Resting murine neutrophils express functional alpha(4) integrins that signal through Src family kinases

There is mounting evidence that alpha (4) (CD49d) integrins are involved in neutrophil recruitment and function during inflammatory responses. We repo rt that all resting murine neutrophils derived from bone marrow or peripher al blood express easily detectable levels of alpha (4) integrins on their s urface. These alpha (4) integrins were functional, as demonstrated by stimu lation of respiratory burst when neutrophils adhered to surfaces coated wit h the murine vascular cell adhesion molecule-1 (mVCAM-1). Adhesion occurred via alpha (4) integrins, as preincubation of neutrophils with an anti-alph a (4)-specific Ab inhibited attachment to mVCAM-1. Direct crosslinking of t he alpha (4) integrin subunit by surface-bound mAbs also elicited superoxid e release and release of the secondary granule marker, lactoferrin, The fun ctional responses that occurred downstream of alpha (4) integrin cross-link ing required signaling by Src family kinases, Neutrophils derived from hck( -/-)fgr(-/-)lyn(-/-) triple-knockout or hck(-/-)fgr(-/-) double-knockout mi ce failed to undergo respiratory burst when plated on mVCAM-1. Triple mutan t neutrophils were also defective in release of both superoxide nd lactofer rin when plated on surfaces coated with mAbs directed against alpha (4). Co rrelated with impaired alpha (4)-induced functional responses, triple-mutan t neutrophils also failed to spread and tightly adhere to anti-alpha (4) mA b-coated surfaces. This is the first direct evidence that functional alpha (4) integrins are expressed by murine PMNs, acid that these surface molecul es can mediate cellular responses such as tight adhesion, spreading, sustai ned respiratory burst, and specific granule release in vitro, Moreover the alpha (4) integrins, like all other integrins tested, use the Src family ki nases to transduce intracellular signals.