PHOSPHORYLATION AND THE ACTIN CYTOSKELETON IN DEFECTIVE NEWBORN NEUTROPHIL CHEMOTAXIS

We have investigated the role of actin polymerization in the defective polymorphonuclear neutrophil (PMN) chemotaxis of the human newborn, a nd its regulation by protein kinase C and by phosphatases 1 and 2A. Is olated PMNs from adult volunteers and healthy term newborns, i.e. umbi lical cord blood, were studied. Chemotaxis was measured by a modified micropore filter assay, and actin polymerization was assessed by flow cytometry. Chemotaxis of newborn PMNs (median 18 mu m, range 9-21 mu m ) was significantly reduced compared with adult PMNs (median 23 mu m, range 17-34 mu m) (P < 0.001). Coincubation with the protein kinase C inhibitor bisindolylmaleimide GF109203X, did not significantly alter c hemotaxis, whereas coincubation with the phosphatase inhibitors calycu lin A or okadaic acid caused parallel dose-dependent inhibition of che motaxis in adult and newborn PMNs. Peak actin polymerization was reduc ed in newborn compared with adult PMNs in response to stimulation with formyl-methionyl-leucyl-phenylalanine and zymosan-activated serum, bu t was normal in response to phorbol myristate acetate. Prior incubatio n for 5 min with bisindolylmaleimide GF109203X, calyculin A, or okadai c acid caused no significant alterations in the actin polymerization r esponse to stimulation with formyl-methionyl-leucyl-phenylalanine. We conclude that: 1) newborn PMNs have reduced actin polymerization in re sponse to stimulation with chemotactic agents which act via cell surfa ce receptors, but not with phorbol myristate acetate, which acts direc tly in the cytoplasm. This suggests that a defect in cell signal trans duction may be an underlying factor in defective newborn PMN chemotaxi s. 2) Phosphatase inhibitors strongly inhibit chemotaxis but not actin polymerization, therefore phosphatases 1 and 2A may be important regu lators of PMN chemotaxis, but this regulation takes place either at a point distal to actin polymerization or via another pathway. 3) Simila r results in adult and newborn PMNs suggest that this is not the site of the underlying defect in newborn PMN chemotaxis.