Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)-related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture. Conversely, infusion of recombinant GDF-15 repressed PMN recruitment after myocardial infarction. In vitro, GDF-15 inhibited PMN adhesion, arrest under flow and transendothelial migration. Mechanistically, GDF-15 counteracted chemokine-triggered conformational activation and clustering of β(2) integrins on PMNs by activating the small GTPase Cdc42 and inhibiting activation of the small GTPase Rap1. Intravital microscopy in vivo in Gdf15-deficient mice showed that Gdf-15 is required to prevent excessive chemokine-activated leukocyte arrest on the endothelium. Genetic ablation of β(2) integrins in myeloid cells rescued the mortality of Gdf15-deficient mice after myocardial infarction. To our knowledge, GDF-15 is the first cytokine identified as an inhibitor of PMN recruitment by direct interference with chemokine signaling and integrin activation. Loss of this anti-inflammatory mechanism leads to fatal cardiac rupture after myocardial infarction.

Summary Endothelial chemokines are instrumental for integrin-mediated lymphocyte adhesion and transendothelial migration (TEM). By dissecting how chemokines trigger lymphocyte integrins to support shear-resistant motility on and across cytokine-stimulated endothelial barriers, we found a critical role for high-affinity (HA) LFA-1 integrin in lymphocyte crawling on activated endothelium. Endothelial-presented chemokines triggered HA-LFA-1 and adhesive filopodia at numerous submicron dots scattered underneath crawling lymphocytes. Shear forces applied to endothelial-bound lymphocytes dramatically enhanced filopodia density underneath crawling lymphocytes. A fraction of the adhesive filopodia invaded the endothelial cells prior to and during TEM and extended large subluminal leading edge containing dots of HA-LFA-1 occupied by subluminal ICAM-1. Memory T cells generated more frequent invasive filopodia and transmigrated more rapidly than their naive counterparts. We propose that shear forces exerted on HA-LFA-1 trigger adhesive and invasive filopodia at apical endothelial surfaces and thereby promote lymphocyte crawling and probing for TEM sites.

Regulation of the affinity of the beta(2) integrin LFA-1 by chemokines is critical to lymphocyte trafficking, but the signaling mechanisms that control this process are not well understood. Here we investigated the signaling events controlling LFA-1 affinity triggering by chemokines in human primary T lymphocytes. We found that the small GTPase Rac1 mediated chemokine-induced LFA-1 affinity triggering and lymphocyte arrest in high endothelial venules. Unexpectedly, another Rho family member, Cdc42, negatively regulated LFA-1 activation. The Rho effectors PLD1 and PIP5KC were also critical to LFA-1 affinity modulation. Notably, PIP5KC was found to specifically control the transition of LFA-1 from an extended low-intermediate state to a high-affinity state, which correlated with lymphocyte arrest. Thus, chemokines control lymphocyte trafficking by triggering a Rho-dependent signaling cascade leading to conformer-specific modulation of LFA-1 affinity.

Podosomes are multimolecular mechanosensory structures with a protrusive actin core and an adhesive ring of integrins and adaptor proteins. Dual-color direct stochastic optical reconstruction microscopy is used to reveal the nanoscale localization of the ring components αMβ2 integrin, talin, and vinculin with respect to the actin core.

Rolling leukocytes are exposed to different adhesion molecules and chemokines. Neutrophil rolling on E-selectin induces integrin αLβ2-mediated slow rolling on intercellular adhesion molecule-1 by activating a phospholipase C (PLC)γ2- and a separate phosphoinositide-3-kinase (PI3K)γ-dependent pathway. E-selectin-signaling cooperates with chemokine signaling to recruit neutrophils into inflamed tissues. However, the distal signaling pathway linking PLCγ2 (Plcg2) to αLβ2-activation is unknown. To identify this pathway, we used different TAT-fusion-mutants and gene-deficient mice in intravital microscopy, autoperfused flow chamber, peritonitis, and biochemical studies. We found that the small GTPase Rap1 is activated following E-selectin engagement and that blocking Rap1a in Pik3cg−/− mice by a dominant-negative TAT-fusion mutant completely abolished E-selectin mediated slow rolling. We identified CalDAG-GEFI (Rasgrp2) and p38 MAPK as key signaling intermediates between PLCγ2 and Rap1a. Gαi-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster muscle were completely abolished in Rasgrp2−/− mice. The physiologic importance of CalDAG-GEFI in E-selectin-dependent integrin activation is shown by complete inhibition of neutrophil recruitment into the inflamed peritoneal cavity of Rasgrp2−/− leukocytes treated with pertussis toxin to block Gαi-signaling. Our data demonstrate that Rap1a activation by p38 MAPK and CalDAG-GEFI is involved in E-selectin-dependent slow rolling and leukocyte recruitment.