Regulation of stimulated integrin surface expression in human neutrophils by tyrosine phosphorylation

Abstract

The control of the adhesive properties of human neutrophils is an essential element of their defense function. One level at which this control is exerted involves the upregulation of the surface expression of beta 2-integrins. In this study, we have examined the potential involvement of tyrosine phosphorylation in the latter process. Two inhibitors of tyrosine kinases with differing modes of action, erbstatin and herbimycin A, were found to inhibit the expression of CD11b and CD18 stimulated by chemotactic factors (fMet-Leu-Phe or leukotriene B4) or growth factors (tumor necrosis factor alpha). This inhibition was not shared by an inactive analog of erbstatin or by the protein kinase C inhibitor Ro 31–8330. Erbstatin also inhibited the unveiling of activation-specific neoepitopes detected by antibody CBRM1/5. Pretreatment of neutrophils (but not of endothelial cells) with erbstatin inhibited the stimulation of neutrophils' adherence to endothelial cells induced by fMet-Leu-Phe. Augmentation of tyrosine phosphorylation by inhibiting tyrosine phosphatases using hydroperoxyvanadate led to an increased surface expression of CD11b and CD18 and enhanced the adhesion of neutrophils to endothelial cells. Finally, the leumedin NPC 15669, which had previously been shown to inhibit stimulated CD11b expression and neutrophil adherence to endothelial cells and to exhibit anti-inflammatory properties in various in vivo models of inflammation, inhibited the stimulation of tyrosine, phosphorylation induced by fMet-Leu-Phe. Taken together, these data establish a strong correlation between tyrosine phosphorylation and integrin upregulation in stimulated human neutrophils.

The Atypical Zeta (ζ) Isoform of Protein Kinase C Regulates CD11b/CD18 Activation in Human Neutrophils

“…This high affinity confirmation can be detected using an activation specific antibody, CBRM1/5 [9]. As expected, stimulation with IL-8 led to an increase in Mac-1 affinity, as measured by the binding of CBRM1/5 ( Figure 5A) [22]. Pretreatment with calphostin C had no effect on IL-8-mediated changes in Mac-1 affinity, but pretreatment of neutrophils with chelerythrine chloride (data not shown) or the PKCζ pseudosubstrate prior to stimulation with IL-8 actually increased Mac-1 affinity above and beyond the level attained with IL-8 alone ( Figure 5A).…”

Human Leukaemic (HMC‐1) and Normal Skin Mast Cells Express β2‐Integrins: Characterization of β2‐Integrins and ICAM‐1 on HMC‐1 Cells

“…Cycloheximide clearly inhibited this process, suggesting a de novo synthesis of these adhesion molecules, in agreement with our finding that transcription levels of the genes encoding the four integrin chains were enhanced in PMA-treated cells. It has been reported that LTB4, a naturally occurring eicosanoid mediator which is mainly known as a chemoattractant for human neutrophils, will up-regulate neutrophil cell surface b2-integrin expression as well as enhance their adhesiveness to endothelial cells [29,30,33,34]. Here, it could be demonstrated that LTB4 also affects cell surface density of LFA-1 on human mast cells of the HMC-1 cell line.…”

Suppression of cytokine-mediated β2-integrin activation on circulating neutrophils in critically ill patients

“…The monoclonal antibody CBRM1/5 does not cross-react with inactive CD11b. It has been consistently shown to react only with activated CD11b on neutrophils [14][15][16] and eosinophils [17]. All flow cytometry samples, both conventional and high sensitivity, included matched isotype controls to control for nonspecific staining.…”