Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists

SUMMARY Newborn calves have a high susceptibility to bacterial infections, which may be related to the impaired neutrophil defense functions in newborns. The oxygen-dependent production of the free radical superoxide anion (O2-) represents an important part of the leukocyte respiratory burst central to neutrophil-directed defenses against bacterial infection. Because protein kinase C (pkc) activation is considered to be an important step in the signal transduction pathway for the O2- generating system, we compared O2- production by newborn and adult bovine neutrophils stimulated with 3 different pkc agonists. When the phorbol ester phorbol 12-myristate 13-acetate (pma) was used, pkc-dependent O2- generation from newborn neutrophils was significantly reduced (P < 0.01) for all concentrations of pma tested (10, 100, and 500 ng/ml). In addition, newborn neutrophils had a significantly (P < 0.01) reduced lag time for O2- generation. Similar significantly (P < 0.01) reduced O2- generation from newborn neutrophils was observed with an additional phorbol ester (phorbol 12, 13-dibutyrate); lag times were not calculated for phorbol 12, 13-dibutyrate. When O2- generation was stimulated with a synthetic diacylglycerol analogue (1,2-dioctanoyl-sn-glycerol), less O2- was generated from both adult and newborn neutrophils than was obtained with the phorbol esters, and newborn neutrophils produced significantly (P < 0.01) less O2- only at 50 μM 1,2-dioctanoyl-sn-glycerol. To assess the importance of pkc and cyclic nucleotide-dependent protein kinases in the O2--related signal-transduction pathways of bovine neutrophils, we determined the influence of a pkc inhibitor, H-7, and a cyclic nucleotide-dependent protein kinase inhibitor, HA-1004, on the respiratory burst of adult and newborn bovine neutrophils. Preincubation of neutrophils (15 minutes) with 100 or 500 μM H-7 inhibited subsequent pma-induced O2- generation from newborn and adult neutrophils in a dose-dependent fashion, but preincubation with HA-1004 did not affect subsequent pma-induced O2- generation from either newborn or adult neutrophils. The pkc inhibitor, H-7 (100 and 500 μM), induced a significantly (P < 0.01) prolonged lag time in adult, but not newborn, neutrophils; HA-1004 had no effect on lag time. These results indicate that pkc, but not cyclic nucleotide-dependent protein kinase, is important in the signal-transduction pathway leading to O2- generation in appropriately stimulated adult and newborn bovine neutrophils, and that deficient respiratory burst activity in newborn neutrophils may be explained by altered activation of pkc.