Formation of focal adhesion-like structures in circulating human neutrophils after severe injury: triggering of a tissue-phase response in the vascular space

Abstract

Neutrophils play a key role in injury to the lung, kidney, liver, and gastrointestinal tract, often seen after major trauma. We evaluated the role of integrin-linked focal adhesions in the primed state, previously identified in peripheral blood neutrophils from severely injured patients. Immunoblot analysis of Triton-insoluble cell fractions revealed that total paxillin content was unchanged in comparison with that found in neutrophils from healthy volunteers, but phosphorylation of paxillin on tyrosine residue 118 was increased by more than 2-fold. Immunoprecipitation with antipaxillin and immunoblotting for proline-rich tyrosine kinase 2 (Pyk2) and for fgr showed significantly more colocalization. Densitometric analysis of total phosphotyrosine profiles also demonstrated significantly more in patient cells as compared with healthy cells. When allowed to adhere to fibronectin-coated plates, healthy and patient cells demonstrate a significant increase in tyrosine phosphorylation from that found in suspension-phase cells. Differential interference contrast microscopy of healthy neutrophils adherent to fibronectin matrices demonstrated rounded cells, without evidence of spreading; spreading was induced by addition of TNF-alpha. Patient neutrophils spread spontaneously, a response not further enhanced by TNF-alpha. Confocal imaging using anti-Pyk2 demonstrated aggregation of Pyk2 into punctate structures in patient but not in healthy cells. We conclude that neutrophils from severely injured patients are in a primed state, characterized by formation of focal adhesion-like structures. The identification of such structures in a clinical disease setting where they likely participate in unwanted consequences provides a novel area for study of regulation of neutrophil function.