Differential activation of signal transduction pathways mediating phagocytosis, oxidative burst, and degranulation by chicken heterophils in responseto stimulation with opsonized Salmonella enteritidis

The activation of signal transduction pathways is required for the expressi on of functional enhancement of cellular activities. Tn the present studies , initial attempts were made to identify the signal transduction factors in volved in activating phagocytosis, generation of an oxidative burst, and de granulation by heterophils isolated from neonatal chickens in response to o psonized Salmonella enteritidis (opsonized SE). Peripheral blood heterophil s were isolated and exposed to known inhibitors of signal transduction path ways for either 20 min (staurosporin, genistein, or verapamil) or 120 min ( pertussis toxin) at 39 degreesC. The cells were then stimulated for 30 min at 39 degreesC with opsonized SE. Phagocytosis, luminol-dependent chemolumi nescence (LDCL), and beta -D glucuronidase release were then evaluated in v itro. The G-protein inhibitor pertussin toxin markedly inhibited (>80%) pha gocytosis of opsonized SE. Both the protein kinase inhibitor (staurosporin) and calcium channel inhibitor (verapamil) reduced phagocytosis in a dose r esponse manner. Genistein, a tyrosine kinase inhibitor, had no effect on ph agocytosis. Staurosporin had a marked inhibitory effect on LDCL (> 90%) whi le genistein had a dose responsive inhibition on LDCL. Both verapamil (40-4 5%) and pertussin toxin (50-55%) had a statistically significant, but less biologically significant effect on LDCL. Genistein significantly reduced th e degranulation (78-81%) of heterophils by opsonized SE. Staurosporin also reduced degranulation by 43-50%, but neither verapamil nor pertussis toxin had a significant effect on degranulation. These findings demonstrate that distinct signal transduction pathways differentially regulate the stimulati on of the functional activities of avian heterophils. Pertussin toxin-sensi tive, Ca++-dependent G-proteins appear to regulate phagocytosis of opsonize d SE, protein kinase C-dependent, tyrosine kinase-dependent protein phospho rylation plays a major role in LDCL, and tyrosine kinase(s)-dependent phosp horylation regulates primary granule release.