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, and its regulation by protein kinase C and by phosphatases 1 and 2A. Isolated PMNs from adult volunteers and healthy term newborns, i.e. umbilical 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 microm, range 9-21 microm) was significantly reduced compared with adult PMNs (median 23 microm, range 17-34 microm) (p < 0.001). Coincubation with the protein kinase C inhibitor bisindolylmaleimide GF109203X, did not significantly alter chemotaxis, whereas coincubation with the phosphatase inhibitors calyculin A or okadaic acid caused parallel dose-dependent inhibition of chemotaxis in adult and newborn PMNs. Peak actin polymerization was reduced in newborn compared with adult PMNs in response to stimulation with formyl-methionyl-leucyl-phenylalanine and zymosan-activated serum, but was normal in response to phorbol myristate acetate. Prior incubation for 5 min with bisindolylmaleimide GF109203X, calyculin A, or okadaic acid caused no significant alterations in the actin polymerization response to stimulation with formyl-methionyl-leucyl-phenylalanine. We conclude that: 1) newborn PMNs have reduced actin polymerization in response to stimulation with chemotactic agents which act via cell surface receptors, but not with phorbol myristate acetate, which acts directly in the cytoplasm. This suggests that a defect in cell signal transduction may be an underlying factor in defective newborn PMN chemotaxis. 2) Phosphatase inhibitors strongly inhibit chemotaxis but not actin polymerization, therefore phosphatases 1 and 2A may be important regulators of PMN chemotaxis, but this regulation takes place either at a point distal to actin polymerization or via another pathway. 3) Similar results in adult and newborn PMNs suggest that this is not the site of the underlying defect in newborn PMN chemotaxis.