Dominant role of L- and P-selectin in mediating CXC chemokine-induced neutrophil migration in vivo

Abstract

The role of selectins in neutrophil emigration in response to the CXC chemokines KC and MIP-2 was investigated in wild type and P-selectin deficient mice. Intrapleural injection of KC or MIP-2 induced a rapid and specific neutrophil accumulation. Emigration 2 h after KC or MIP-2 was reduced 83 - 88% by anti-L-selectin mAb and 53 - 63% by anti-P-selectin mAb. Co-administration of anti-L- and P-selectin mAbs abolished neutrophil migration induced by either chemokine. An anti-E-selectin mAb tested alone did not affect KC-induced neutrophil migration after 2 or 4 h. Moreover, anti-E-selectin did not have an additive inhibitory effect on KC-induced neutrophil migration compared with P-selectin blockade alone. This was found when neutrophil migration was measured at 2 and 4 h after KC. Despite a blood neutrophilia, neutrophil migration at 2 and 4 h after KC was markedly smaller (by approximately 90%) in P-selectin deficient mice compared with wild type animals. Responses at both time points were not decreased further in animals given E-selectin mAb but were reduced to the PBS control level in the presence of anti-L-selectin. In vitro study of cultured murine endothelial cells demonstrated that KC can directly increase cell surface P-selectin expression. These data suggest that CXC chemokine-induced neutrophil accumulation is dependent on both neutrophil L-selectin and a rapid upregulation of endothelial P-selectin but there is no evidence for E-selectin induction.

Figure 1

Dose dependency and kinetics of neutrophil accumulation in the pleural cavity in response to KC or MIP-2. (a) increasing doses of KC or MIP-2 (0.01–0.1 μg) induced a significant neutrophil accumulation at 2 h when compared with PBS-treated controls. (b) Time course of neutrophil accumulation. Values represent the mean±s.e.mean of six mice. *P<.05 and **P<0.01 compared with PBS, and ^#P<0.05 compared to KC 0.01 μg by one-way ANOVA followed by Student-Newmann-Keuls post-test.

Figure 2

Role of L- and P-selectin in KC- and MIP-2 induced neutrophil accumulation. Mice were treated with an i.v. injection of control IgG, anti-L-selectin alone, anti-P-selectin alone or anti-L- and -P-selectin in combination. KC (a) or MIP-2 (b) was administered (0.1 μg each) into the pleural cavity and neutrophil accumulation measured after 2 h. Values represent mean±s.e.mean of six mice. *P<0.05 and **P<0.01 compared with KC or MIP-2 by one-way ANOVA followed by Student-Newmann-Keuls post-test.

Figure 3

Role of E-selectin in KC-induced neutrophil accumulation. Mice were treated with an i.v. injection of control IgG (non-blocking P- and E-selectin mAbs), anti-P-selectin mAb alone or anti-P- and -E-selectin mAbs in combination. KC (0.1 μg) was administered into the pleural cavity and neutrophil accumulation measured after (a) 2 h or (b) 4 h. Values represent mean±s.e.mean of 4–6 mice. *P<0.05 compared with KC by one-way ANOVA followed by Student-Newmann-Keuls post-test.

Figure 4

KC-induced neutrophil accumulation in P-selectin −/− mice. P-selectin +/+ or −/− mice were treated with an i.v. injection of control IgG, anti-E-selectin or anti-L-selectin mAbs. KC (0.1 μg) was administered into the pleural cavity and neutrophil accumulation measured after (a) 2 h or (b) 4 h. Values represent mean±s.e.mean of four mice in each group. The dashed line represents the response to PBS. *P<0.05 and **P<0.01 compared with response to KC in −/− mouse by one-way ANOVA followed by Student-Newmann-Keuls post-test.

Figure 5

KC-induced P-selectin upregulation on murine endothelial cells in vitro. Cultures of the murine endothelioma cell line bEnd5 were treated with PBS without P-selectin mAb (top left), with PBS plus P-selectin mAb (top right), thrombin (30 u ml⁻¹) plus P-selectin mAb (bottom left) or KC (10⁻⁶ M) plus P-selectin mAb (bottom right). After 30 min, cells were washed and P-selectin expression detected with Alexa Fluor® 488-labelled secondary antibody. The calibration bar represents 20 μm.