scholarly journals Binding of Rap1 and Riam to Talin1 Fine-Tune β2 Integrin Activity During Leukocyte Trafficking

2021 ◽  
Vol 12 ◽  
Thomas Bromberger ◽  
Sarah Klapproth ◽  
Ina Rohwedder ◽  
Jasmin Weber ◽  
Robert Pick ◽  

β2 integrins mediate key processes during leukocyte trafficking. Upon leukocyte activation, the structurally bent β2 integrins change their conformation towards an extended, intermediate and eventually high affinity conformation, which mediate slow leukocyte rolling and firm arrest, respectively. Translocation of talin1 to integrin adhesion sites by interactions with the small GTPase Rap1 and the Rap1 effector Riam precede these processes. Using Rap1 binding mutant talin1 and Riam deficient mice we show a strong Riam-dependent T cell homing process to lymph nodes in adoptive transfer experiments and by intravital microscopy. Moreover, neutrophils from compound mutant mice exhibit strongly increased rolling velocities to inflamed cremaster muscle venules compared to single mutants. Using Hoxb8 cell derived neutrophils generated from the mutant mouse strains, we show that both pathways regulate leukocyte rolling and adhesion synergistically by inducing conformational changes of the β2 integrin ectodomain. Importantly, a simultaneous loss of both pathways results in a rolling phenotype similar to talin1 deficient neutrophils suggesting that β2 integrin regulation primarily occurs via these two pathways.

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3257-3264 ◽  
Dori L. Borjesson ◽  
Scott I. Simon ◽  
Emir Hodzic ◽  
Hilde E. V. DeCock ◽  
Christie M. Ballantyne ◽  

AbstractTick saliva contains anti-inflammatory and immunosuppressive substances that facilitate blood feeding and enhance tick-vectored pathogen transmission, including Anaplasma phagocytophila,an etiologic agent of granulocytic ehrlichiosis. As such, inflammation at a tick-feeding site is strikingly different than that typically observed at other sites of inflammation. Up-regulation of CD11b/CD18 occurs in host granulocytes following interaction or infection withA phagocytophila, and the absence of CD11b/CD18 results in early increases in bacteremia. We hypothesized that β2 integrin–dependent infection kinetics and leukocyte extravasation are important determinants of neutrophil trafficking to, and pathogen acquisition at, tick-feeding sites.A phagocytophila infection kinetics were evaluated in CD11a/CD18, CD11b/CD18, and CD18 knock-out mice using quantitative polymerase chain reaction (PCR) of blood, ticks, and skin biopsies in conjunction with histopathology. A marked increase in the rate ofA phagocytophila infection of neutrophils and pathogen burden in blood followed tick feeding. Infection kinetics were modified by β2 integrin expression and systemic neutrophil counts. Significant neutrophil-pathogen trafficking was observed to both suture and tick sites. Despite the prominent role for β2 integrins in neutrophil arrest in flowing blood, successful pathogen acquisition by ticks occurred in the absence of β2 integrins. Establishment of feeding pools that rely less on leukocyte trafficking and more on small hemorrhages may explain the ready amplification of A phagocytophila DNA from ticks infested on CD11/CD18-deficient mouse strains.

2019 ◽  
Vol 3 (3) ◽  
pp. 256-267 ◽  
Pierre Cunin ◽  
Pui Y. Lee ◽  
Edy Kim ◽  
Angela B. Schmider ◽  
Nathalie Cloutier ◽  

Abstract Antibody ligation of the murine neutrophil surface protein Ly6G disrupts neutrophil migration in some contexts but not others. We tested whether this variability reflected divergent dependence of neutrophil migration on β2 integrins, adhesion molecules that interact with Ly6G at the neutrophil surface. In integrin-dependent murine arthritis, Ly6G ligation attenuated joint inflammation, even though mice lacking Ly6G altogether developed arthritis normally. By contrast, Ly6G ligation had no impact on integrin-independent neutrophil migration into inflamed lung. In peritoneum, the role of β2 integrins varied with stimulus, proving dispensable for neutrophil entry in Escherichia coli peritonitis but contributory in interleukin 1 (IL-1)–mediated sterile peritonitis. Correspondingly, Ly6G ligation attenuated only IL-1 peritonitis, disrupting the molecular association between integrins and Ly6G and inducing cell-intrinsic blockade restricted to integrin-dependent migration. Consistent with this observation, Ly6G ligation impaired integrin-mediated postadhesion strengthening for neutrophils arresting on activated cremaster endothelium in vivo. Together, these findings identify selective inhibition of integrin-mediated neutrophil emigration through Ly6G ligation, highlighting the marked site and stimulus specificity of β2 integrin dependence in neutrophil migration.

2007 ◽  
Vol 98 (08) ◽  
pp. 262-273 ◽  
Jürgen Schymeinsky ◽  
Attila Mócsai ◽  
Barbara Walzog

SummaryPolymorphonuclear neutrophils (PMN) are key components of the innate immunity and their efficient recruitment to the sites of lesion is a prerequisite for acute inflammation. Signaling via adhesion molecules of the β2 integrin family (CD11/CD18) plays an essential role for PMN recruitment and activation during inflammation. In this review, we will focus on the non-receptor tyrosine kinase Syk, an important downstream signaling component of β2 integrins that is required for the control of different PMN functions including adhesion,migration and phagocytosis. The exploration of β2 integrin-mediated Syk activation provided not only novel insights into the control of PMN functions but also led to the identification of Syk as a new molecular target for therapeutic intervention during inflammatory diseases.

2017 ◽  
Vol 114 (30) ◽  
pp. E6250-E6259 ◽  
Ana Maria Manso ◽  
Hideshi Okada ◽  
Francesca M. Sakamoto ◽  
Emily Moreno ◽  
Susan J. Monkley ◽  

Continuous contraction–relaxation cycles of the heart require strong and stable connections of cardiac myocytes (CMs) with the extracellular matrix (ECM) to preserve sarcolemmal integrity. CM attachment to the ECM is mediated by integrin complexes localized at the muscle adhesion sites termed costameres. The ubiquitously expressed cytoskeletal protein talin (Tln) is a component of muscle costameres that links integrins ultimately to the sarcomere. There are two talin genes, Tln1 and Tln2. Here, we tested the function of these two Tln forms in myocardium where Tln2 is the dominant isoform in postnatal CMs. Surprisingly, global deletion of Tln2 in mice caused no structural or functional changes in heart, presumably because CM Tln1 became up-regulated. Tln2 loss increased integrin activation, although levels of the muscle-specific β1D-integrin isoform were reduced by 50%. With this result, we produced mice that had simultaneous loss of both CM Tln1 and Tln2 and found that cardiac dysfunction occurred by 4 wk with 100% mortality by 6 mo. β1D integrin and other costameric proteins were lost from the CMs, and membrane integrity was compromised. Given that integrin protein reduction occurred with Tln loss, rescue of the phenotype was attempted through transgenic integrin overexpression, but this could not restore WT CM integrin levels nor improve heart function. Our results show that CM Tln2 is essential for proper β1D-integrin expression and that Tln1 can substitute for Tln2 in preserving heart function, but that loss of all Tln forms from the heart-muscle cell leads to myocyte instability and a dilated cardiomyopathy.

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5266-5276 ◽  
Anca Sindrilaru ◽  
Thorsten Peters ◽  
Jürgen Schymeinsky ◽  
Tsvetelina Oreshkova ◽  
Honglin Wang ◽  

Abstract Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relaying signals from immune response receptors and integrins to Rho-GTPases. We here provide first evidence for a role of Vav3 for β2-integrins–mediated macrophage functions during wound healing. Vav3−/− and Vav1−/−/Vav3−/− mice revealed significantly delayed healing of full-thickness excisional wounds. Furthermore, Vav3−/− bone marrow chimeras showed an identical healing defect, suggesting that Vav3 deficiency in leukocytes, but not in other cells, is causal for the impaired wound healing. Vav3 was required for the phagocytotic cup formation preceding macrophage phagocytosis of apoptotic neutrophils. Immunoprecipitation and confocal microscopy revealed Vav3 activation and colocalization with β2-integrins at the macrophage membrane upon adhesion to ICAM-1. Moreover, local injection of Vav3−/−or β2-integrin(CD18)−/− macrophages into wound margins failed to restore the healing defect of Vav3−/− mice, suggesting Vav3 to control the β2-integrin–dependent formation of a functional phagocytic synapse. Impaired phagocytosis of apoptotic neutrophils by Vav3−/− macrophages was causal for their reduced release of active transforming growth factor (TGF)-β1, for decreased myofibroblasts differentiation and myofibroblast-driven wound contraction. TGF-β1 deficiency in Vav3−/− macrophages was causally responsible for the healing defect, as local injection of either Vav3-competent macrophages or recombinant TGF-β1 into wounds of Vav3−/− mice fully rescued the delayed wound healing.

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 2035-2045 ◽  
Jonathan J. Miner ◽  
Lijun Xia ◽  
Tadayuki Yago ◽  
János Kappelmayer ◽  
Zhenghui Liu ◽  

Abstract In inflamed venules, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to roll on P-selectin and E-selectin and to activate integrin αLβ2 (lymphocyte function-associated antigen-1, LFA-1) to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Studies in cell lines have suggested that PSGL-1 requires its cytoplasmic domain to localize in membrane domains, to support rolling on P-selectin, and to signal through spleen tyrosine kinase (Syk). We generated “ΔCD” mice that express PSGL-1 without the cytoplasmic domain. Unexpectedly, neutrophils from these mice localized PSGL-1 normally in microvilli, uropods, and lipid rafts. ΔCD neutrophils expressed less PSGL-1 on their surfaces because of inefficient export from the endoplasmic reticulum. Limited digestion of wild-type neutrophils with O-sialoglycoprotein endopeptidase was used to reduce the PSGL-1 density to that on ΔCD neutrophils. At matched PSGL-1 densities, both ΔCD and wild-type neutrophils rolled similarly on P-selectin. However, ΔCD neutrophils rolling on P-selectin did not trigger Syk-dependent activation of LFA-1 to slow rolling on ICAM-1. These data demonstrate that the PSGL-1 cytoplasmic domain is dispensable for leukocyte rolling on P-selectin but is essential to activate β2 integrins to slow rolling on ICAM-1.

Blood ◽  
2015 ◽  
Vol 126 (25) ◽  
pp. 2704-2712 ◽  
Sarah Klapproth ◽  
Markus Sperandio ◽  
Elaine M. Pinheiro ◽  
Monika Prünster ◽  
Oliver Soehnlein ◽  

Key Points RIAM is an essential regulator of β2 integrins on leukocytes. Leukocyte α4β1 integrin is activated in a RIAM-independent manner.

2020 ◽  
Vol 88 (6) ◽  
Koichi Yuki ◽  
Lifei Hou

ABSTRACT Sepsis remains medically challenging, with high morbidity and mortality. A novel intervention is urgently needed in the absence of specific, targeted therapy. Neutrophils act as double-edged swords in sepsis; they can help to eradicate microbes, but they also contribute to tissue injury. β2 integrins are critical adhesion molecules that regulate a number of neutrophil functions. β2 integrins consist of four members, namely, αLβ2, αMβ2, αXβ2, and αDβ2. Here, we review the role of each β2 integrin in neutrophils and sepsis and consider future direction for therapeutic intervention.

2004 ◽  
Vol 380 (1) ◽  
pp. 57-65 ◽  
Stefano MANARINI ◽  
Lorenzo FEDERICO ◽  
Zsuzsa BAGOLY ◽  
Romina PECCE ◽  

In human PMN (polymorphonuclear cells), challenged by P-selectin, the β2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108–116]. This suggested that an SRC-dependent outside-in signalling strengthens the β2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock β2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor β2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common β2 chain, by a combination of antibodies against αL and αM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by β2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by β2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize β2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.

2006 ◽  
Vol 396 (1) ◽  
pp. 89-98 ◽  
Licia Totani ◽  
Antonio Piccoli ◽  
Stefano Manarini ◽  
Lorenzo Federico ◽  
Romina Pecce ◽  

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin–IgG fusion protein, by a mechanism that involved β2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(β2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an ‘outside-in’ signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented β2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin–IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck−/−fgr−/− and hck−/−fgr−/−lyn−/− mice showed significant impairment of β2-integrin-mediated adhesion to CHO-E. Moreover, the expression of β2 integrin activation epitopes at the sites of cell–cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a β2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for β2 integrin function in PMN tethered by E-selectin.

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