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Leukocytes arrested on inflamed endothelium via integrins are subjected to force imparted by flowing blood. How leukocytes respond to this force and resist detachment is poorly understood. Live-cell imaging with Lifeact-transfected U937 cells revealed that force triggers actin polymerization at upstream α4β1 integrin adhesion sites and the adjacent cortical cytoskeleton. Scanning electron microscopy revealed that this culminates in the formation of structures that anchor monocyte adhesion. Inhibition of actin polymerization resulted in cell deformation, displacement, and detachment. Transfection of dominant-negative constructs and inhibition of function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. These included activation of Rap1, phosphoinositide 3-kinase γ isoform, and Rac but not Cdc42. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces.

Chemokine/chemoattractant G protein-coupled receptors trigger an inside-out signaling network that rapidly activates integrins, a key step in inflammatory leukocyte recruitment. Integrins mediate leukocyte arrest and adhesion to endothelium through multivalent binding, and they transmit outside-in signals to stabilize adhesion and coordinate cell spreading and migration. In the present study, we used RNA interference in the U937 monocytic cell line to investigate the role of talin-1, kindlin-3, and α-actinin-1 in the fMLF- and SDF-1α-induced upregulation of α(4)β(1) integrin affinity and consequent adhesive events. Affinity upregulation of α(4)β(1) integrin was not impaired by small interfering RNA knockdown of talin-1, kindlin-3, or α-actinin-1. Only kindlin-3 knockdown increased flow-induced detachment from VCAM-1-coated surfaces in response to fluid flow, whereas knockdown of either talin-1 or kindlin-3 increased detachment from ICAM-1-coated surfaces. Biochemical analyses revealed that α(4)β(1) expression was highly enriched in U937 cell microridges and murine lymphocyte microvilli. Kindlin-3 was present throughout the cell, whereas talin-1 was largely excluded from microridges/microvilli. The subcellular colocalization of α(4)β(1) and kindlin-3 in microridges may explain why kindlin-3 rapidly associates with α(4)β(1) after G protein-coupled receptor signaling and contributes to adhesion strengthening. Talin-1 contributed to α(4)β(1)-dependent chemotaxis, suggesting that it participates in a later stage of the leukocyte adhesion cascade when the leukocyte cytoskeleton undergoes dramatic rearrangement.

Monocyte recruitment or emigration to tissues is an essential component of host defense in both acute and chronic inflammatory responses. Sequential molecular interactions mediate a cascade of tethering, rolling, arrest, stable adhesion, and intravascular crawling that culminates in monocyte diapedesis across the vascular endothelium and migration through the basement membrane of postcapillary venules. Integrins are complex adhesion and signaling molecules. Dynamic alterations in their conformation and distribution on the monocyte cell surface are required for many steps of monocyte emigration. Intracellular signaling initiated by chemokine receptors induces conformational changes in integrins that upregulate their affinity for ligands, and this is essential for monocyte arrest. This review focuses on the activation of monocyte alpha4beta1 integrins by endothelial chemokines, which is required for the arrest of monocytes rolling on vascular cell adhesion molecule 1 under shear flow. Using soluble ligand-binding assays and adhesion assays in parallel-plate flow chambers, critical signaling mediators in chemokine-induced alpha4beta1 integrin affinity upregulation and monocyte arrest have been identified, including phospholipase C, calcium, and calmodulin.

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 alpha4beta1 integrin affinity up-regulation using formyl peptide receptor-transfected U937 cells stimulated with fMLP or stromal-derived factor-1alpha and human peripheral blood monocytes stimulated with multiple chemokines or chemoattractants. The up-regulation 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 alpha4 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, phosphoinositide 3-kinase, or protein kinase C (PKC) signaling did not inhibit chemoattractant-induced LDV or VCAM-1 binding. However, activation of PKC by phorbol ester up-regulated alpha4beta1 affinity with kinetics distinct from those of GPCR signaling. A critical role for PLC and calmodulin was also established for leukocyte arrest and adhesion strengthening.

Leukocyte alpha4beta1 integrins regulate hematopoietic and lymphoid development, as well as the emigration of circulating cells to sites of inflammation. Because vascular cell adhesion molecule-1 (VCAM-1) binding to high-affinity alpha4beta1 is stable, these integrins can be detected and selectively precipitated from cell lysates using VCAM-1/Fc. With this approach, high-affinity alpha4beta1 integrin expression was demonstrated on lymphocytes in the bone marrow, thymus, spleen, and the peritoneal cavity of normal mice, but not in peripheral lymph nodes. Immature lymphocytes preferentially expressed high-affinity alpha4beta1 in the bone marrow and thymus. Paxillin is a cytoplasmic adaptor molecule that can bind to the alpha4 tail and initiate signaling. Paxillin was associated selectively with high-affinity integrins that were isolated from human Jurkat T cells or from murine tissues, and blotting with a phospho-specific antibody demonstrated that Ser988 in the alpha4 cytoplasmic tail was dephosphorylated in high-affinity but not low-affinity integrins. A rapid and transient alpha4beta1 affinity up-regulation in formyl peptide receptor-transfected U937 cells stimulated with N-formyl-methyonyl-leucyl-phenylalanine (fMLP) correlated temporally with induced paxillin binding to alpha4 integrins. These data suggest that ligand binding to high-affinity alpha4beta1 integrins may initiate outside-in signaling cascades through paxillin that regulate leukocyte maturation and emigration.