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

Abstract

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.

Neutrophil recruitment in immunized mice depends on MIP‐2 inducing the sequential release of MIP‐1α, TNF‐α and LTB4

“…Other authors have also demonstrated that both MIP-2 and KC were produced in a delayed-type hypersensitivity (DTH) model, but only neutralizing antibodies against MIP-2 were able to reduce neutrophil accumulation and suppress the DTH reaction [24]. MIP-2 and other CXC-ELR + chemokines bind with high affinity to both CXCR1 and CXCR2 expressed on several cell types including T lymphocytes, NK cells, neutrophils, eosinophils, basophils, dendritic cells and platelets [25][26][27]. Whereas human neutrophils express both CXCR1 and CXCR2, only CXCR2 has been detected in mice [8,9,23,28].…”

“…CXCR2 mRNA expression in peritoneal cells from antigen (OVA)-challenged immunized mice were detected by RT-PCR, based on a previously described protocol [25,40]. Total cellular RNA from 1-h antigen-challenged peritoneal cells was extracted using the Trizol reagent (Gibco BRL, Life Technologies, Rockville, MD) according to the directions supplied by the manufacturer.…”

Angiotensin II Induces Neutrophil Accumulation In Vivo Through Generation and Release of CXC Chemokines

“…23 Like IL-8, P-selectin is presynthesized and stored in endothelial cell Weibel-Palade bodies 14 and is rapidly mobilized to the cell surface after exposure to Ang II. 15 Because CXC chemokines induce P-selectin upregulation, 24 it is possible that the release of IL-8 in response to Ang II augments the P-selectin upregulation. Therefore, in addition to their role in Ang II-induced neutrophil adhesion and migration, CXC chemokines may also contribute to the rolling response elicited by this peptide hormone.…”

Divergent Signaling Pathways in Phagocytic Cells during Sepsis

“…This discrepancy might be important for CXC chemokines to create a concentration difference between two compartments, which is necessary for mobilization of neutrophil migration. CXC chemokines have been implicated in all steps in the extravasation process of leukocytes, including rolling, adhesion, and transmigration in vivo (27). Thus, the role of blood CXC chemokines may primarily focus on the activation of neutrophils and endothelial cells, setting the stage for neutrophil migration, whereas the local CXC chemokines chiefly function chemotactically during sepsis.…”