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PSGL-1 engagement by E-selectin signals through Src kinase Fgr and ITAM adapters DAP12 and FcR gamma to induce slow leukocyte rolling.

ABSTRACT: E-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1) can activate the beta(2) integrin lymphocyte function-associated antigen-1 by signaling through spleen tyrosine kinase (Syk). This signaling is independent of G alpha(i)-protein-coupled receptors, results in slow rolling, and promotes neutrophil recruitment to sites of inflammation. However, the signaling pathways linking E-selectin engagement of PSGL-1 to Syk activation are unknown. To test the role of Src family kinases and immunoreceptor tyrosine-based activating motif (ITAM)-containing adaptor proteins, we used different gene-deficient mice in flow chamber, intravital microscopy, and peritonitis studies. E-selectin-mediated phosphorylation of Syk and slow rolling was abolished in neutrophils from fgr(-/-) or hck(-/-) lyn(-/-) fgr(-/-) mice. Neutrophils from Tyrobp(-/-) Fcrg(-/-) mice lacking both DAP12 and FcRgamma were incapable of sustaining slow neutrophil rolling on E-selectin and intercellular adhesion molecule-1 and were unable to phosphorylate Syk and p38 MAPK. This defect was confirmed in vivo by using mixed chimeric mice. G alpha(i)-independent neutrophil recruitment into the inflamed peritoneal cavity was sharply suppressed in Tyrobp(-/-) Fcrg(-/-) mice. Our data demonstrate that an ITAM-dependent pathway involving the Src-family kinase Fgr and the ITAM-containing adaptor proteins DAP12 and FcRgamma is involved in the initial signaling events downstream of PSGL-1 that are required to initiate neutrophil slow rolling.

SUBMITTER: Zarbock A

PROVIDER: S-EPMC2556779 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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PSGL-1 engagement by E-selectin signals through Src kinase Fgr and ITAM adapters DAP12 and FcR gamma to induce slow leukocyte rolling.

Zarbock Alexander A Abram Clare L CL Hundt Matthias M Altman Amnon A Lowell Clifford A CA Ley Klaus K

The Journal of experimental medicine 20080915 10

E-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1) can activate the beta(2) integrin lymphocyte function-associated antigen-1 by signaling through spleen tyrosine kinase (Syk). This signaling is independent of G alpha(i)-protein-coupled receptors, results in slow rolling, and promotes neutrophil recruitment to sites of inflammation. However, the signaling pathways linking E-selectin engagement of PSGL-1 to Syk activation are unknown. To test the role of Src family kinases and immuno ...[more]

PMID: 18794338

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Project description:Model substrates presenting biochemical cues immobilized in a controlled and well-defined manner are of great interest for their applications in biointerface studies that elucidate the molecular basis of cell receptor-ligand interactions. Herein, we describe a direct, photochemical method to generate surface-immobilized biomolecular gradients that are applied to the study of selectin-mediated leukocyte rolling. The technique employs benzophenone-modified glass substrates, which upon controlled exposure to UV light (350-365 nm) in the presence of protein-containing solutions facilitate the generation of covalently immobilized protein gradients. Conditions were optimized to generate gradient substrates presenting P-selectin and PSGL-1 (P-selectin glycoprotein ligand-1) immobilized at site densities over a 5- to 10-fold range (from as low as ∼200 molecules μm(-2) to as high as 6000 molecules μm(-2)). The resulting substrates were quantitatively characterized via fluorescence analysis and radioimmunoassays before their use in the leukocyte rolling assays. HL-60 promyelocytes and Jurkat T lymphocytes were assessed for their ability to tether to and roll on substrates presenting immobilized P-selectin and PSGL-1 under conditions of physiologically relevant shear stress. The results of these flow assays reveal the combined effect of immobilized protein site density and applied wall shear stress on cell rolling behavior. Two-component substrates presenting P-selectin and ICAM-1 (intercellular adhesion molecule-1) were also generated to assess the interplay between these two proteins and their effect on cell rolling and adhesion. These proof-of-principle studies verify that the described gradient generation approach yields well-defined gradient substrates that present immobilized proteins over a large range of site densities that are applicable for investigation of cell-materials interactions, including multi-parameter leukocyte flow studies. Future applications of this enabling methodology may lead to new insights into the biophysical phenomena and molecular mechanism underlying complex biological processes such as leukocyte recruitment and the inflammatory response.

Dataset Information

PSGL-1 engagement by E-selectin signals through Src kinase Fgr and ITAM adapters DAP12 and FcR gamma to induce slow leukocyte rolling.

Publications

PSGL-1 engagement by E-selectin signals through Src kinase Fgr and ITAM adapters DAP12 and FcR gamma to induce slow leukocyte rolling.

Similar Datasets

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