beta1 integrins are critically involved in neutrophil locomotion in extravascular tissue In vivo

Abstract

Recruitment of leukocytes from blood to tissue in inflammation requires the function of specific cell surface adhesion molecules. The objective of this study was to identify adhesion molecules that are involved in polymorphonuclear leukocyte (PMN) locomotion in extravascular tissue in vivo. Extravasation and interstitial tissue migration of PMNs was induced in the rat mesentery by chemotactic stimulation with platelet-activating factor (PAF; 10(-7) M). Intravital time-lapse videomicroscopy was used to analyze migration velocity of the activated PMNs, and the modulatory influence on locomotion of locally administered antibodies or peptides recognizing various integrin molecules was examined. Immunofluorescence flow cytometry revealed increased expression of alpha4, beta1, and beta2 integrins on extravasated PMNs compared with blood PMNs. Median migration velocity in response to PAF stimulation was 15.5 +/- 4.5 micron/min (mean +/- SD). Marked reduction (67 +/- 7%) in motility was observed after treatment with mAb blocking beta1 integrin function (VLA integrins), whereas there was little, although significant, reduction (22 +/- 13%) with beta2 integrin mAb. Antibodies or integrin-binding peptides recognizing alpha4beta1, alpha5beta1, or alphavbeta3 were ineffective in modulating migration velocity. Our data demonstrate that cell surface expression of beta1 integrins, although limited on blood PMNs, is induced in extravasated PMNs, and that members of the beta1 integrin family other than alpha4beta1 and alpha5beta1 are critically involved in the chemokinetic movement of PMNs in rat extravascular tissue in vivo.

Figure 1

Immunofluorescent staining of integrins on blood PMNs (thin line) and on extravasated PMNs collected from the peritoneal cavity (thick line). Thin vertical line indicates the 99th percentile of fluorescence events for cells stained with isotype matched control antibody. Histograms are representative tracings of three to five analyses for each antibody.

Figure 2

Micrographs showing migrating PMNs in a tissue section of the rat mesentery after stimulation with PAF (10⁻⁷ M) for 40 min (A), and immunofluorescent staining for α₄ integrins in the same cells (B). The fluorescence is concentrated and localized to spots in most PMNs, indicating a polarized integrin expression (arrows), whereas in some cells a more scattered distribution is seen. Bar: 10 μm.

Figure 3

Frequency distribution of PMN migration velocity in extravascular tissue of the rat mesentery in response to chemotactic stimulation with PAF (10⁻⁷ M). (A) Migration velocities during control period (PAF alone). (B) Migration velocities after topical treatment with the anti-β₁ mAb HMβ1-1.

Figure 4

Effect of local treatment with various integrin antibodies and integrin-binding peptides on PMN migration velocity in the rat mesentery stimulated with PAF (10⁻⁷ M). Data are expressed as percent of migration velocity before treatment, and represent mean ± SD of five experiments for each reagent tested. Asterisk, denotes significant difference from control (P <0.05).

Figure 5

Time course of inhibitory effect of the anti-β₁ mAb HMβ1-1 on PMN migration velocity in the rat mesentery (•) compared with control antibody (○). Data are based on calculation of mean migration velocity during defined 10 or 20 min intervals, and presented as means ± SD of five separate experiments for each antibody. Note that the inhibitory effect on PMN locomotion persisted throughout the observation period.