Skap2 is required for β2 integrin-mediated neutrophil recruitment and functions.

Integrin activation is required for neutrophil functions. Impaired integrin activation on neutrophils is the hallmark of leukocyte adhesion deficiency (LAD) syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. The Src kinase-associated phosphoprotein 2 (Skap2) is involved in integrin functions in different leukocyte subtypes. However, the role of Skap2 in β2 integrin activation and neutrophil recruitment is unknown. In this study, we demonstrate the crucial role of Skap2 in regulating actin polymerization and binding of talin-1 and kindlin-3 to the β2 integrin cytoplasmic domain, thereby being indispensable for β2 integrin activation and neutrophil recruitment. The direct interaction of Skap2 with the Wiskott-Aldrich syndrome protein via its SH3 domain is critical for integrin activation and neutrophil recruitment in vivo. Furthermore, Skap2 regulates integrin-mediated outside-in signaling events and neutrophil functions. Thus, Skap2 is essential to activate the β2 integrins, and loss of Skap2 function is sufficient to cause a LAD-like phenotype in mice

The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation.

Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown. By using spinning-disk confocal intravital microscopy, we examined the molecular mechanisms of leukocyte recruitment to sites of focal hepatic necrosis in vivo. We demonstrated that the Bruton's tyrosine kinase (Btk) was required for multiple Mac-1 activation events involved in neutrophil recruitment and functions during sterile inflammation triggered by fMLF. The Src family kinase Hck, Wiskott-Aldrich-syndrome protein, and phospholipase Cγ2 were also involved in this pathway required for fMLF-triggered Mac-1 activation and neutrophil recruitment. Thus, we have identified a neutrophil Btk signalosome that is involved in a signaling pathway triggered by formylated peptides leading to the selective activation of Mac-1 and neutrophil recruitment during sterile inflammation

Cross-Talk between Shp1 and PIPKIγ Controls Leukocyte Recruitment.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. However, overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to the release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of αLβ2 (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly). Despite the clinical importance, cell-intrinsic molecular mechanisms of negative regulation of integrin adhesiveness and neutrophil recruitment are poorly understood. Mice deficient in the tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase 1 (Shp1) show increased leukocyte adhesion, but the interpretation of these data is limited by the severe global phenotype of these mice. In this study, we used mice with global and myeloid-restricted deletion of Shp1 to study neutrophil arrest, adhesion, crawling, and transendothelial migration in vitro and in vivo. Shp1 deficiency results in increased neutrophil adhesion in vivo; however, neutrophil crawling, transmigration, and chemotaxis were reduced in these mice. Mechanistically, Shp1 binds and controls PIPKIγ activity and, thereby, modulates phosphatidylinositol (4,5)-bisphosphate levels and adhesion. Thus, Shp1 is involved in the deactivation of integrins and regulation of neutrophil recruitment into inflamed tissue

Cross-Talk between Shp1 and PIPKIγ Controls Leukocyte Recruitment.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. However, overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to the release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of αLβ2 (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly). Despite the clinical importance, cell-intrinsic molecular mechanisms of negative regulation of integrin adhesiveness and neutrophil recruitment are poorly understood. Mice deficient in the tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase 1 (Shp1) show increased leukocyte adhesion, but the interpretation of these data is limited by the severe global phenotype of these mice. In this study, we used mice with global and myeloid-restricted deletion of Shp1 to study neutrophil arrest, adhesion, crawling, and transendothelial migration in vitro and in vivo. Shp1 deficiency results in increased neutrophil adhesion in vivo; however, neutrophil crawling, transmigration, and chemotaxis were reduced in these mice. Mechanistically, Shp1 binds and controls PIPKIγ activity and, thereby, modulates phosphatidylinositol (4,5)-bisphosphate levels and adhesion. Thus, Shp1 is involved in the deactivation of integrins and regulation of neutrophil recruitment into inflamed tissue

Cross-Talk between Shp1 and PIPKIγ Controls Leukocyte Recruitment

Neutrophil recruitment to site of inflammation plays a pivotal role in host defense. However, an overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of α(L)β(2) (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly). Despite the clinical importance, cell-intrinsic molecular mechanisms of negative regulation of integrin adhesiveness and neutrophil recruitment are poorly understood. Mice deficient in the tyrosine phosphatase Shp1 show increased leukocyte adhesion, but interpretation of these data is limited by the severe global phenotype of these mice. Here, we used mice with global and myeloid-restricted deletion of Shp1 to study neutrophil arrest, adhesion, crawling and transendothelial migration in vitro and in vivo. Shp1 deficiency results in an increased neutrophil adhesion in vivo. However, neutrophil crawling, transmigration and chemotaxis were reduced in these mice. Mechanistically, Shp1 binds and controls PIPKIγ-activity and thereby modulates PtdIns(4,5)P(2) levels and adhesion. Thus, Shp1 is involved in the deactivation of integrins and regulation of neutrophil recruitment into inflamed tissue

PRN473, an inhibitor of Bruton's tyrosine kinase, inhibits neutrophil recruitment via inhibition of macrophage antigen‐1 signalling

Background and purposeFollowing inflammatory stimuli, neutrophils are recruited to sites of inflammation and exert effector functions that often have deleterious effects on tissue integrity, which can lead to organ failure. Bruton's tyrosine kinase (Btk) is expressed in neutrophils and constitutes a promising pharmacological target for neutrophil-mediated tissue damage. Here, we evaluate a selective reversible inhibitor of Btk, PRN473, for its ability to dampen neutrophil influx via inhibition of adhesion receptor signalling pathways.Experimental approachIn vitro assays were used to assess fMLP receptor 1 (Fpr-1)-mediated binding of ligands to the adhesion receptors macrophage antigen-1 (Mac-1) and lymphocyte function antigen-1. Intravital microscopy of the murine cremaster was used to evaluate post-adhesion strengthening and endoluminal crawling. Finally, neutrophil influx was visualized in a clinically relevant model of sterile liver injury in vivo. Btk knockout animals were used as points of reference for Btk functions.Key resultsPharmacological inhibition of Btk by PRN473 reduced fMLP-induced phosphorylation of Btk and Mac-1 activation. Biochemical experiments demonstrated the specificity of the inhibitor. PRN473 (20 mg·kg-1 ) significantly reduced intravascular crawling and neutrophil recruitment into inflamed tissue in a model of sterile liver injury, down to levels seen in Btk-deficient animals. A higher dose did not provide additional reduction of intravascular crawling and neutrophil recruitment.Conclusions and implicationsPRN473, a highly selective inhibitor of Btk, potently attenuates sterile liver injury by inhibiting the activation of the β2 -integrin Mac-1 and subsequently neutrophil recruitment into inflamed tissue

The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation

Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown. By using spinning-disk confocal intravital microscopy, we examined the molecular mechanisms of leukocyte recruitment to sites of focal hepatic necrosis in vivo. We demonstrated that the Bruton’s tyrosine kinase (Btk) was required for multiple Mac-1 activation events involved in neutrophil recruitment and functions during sterile inflammation triggered by fMLF. The Src family kinase Hck, Wiskott-Aldrich-syndrome protein, and phospholipase Cγ2 were also involved in this pathway required for fMLF-triggered Mac-1 activation and neutrophil recruitment. Thus, we have identified a neutrophil Btk signalosome that is involved in a signaling pathway triggered by formylated peptides leading to the selective activation of Mac-1 and neutrophil recruitment during sterile inflammation