Functions of integrin receptors in extravascular neutrophil migration and respiratory burst

Leukocytes circulate in the vasculature, but, must leave the blood stream and migrate through the surrounding tissue to exert their protective functions in inflammation. Numerous studies have revealed that intravascular leukocyte-endothelial cell interactions involve functions of adhesion molecules belonging to the selectin and integrin receptor families. On the other hand, little is known about the molecular mechanisms regulating the extravascular phase of leukocyte recruitment. This study aimed at investigating the involvement of cell adhesion molecules in extravascular neutrophil migration and respiratory burst, two fundamental events in the inflammatory response. A novel intravital microscopy model was used to analyze migration of activated neutrophils in the rat mesenteric tissue. Function blocking antibodies and peptides against integrin receptors were administered topically to the tissue and the effect on migration velocity of individual neutrophils was assessed. Blockage of the collagen binding [alpha]2[beta]1 integrin significantly reduced the neutrophil migration velocity (from 14.7 ± 1.4 [my]m/min to 3.9 ± 0.9 [my]m/min), whereas blockage of fibronectin binding [alpha]4[beta]1 and [alpha]5[beta]1 integrins was without effect on neutrophil migration. Limited reduction in velocity (~20 % inhibition) was also seen after blockage of the common [beta] chain of the [beta]2 integrins. Similarly, as demonstrated in the mouse air pouch model of acute inflammation chemoattractant-induced recruitment of neutrophils to tissues in vivo was impaired after function blockage of [alpha]2[beta]1 integrin with antibodies or peptides.

Studies of chemotactic neutrophil migration in gels composed of various amounts of the matrix proteins collagen, fibronectin and laminin revealed an inhibitory function of fibronectin on neutrophil migration. It was demonstrated that the suppressive effect on neutrophil motility exerted by this protein is mediated predominantely through an adhesive interaction of [alpha]4[beta]1 integrin with fibronectin. Collectively, these data obtained from combined in vitro and in vivo experiments show a critical role of [alpha]2[beta]1 integrin in neutrophil migration and suggest that specific [beta]1 integrins may both promote [alpha]2[beta]1 integrin) and suppress [alpha]4[beta]1 integrin) forward motion of neutrophils in the extravascular tissue dependent on the relative abundance of matrix proteins. Activation of [beta]1 and [beta]2 integrin receptors through antibody-induced receptor cross-linking further revealed mechanisms of receptor cross-talk in neutrophil migration. Activation of neutrophils; through ligation of [beta]2 integrins combined with chemotactic stimulation resulted in tyrosine kinase-dependent upregulation of functional [beta]1 integrins. To investigate the dynamic surface expression of integrin receptors in neutrophils we used immunofluorescence flow cytometry and laser scanning confocal microscopy. Interestingly, whereas circulating blood neutrophils express only limited amounts Of [alpha]4[beta]1, [alpha]5[beta]1 and [alpha]6[beta]1 integrin and are negative for [alpha]2[beta]1 integrin, upregulation Of [alpha]4[beta]1 and [alpha]5[beta]1, and induction of [alpha]2[beta]1 expression was seen in extravasated neutrophils collected from skin blister chamber fluid in humans or peritoneal lavage fluid in the rat. In migrating neutrophils, [alpha]2[beta]1 integrin was localized to the anterior lamellopodium of the cell, which is typical for receptors involved in cell motility. Taken together, activation Of [beta]2 integrins through artificial ligation (antibody cross-linking) or physiological ligation (transendothelial migration) resulted in upregulation of [beta]1 integrin expression in neutrophils. The association between neutrophil emigration and [beta]1 integin cell surface expression clearly illustrates a phenotypic adaption to a different environment when leaving the vasculature.

Since [beta]1 integrins are upregulated in extravasated neutrophils and these receptors specifically interact with matrix proteins it was of interest to explore whether [beta]1 integrin ligation can modulate extravascular respiratory burst of neutrophils. [beta]1 integrin ligation was achieved through antibody-induced receptor cross-linking or by exposure of neutrophils to solid phase matrix proteins, acting as physiological ligands. Occupancy of [beta]1 integrins enhanced respiratory burst in neutrophils predominantly dependent oil [alpha]4[beta]1 integrin mediated cell activation. The magnitude of respiratory burst evoked by [beta]1 integrin ligation was less than after [beta]2 integrin activation. However, [beta]1 integrin mediated respiratory burst was longer lasting compared with that seen after [beta]2 integrin ligation. In cells activated via combined ligation of [beta]1 and [beta]2 integrins, high levels of superoxide production, comparable to the peak values seen after separate ligation of [beta]2 integrins, were maintained throughout a two hour observation period. Under physiological conditions, when neutrophils emigrate from the vasculature, combined ligation of both [beta]1 and [beta]2 integrins; is likely to occur. Accordingly, these data indicate that, initial O2--production may be dependent primarily on [beta]2 integrins, whereas sustained respiratory burst relies also on neutrophil activation via [beta]1 integrins.

List of scientific papers

I. Werr J, Xie X, Hedqvist P, Ruoslahti E, Lindbom L (1998). beta1 integrins are critically involved in neutrophil locomotion in extravascular tissue In vivo. J Exp Med. 187(12):2091-6.
https://pubmed.ncbi.nlm.nih.gov/9625769

II. Werr J, Johansson J, Eriksson EE, Hedqvist P, Ruoslahti E, Lindbom L (2000). Integrin alpha(2)beta(1) (VLA-2) is a principal receptor used by neutrophils for locomotion in extravascular tissue. Blood. 95(5):1804-9.
https://pubmed.ncbi.nlm.nih.gov/10688841

III. Werr J, Eriksson EE, Hedqvist P, Lindbom L (2000). Engagement of beta2 integrins induces surface expression of beta1 integrin receptors in human neutrophils. J Leukoc Biol. 68(4):553-60.
https://pubmed.ncbi.nlm.nih.gov/11037978

IV. Werr J, Eriksson EE, Hedqvist P, Lindblom L (2000). New roles of beta1 and beta2 integrins in neutrophil motility revealed through studies of neutrophil chemotaxis in collagen gels. [Manuscript]

V. Werr J, Eriksson EE, Textorius L, Hedqvist P, Lindblom L (2000). Ligation of beta1 integrin receptors upregulates and prolongs respiratory burst in neutrophils. [Manuscript]