CXC chemokines, MIP-2 and KC, induce P-selectin-dependent neutrophil rolling and extravascular migration in vivo.

The purpose of this study was to examine the impact of CXC chemokines, i.e. macrophage inflammatory 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. 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) significantly inhibited MIP-2- and KC-induced leukocyte rolling by more than 96%. This marked decrease in rolling abolished firm adhesion and extravascular accumulation of neutrophils (>89% 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. 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 onP-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. We could not detect gene expression of CXCR2 in murine endothelial cells, whereas neutrophils were positive, indicating that the stimulatory effect of CXC chemokines on leukocyte-endothelium interactions is not a direct effect on the endothelium but rather an indirect effect via activation of an intermediary tissue cell. However, challenge with MIP-2 and KC did not increase the number of degranulated mast cells. 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 infiltration in vivo.