-activated tissues. Taken together, our data demonstrate that P-selectin function plays an important role in TNF-alpha-induced inflammatory cell recruitment by mediating leukocyte rolling as a precondition for cytokine-provoked firm adhesion and transmigration in vivo. These findings, thus, suggest that inhibition of P-selectin may be a central target for pharmacological intervention in inflammatory diseases.