Phospholipase C, calcium, and calmodulin are critical for α4β1 integrin affinity up-regulation and monocyte arrest triggered by chemoattractants

Abstract

During inflammation, monocytes roll on activated endothelium and arrest after stimulation by proteoglycan-bound chemokines and other chemoattractants. We investigated signaling pathways downstream of G protein-coupled receptors (GPCRs) that are relevant to ␣4␤1 integrin affinity up-regulation using formyl peptide receptor-transfected U937 cells stimulated with fMLP or stromal-derived factor-1␣ and human peripheral blood monocytes stimulated with multiple chemokines or chemoattractants. The upregulation of soluble LDV peptide or vascular cell adhesion molecule-1 (VCAM-1) binding by these stimuli was critically dependent on activation of phospholipase C (PLC), inositol 1,4,5-triphosphate receptors, increased intracellular calcium, influx of extracellular calcium, and calmodulin, suggesting that this signaling pathway is required for ␣4 integrins to assume a high-affinity conformation. In fact, a rise in intracellular calcium following treatment with thapsigargin or ionomycin was sufficient to induce binding of ligand. Blockade of p44/42 and p38 mitogen-activated protein (MAP) kinases, IntroductionDuring emigration, integrin activation by chemoattractants or chemokines is a key step resulting in the arrest of rolling leukocytes. 1,2 This activation step results in the recruitment of specific leukocytes from the blood and is important for both inflammatory and homeostatic physiology. Two models have been proposed to explain integrin activation: affinity and valency. 3,4 Alterations in integrin conformation can increase the intrinsic affinity of each molecule for its cognate ligands and result in stable or persistent binding. 5,6 Binding of monomeric soluble ligand is dependent on individual integrin-ligand interactions and can be used to assess integrin affinity. 7 Thus far, several integrins in the ␤1, ␤2, ␤3, and ␤7 families have been shown to modulate their affinity in response to inside-out signals. [8][9][10] In contrast, the ability of a cell to increase the number of cotemporaneous integrin-ligand bonds is multivalent ligand binding. 11 This can be the result of increased integrin lateral mobility in the plasma membrane or clustering and cannot be detected by soluble monomeric ligand. In this model, lateral mobility of integrins within the plasma membrane directly correlates with increased cell-substrate adhesion. 12,13 It is unlikely that these 2 modes of modulating integrin-ligand binding are mutually exclusive, and indeed they may be interdependent, 14 because studies suggest that integrin microclustering is dependent on affinity up-regulation and ligand binding. 15 Studies have indicated that rapid up-regulation of ␣4␤1 and ␣L␤2 integrin affinity is required for monocyte arrest on vascular cell adhesion molecule-1 (VCAM-1) and T-lymphocyte arrest on intercellular adhesion molecule-1 (ICAM-1), respectively, [16][17][18] although multivalent binding also may be involved. 17,19 Signaling molecules downstream of G protein-coupled receptors (GPCRs) that are involved in the up-regulation of integrin affinity b...

Talin 1 and Paxillin Facilitate Distinct Steps in Rapid VLA-4-mediated Adhesion Strengthening to Vascular Cell Adhesion Molecule 1

“…an outside-in integrin activation process). These results contrast the ability of certain chemokines and chemoattractants to stimulate through inside-out signaling VLA-4 affinity to soluble VCAM-1 and conformationally switch ␤ 1 integrins into highly active states in monocyte lines and primary monocytes (58). Nevertheless, in the absence of such inside-out conformational switches of T cell VLA-4, talin 1 can still contribute to this outside-in activation of VLA-4 by chemokine, possibly by enriching a priori the number of high affinity VLA-4 available for chemokine stimulation.…”

Integrin Regulation during Leukocyte Recruitment

“…Furthermore, the published data suggest that PLC activation seems to be a key event in GPCR-mediated integrin activation (32,37). The available data indicate that the involved GEFs and GTPases not only vary among cell types but also among signaling pathways targeting different integrins.…”

“…The elevation of intracellular calcium, the calcium-binding messenger protein calmodulin, and inositol-1,4,5-triphosphate receptors as downstream events of PLC activation were found to be involved in GPCR-mediated VLA-4 activation following stimulation with fMLP or CXCL12 in monocytes (37). Neither p38 MAPKs, PI3K, or, most interestingly, PKC was necessary to achieve VLA-4 activation and subsequent binding to VCAM-1 (37). In contrast, neutrophil response to chemokines uses distinct PI3K isoforms over time: early responses were found to be PI3Kg dependent, whereas prolonged recruitment of neutrophils following MIP-2 or TNF-a superfusion were PI3Kd dependent (23).…”

“…The elevation of intracellular calcium, the calcium-binding messenger protein calmodulin, and inositol-1,4,5-triphosphate receptors as downstream events of PLC activation were found to be involved in GPCR-mediated VLA-4 activation following stimulation with fMLP or CXCL12 in monocytes (37). Neither p38 MAPKs, PI3K, or, most interestingly, PKC was necessary to achieve VLA-4 activation and subsequent binding to VCAM-1 (37).…”

Chemokine Regulation of Neutrophil Infiltration of Skin Wounds

“…To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound receptor-dependent inside-out modulation of integrin activation are currently under vigorous investigation and have been known to be mediated by an early increase in intracellular Ca 2 + followed by a late activation of the small GTPase Rap1. 20,21 Furthermore, the paired immunoglobulin-like type 2 receptor (PILRa) has been found to negatively regulate the chemoattractant binding-induced integrin activation. 22 The PILRa -/ -neutrophils show robust transmigration activities in a number of acute inflammation models.…”