An anti-platelet-endothelial cell adhesion molecule-1 antibody inhibits leukocyte extravasation from mesenteric microvessels in vivo by blocking the passage through the basement membrane.

Platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) plays an active role in the process of leukocyte migration through cultured endothelial cells in vitro and anti-PECAM-1 antibodies (Abs) inhibit accumulation of leukocytes into sites of inflammation in vivo. Despite the latter, it is still not clear at which stage of leukocyte emigration in vivo PECAM-1 is involved. To address this point directly, we studied the effect of an anti-PECAM-1 Ab, recognizing rat PECAM-1, on leukocyte responses within rat mesenteric microvessels using intravital microscopy. In mesenteric preparations activated by interleukin (IL)-1 beta, the anti-PECAM-1 Ab had no significant effect on the rolling or adhesion of leukocytes, but inhibited their migration into the surrounding extravascular tissue in a dose-dependent manner. Although in some vessel segments these leukocytes had come to a halt within the vascular lumen, often the leukocytes appeared to be trapped within the vessel wall. Analysis of these sections by electron microscopy revealed that the leukocytes had passed through endothelial cell junctions but not the basement membrane. In contrast to the effect of the Ab in mesenteric preparations treated with IL-1 beta, leukocyte extravasation induced by topical or intraperitoneal administration of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine was not inhibited by the anti-PECAM-1 Ab. These results directly demonstrate a role for PECAM-1 in leukocyte extravasation in vivo and indicate that this involvement is selective for leukocyte extravasation elicited by certain inflammatory mediators. Further, our findings provide the first in vivo indication that PECAM-1 may have an important role in triggering the passage of leukocytes through the perivascular basement membrane.

Fc gamma RIII mediates neutrophil recruitment to immune complexes. a mechanism for neutrophil accumulation in immune-mediated inflammation.

Neutrophil accumulation is a hallmark of immune complex-mediated inflammatory disorders. Current models of neutrophil recruitment envision the capture of circulating neutrophils by activated endothelial cells. We now demonstrate that immobilized immune complexes alone support the rapid attachment of neutrophils, under physiologic flow conditions. Initial cell tethering requires the low-affinity Fc gamma receptor IIIB (Fc gamma RIIIB), and the beta(2) integrins are additionally required for the subsequent shear-resistant adhesion. The attachment function of Fc gamma RIIIB may be facilitated by its observed presentation on neutrophil microvilli. In vivo, in a model of acute antiglomerular basement membrane nephritis in which immune complexes are accessible to circulating neutrophils, Fc gamma RIII-deficient mice had a significant reduction in neutrophil recruitment. Thus, the interaction of immune complexes with Fc gamma RIII may mediate early neutrophil recruitment in immune complex-mediated inflammation.

Alpha4beta1-integrin activation is necessary for high-efficiency T-cell subset interactions with VCAM-1 under flow.

OBJECTIVE: The purpose of this study was to examine the relationship between alpha4beta1-integrin state of activation on CD4+ T-cell subsets and their adhesive interaction to VCAM-1 under flow. METHODS: Human CD4+ memory and naive T-cells were freshly isolated and effector-helper T-cell subsets. Th1 and Th2 cells, were differentiated in vitro from CD4+ naive T-cells. The expression of activation/ligand induced epitopes on beta1-integrins of each T-cell subset was assessed using mAb HUTS21 and mAb 15/7. T-cell subsets attachment and rolling on VCAM-1 was determined under defined flow conditions and the rates of attachment (ka), accumulation, and instantaneous rolling velocities were correlated to their beta1-integrin activation epitope expression. RESULTS: A subset of memory T-cells constitutively express activation/ligand induced epitopes on beta1-integrins recognized by mAb HUTS21 and 15/7, whereas expression levels on naive T-cells is low or not detectable. Consistent with an activated phenotype, memory T-cells exhibit significantly higher rates of attachment and accumulation on VCAM-1 under flow as compared to naive T-cells. Interestingly, the expression of activation/ligand induced epitopes on beta1-integrins on Th2 cells and the ability of these cells to interact with VCAM-1 are comparable to memory T-cells. In contrast, Th1 cells did not interact as efficiently with VCAM-1, which correlated with lower expression of activation/ligand induced epitopes on these cells. VCAM-1 interactions are inhibited completely by pretreatment of the T-cells with blocking mAb to alpha4-integrins or beta1-integrins, indicating that alpha4beta1 is the predominant T-cell integrin involved. CONCLUSIONS: Memory T-cells express constitutively active alpha4beta1-integrins, as compared to naive T-cells, which mediate high rates of initial attachment and sustained high-affinity adhesive interactions with VCAM-1 under flow conditions in vitro. Similarly, in vitro differentiated Th2 cells but not Th1 cells, which also express elevated levels of activated alpha4beta1-integrins, are capable of sustaining high-affinity adhesive interactions with VCAM-1. The differences observed in beta1-integrin activation on T-cell subsets may underlie selective recruitment patterns of T-cell subsets in vivo.

Divergent effects of platelet-endothelial cell adhesion molecule-1 and beta 3 integrin blockade on leukocyte transmigration in vivo.

The final stage in the migration of leukocytes to sites of inflammation involves movement of leukocytes through the endothelial cell layer and the perivascular basement membrane. Both platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) and the integrin alphavbeta3 have been implicated in this process, and in vitro studies have identified alphavbeta3 as a heterotypic ligand for PECAM-1. In the present study we have addressed the roles of these molecules by investigating and comparing the effects of PECAM-1 and alphavbeta3 blockade on leukocyte migration in vivo. For this purpose we have examined the effects of neutralizing Abs directed against PECAM-1 (domain 1-specific, mAb 37) and beta3 integrins (mAbs 7E3 and F11) on leukocyte responses in the mesenteric microcirculation of anesthetized rats using intravital microscopy. The anti-PECAM-1 mAb suppressed leukocyte extravasation, but not leukocyte rolling or firm adhesion, elicited by IL-1beta in a dose-dependent manner (e.g., 67% inhibition at 10 mg/kg 37 Fab), but had no effect on FMLP-induced leukocyte responses. Analysis by electron microscopy suggested that this suppression was due to an inhibition of neutrophil migration through the endothelial cell barrier. By contrast, both anti-beta3 integrin mAbs, 7E3 F(ab')2 (5 mg/kg) and F11 F(ab')2 (5 mg/kg), selectively reduced leukocyte extravasation induced by FMLP (38 and 46%, respectively), but neither mAb had an effect on IL-1beta-induced leukocyte responses. These findings indicate roles for both PECAM-1 and beta3 integrins in leukocyte extravasation, but do not support the concept that these molecules act as counter-receptors in mediating leukocyte transmigration.