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Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.

ABSTRACT: Selectins and their ligands mediate leukocyte rolling, allowing interactions with chemokines that lead to integrin activation and arrest. Here we show that E-selectin is crucial for generating a secondary wave of activating signals, transduced specifically by E-selectin ligand-1, that induces polarized, activated alpha(M)beta(2) integrin clusters at the leading edge of crawling neutrophils, allowing capture of circulating erythrocytes or platelets. In a humanized mouse model of sickle cell disease, the capture of erythrocytes by alpha(M)beta(2) microdomains leads to acute lethal vascular occlusions. In a model of transfusion-related acute lung injury, polarized neutrophils capture circulating platelets, resulting in the generation of oxidative species that produce vascular damage and lung injury. Inactivation of E-selectin or alpha(M)beta(2) prevents tissue injury in both inflammatory models, suggesting broad implications of this paradigm in thromboinflammatory diseases. These results indicate that endothelial selectins can influence neutrophil behavior beyond its canonical rolling step through delayed, organ-damaging, polarized activation.

SUBMITTER: Hidalgo A

PROVIDER: S-EPMC2772164 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.

Hidalgo Andrés A Chang Jungshan J Jang Jung-Eun JE Peired Anna J AJ Chiang Elaine Y EY Frenette Paul S PS

Nature medicine 20090322 4

Selectins and their ligands mediate leukocyte rolling, allowing interactions with chemokines that lead to integrin activation and arrest. Here we show that E-selectin is crucial for generating a secondary wave of activating signals, transduced specifically by E-selectin ligand-1, that induces polarized, activated alpha(M)beta(2) integrin clusters at the leading edge of crawling neutrophils, allowing capture of circulating erythrocytes or platelets. In a humanized mouse model of sickle cell disea ...[more]

PMID: 19305412

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Project description:BACKGROUND:Platelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ib? (GPIb?), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIb? and enhancing the ligand-binding activity under thromboinflammatory conditions. METHODS:Bioinformatic analysis identified 2 potential allosteric disulfide bonds in GPIb?. Agglutination assays, flow cytometry, surface plasmon resonance analysis, a protein-protein docking model, proximity ligation assays, and mass spectrometry were used to demonstrate a direct interaction between PDI and GPIb? and to determine a role for PDI in regulating GPIb? function and platelet-neutrophil interactions. Also, real-time microscopy and animal disease models were used to study the pathophysiological role of PDI-GPIb? signaling under thromboinflammatory conditions. RESULTS:Deletion or inhibition of platelet PDI significantly reduced GPIb?-mediated platelet agglutination. Studies using PDI-null platelets and recombinant PDI or Anfibatide, a clinical-stage GPIb? inhibitor, revealed that the oxidoreductase activity of platelet surface-bound PDI was required for the ligand-binding function of GPIb?. PDI directly bound to the extracellular domain of GPIb? on the platelet surface and reduced the Cys4-Cys17 and Cys209-Cys248 disulfide bonds. Real-time microscopy with platelet-specific PDI conditional knockout and sickle cell disease mice demonstrated that PDI-regulated GPIb? function was essential for platelet-neutrophil interactions and vascular occlusion under thromboinflammatory conditions. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that PDI-GPIb? signaling played a crucial role in tissue damage. CONCLUSIONS:Our results demonstrate that PDI-facilitated cleavage of the allosteric disulfide bonds tightly regulates GPIb? function, promoting platelet-neutrophil interactions, vascular occlusion, and tissue damage under thromboinflammatory conditions.

Project description:Essential thrombocythemia (ET) is comprised among chronic myeloproliferative neoplasms (MPN) and is caused by driver mutations in JAK2, CALR, and MPL, which lead to megakaryocyte proliferation and prominent thrombocytosis. Thrombosis remains the main cause of morbidity in ET and is driven by the interplay between blood cells, the endothelium, the clotting cascade, and host-derived inflammatory mediators. Platelet activation plays a key role in the thrombotic predisposition, although the underlying mechanisms remain poorly defined. In addition to their role in hemostasis, platelets participate in innate immunity and inflammation owing to the expression of toll-like receptors (TLR), which recognize inflammatory signals, triggering platelet functional responses. Considering the impact of inflammation on ET procoagulant state, we assessed the contribution of TLR2 and TLR4 to platelet hemostatic and inflammatory properties in ET patients, by using Pam3CSK4 and lipopolysaccharide (LPS) as specific TLR2 and TLR4 ligands, respectively. TLR2 ligation induced increased surface translocation of ?-granule-derived P-selectin and CD40L, which mediate platelet interaction with leukocytes and endothelial cells, respectively, and higher levels of dense granule-derived CD63 in patients, whereas PAC-1 binding was not increased and LPS had no effect on these platelet responses. Platelet-neutrophil aggregate formation was elevated in ET at baseline and after stimulation of both TLR2 and TLR4. In addition, ET patients displayed higher TLR2- and TLR4-triggered platelet secretion of the chemokine RANTES (CCL5), whereas von Willebrand factor release was not enhanced, revealing a differential releasate pattern for ?-granule-stored inflammatory molecules. TLR-mediated hyperresponsiveness contrasted with impaired or preserved responses to classic platelet hemostatic agonists, such as TRAP-6 and thrombin. TLR2 and TLR4 expression on the platelet surface was normal, whereas phosphorylation of downstream effector ERK1/2 was higher in patients at baseline and after incubation with Pam3CSK4, which may partly explain the enhanced TLR2 response. In conclusion, exacerbated response to TLR stimulation may promote platelet activation in ET, boosting platelet/leukocyte/endothelial interactions and secretion of inflammatory mediators, overall reinforcing the thromboinflammatory state. These findings highlight the role of platelets as inflammatory sentinels in MPN prothrombotic scenario and provide additional evidence for the close intertwining between thrombosis and inflammation in this setting.

Dataset Information

Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.

Publications

Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.

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