Resting Murine Neutrophils Express Functional α4 Integrins that Signal Through Src Family Kinases

Abstract

There is mounting evidence that α4 (CD49d) integrins are involved in neutrophil recruitment and function during inflammatory responses. We report that all resting murine neutrophils derived from bone marrow or peripheral blood express easily detectable levels of α4 integrins on their surface. These α4 integrins were functional, as demonstrated by stimulation of respiratory burst when neutrophils adhered to surfaces coated with the murine vascular cell adhesion molecule-1 (mVCAM-1). Adhesion occurred via α4 integrins, as preincubation of neutrophils with an anti-α4-specific Ab inhibited attachment to mVCAM-1. Direct cross-linking of the α4 integrin subunit by surface-bound mAbs also elicited superoxide release and release of the secondary granule marker, lactoferrin. The functional responses that occurred downstream of α4 integrin cross-linking required signaling by Src family kinases. Neutrophils derived from hck−/−fgr−/−lyn−/− triple-knockout or hck−/−fgr−/− double-knockout mice failed to undergo respiratory burst when plated on mVCAM-1. Triple mutant neutrophils were also defective in release of both superoxide and lactoferrin when plated on surfaces coated with mAbs directed against α4. Correlated with impaired α4-induced functional responses, triple-mutant neutrophils also failed to spread and tightly adhere to anti-α4 mAb-coated surfaces. This is the first direct evidence that functional α4 integrins are expressed by murine PMNs, and that these surface molecules can mediate cellular responses such as tight adhesion, spreading, sustained respiratory burst, and specific granule release in vitro. Moreover the α4 integrins, like all other integrins tested, use the Src family kinases to transduce intracellular signals.