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

Abstract

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.

Figure 1

Time-dependent effect of intrascrotal challenge with 500 ng of MIP-2 (solid line) and KC (dashed line) on leukocyte (a) rolling (b) firm adhesion and (c) extravascular recruitment in the mouse cremaster muscle. Data represents mean±s.e.mean and n=3–5.

Figure 2

Effect of an anti-P-selectin antibody (Anti-P, 40 μg per mouse) and an isotype-matched control antibody (Control antibody, 40 μg per mouse) on (a) rolling (b) firm adhesion and (c) extravascular recruitment of neutrophils in the mouse cremaster muscle induced by intrascrotal challenge with MIP-2 (open bars) and KC (filled bars) for 2 h. In separate experiments, mice were treated intrascrotally with PBS alone (grey bars). Data represents mean±s.e.mean. ^*P<0.05 vs control antibody and ^#P<0.05 vs PBS, n=5.

Figure 3

Number of extravascular neutrophils (PMNLs) per mm² in the mouse cremaster muscle whole mounts without prior intravital microscopic observation (see Methods for detailed explanation). Mice were challenged intrascrotally with PBS (Control), MIP-2 (500 ng) KC (500 ng) and fMLP (10⁻⁷ and 10⁻⁶ M) for 2 h before harvesting of the cremaster muscle tissue. In separate experiments, mice were pretreated intravenously with the anti-P-selectin antibody RB40.34 (Anti-P, 40 μg per mouse) immediately prior to challenge with 500 ng of MIP-2 and KC. Data represents mean±s.e.mean and n=4–5. ^*P<0.05 vs control, #P<0.05 vs MIP-2 alone and ¤P<0.05 vs KC alone.

Figure 4

Expression of CXCR2 mRNA in murine neutrophils (PMNL) endothelial cells (EC). β-actin serves as an housekeeping gene. The results presented are from one experiment, which is representative of three others performed.

Figure 5

Changes in the number of circulating neutrophils (PMNLs) at different time-points after intrascrotal administration of 500 ng of (a) MIP-2 and (b) KC. Blood was collected from the tail and analysed in a haemocytometer. Data are mean±s.e.mean and represent 10⁶ cells ml⁻¹. ^*P<0.05 vs 0 and n=5.