DEFECTIVE MEMBRANE POTENTIAL CHANGES IN NEUTROPHILS FROM HUMAN NEONATESBe FULVIO SACCHI AND HARRY R. HILLA number of studies have been carried out to determine the functional activity of polymorphonuclear leukocytes (PMN) from human neonates in an attempt to explain their unusual susceptibility to infection. Although neonatal PMN are capable of ingesting and killing microorganisms (1, 2), chemotaxis of these cells is markedly impaired in vitro and in vivo (3-7). The mechanism(s) involved in the pathogenesis of abnormal neonatal PMN chemotaxis are unknown. Miller (6) has reported that neonatal PMN have a decreased ability to deform following exposure to chemotactic stimuli, while Anderson and co-workers (7) found that neonatal PMN fail to form uropods and to redistribute adhesion sites. The decreased ability of these cells to change shape and form surface projections suggests that the functional defect may be at the level of the cell membrane. There does not appear to be a significant abnormality in the interaction between chemotactic factor and cell membrane-bound chemotactic factor receptors on resting neonatal PMN at 37°C (7,8). The defect may be, therefore, at a level beyond this initial interaction.The recent introduction of spectrofluorometric techniques for measuring critical ionic changes at the membrane and in the intracellular milieu has led to a better understanding of the events associated with phagocyte activation (9-14). Such probes include the cyanine dye 3-3 dipentyloxacarbocyanine [DiOCs(3)], which is sensitive to changes in membrane potential (11,12,15), and Quin 2/AM (13, 16), which assesses changes in intracellular free calcium. We have used these two fluorescent probes to assess the cellular events following chemotactic factor-receptor interaction in PMN from human neonates and compared the results with those obtained in PMN from adults. Materials and Methods PMN Preparations.Whole blood was obtained from peripheral veins of healthy adults and from the umbilical cords of normal-term neonates in acid citrate dextrose (Becton Dickinson and Co., Rutherford, N J). The blood was allowed to settle in 1% dextran (Pharmacia, Piscataway, N J) and the PMN were separated on Ficoil-Hypaque (Pharmacia) gradients. Contaminating erythrocytes were hypotonicaily lysed, and the PMN suspensions (98%) were washed in phosphate-buffered saline (PBS, pH 7.4) and suspended in a modified Hanks' balanced salt solution (HBSS) containing 124 mM NaCI, 4 mM KCI, 0.64 mM NaH~PO4, 0.66 mM K2HPO4, 15.2 mM NaHCOs, 10 mM Hepes buffer, 5.56 mM glucose, and 1.6 mM CaCI2 or 2 mM EGTA. The pH was adjusted to 7.4.

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In an attempt to determine the mechanism of the profound defect in chemotaxis observed in the neutrophils of human neonates, we have examined the generation of polymerized or filamentous actin (F actin) following stimulation of the cells with chemotactic factors. We have also examined the changes in the intracellular levels of free calcium in neonatal neutrophils and compared the results with those in adult neutrophils. Following exposure to formyl-methionyl-leucyl-phenylalanine (FMLP) or zymosan-activated serum (ZyAS), neutrophils from adult donors showed an increase in intracellular free calcium, as determined by Quin 2/AM fluorescence, and in actin polymerization (45–55%), as measured by nitrobenzoxadiazole phallicidin fluorescence. These responses were abolished by preincubation with the calcium antagonist verapamil (0.1 mM), which inhibits both calcium influx and release from intracellular stores. In marked contrast to the results obtained with neutrophils from adults, neutrophils from newborn infants, which have defective chemotactic responses, failed to generate F actin following FMLP or ZyAS stimulation and developed significantly lower levels of free intracellular calcium.