Defective membrane potential changes in neutrophils from human neonates.

Abstract

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.

Impaired Leukotriene B4 Release by Neonatal Polymorphonuclear Leukocytes

“…This may explain our results if up-regulation is critical for the PMN response, which is not in agreement with other results previously reported (31). Other altered stimulus-response coupling mechanisms in N-PMN, such as the changes in membrane potential after chemotactic stimulation (32), could play a role in the low STZstimulated LTB, production.…”

Biochemical, Structural, and Functional Abnormalities of Polymorphonuclear Leukocytes in the Neonate

“…Strauss and Snyder (74) also reported employing the potential sensitive dye DiOC5(3). We (23) found that the binding of FMLP to PMNs from neonates was normal that the PMNs of neonates with defective chemotactic function at both 22 and 4" C, and that the affinity of the binding was failed to show the hyperpolarization response observed following normal at both temperatures. Based on the results of their studies, chemotactic factor stimulation of PMNs from adults (Fig.…”

“…Employing the low levels of this critical energy source (81). Thus, both an calcium-sensitive probe Quin 2/AM, we (23) found that the level of free calcium reached within the PMNs of neonates was lower 40 than that of the PMNs from adults following chemotactic factor stimulation (Fig. 2).…”

“…Following ligand-receptor interaction, a complicated series of events occurs that subsequently results in functional activity. These events include changes in membrane potential (2 1-23), the breakdown of phosphoinositol into inositol triphosphate and diacyl-glycerol (24), an increase in free intracellular calcium ion (Ca++) (23,25,26) provoked, in part, by inositol triphosphate, diacyl-glycerol-induced phosphokinase C activation (24), sodium ion (Na+) influx (27), alkalinization of the cytoplasm (28), and an increase in cell volume (27). Although, probably not directly involved in signal transduction of the PMN (29), there is also activation of adenylate cyclase and the generation of a transient elevation in cyclic 3'5' adenosine monophosphate (30, 3 1).…”

Impaired Surface Membrane Expression of C3bi but Not C3b Receptors on Neonatal Neutrophils

“…The normal increase in intracellular free calcium concentration that occurs upon stimulation of adult PMNs with f-MLP is also reduced in newborns' PMNs. (3). In addition, phagocytosis by neonatal cells has been shown to be deficient when limiting amounts of serum were used for opsonization of the test organisms (5,6).…”