Abstract Signaling through integrin receptors is required for optimal reactive oxygen species (ROS) production by polymorphonuclear neutrophils (PMN). The E3 ubiquitin ligase and adapter protein C-Cbl has been implicated in integrin-mediated adhesion, and is a substrate for integrin-stimulated protein tyrosine kinases. We hypothesized that c-Cbl regulates integrin-driven PMN functions. We observed tyrosine phosphorylation of c-Cbl after beta-2 integrin cross-linking in the NB4 myeloid cell line. To address the requirement for c-Cbl in primary PMN signaling, we utilized c-Cbl deficient mice. We found that while c-Cbl is dispensable for PMN maturation, signaling through both immunoreceptors and integrins on PMNs is modulated by c-Cbl. Upon stimulation of c-Cbl-deficient bone marrow-derived PMNs with beta-2 integrin ligands (poly-RGD or fibrinogen plus TNF-alpha), we observed an 80% decrease in ROS generation compared to stimulated control cells. Phorbol ester-driven ROS generation is unaffected by c-Cbl deficiency. We next addressed the biochemical mechanism of c-Cbl action, investigating MAPK, PI3K, and Ca2+dependent pathways downstream of integrins. We observed no defect in either ERK or Akt phosphorylation after integrin engagement in c-Cbl deficient PMNs; furthermore, we observed that poly-RGD stimulated ROS production is unimpaired in PMNs lacking the PI3K p110gamma subunit. However, c-Cbl-null PMNs displayed decreased phospholipase C phosphorylation. Together, these data strongly suggest that c-Cbl selectively regulates Ca2+driven signaling downstream of beta2 integrins and upstream of ROS generation in PMNs. Supported by the Minnesota Medical Foundation