ABSTRACT. Newborn infants are more susceptible to bacterial infections than adults. This susceptibility has been attributed to defects in humoral and cellular activity. Host cellular activity can be modified by factors produced by bacteria or the host in response to infection. We assessed the effect of two factors associated with gramnegative bacterial infection, lipopolysaccharide (LPS) and TNF-a, on polymorphonuclear neutrophilic granulocytes (PMN) obtained from adult or newborns (umbilical cord blood). PILIN were primed in vitro with L P S (10 pg/L) or TNF-a (lo-' ILI) for 45 min and then assessed, using a chemiluminescence (CL) assay a s a n indicator of oxidative radical production with formyl-methionyl-leucyl-phenylalanine as the trigger for C L initiation. C L activity of unprimed P h I N was similar for adults and newborns (13.3 and 13.7 C L units, respectively). After priming with LPS, C L activity was increased to 43.4 C L units for P b I N from adults but to only 17.6 C L units for P b I N from newborns ( p < 0.001, adults versus newborn increment). Priming of P h I N with L P S was most effective when autologous plasma was present. Using FITC-conjugated L P S and a flow cytometry assay, we could demonstrate no difference between the binding affinity of L P S for adult and newborn PMN. IIowever, formyl-methionyl-leucyl-phenylalanine binding studies indicated that adult P h I N had a higher number of binding sites. TNF-a priming of newborn PILIN was also ineffective. Adult P M N increased CL activity by 3.9-fold when primed with TNF-a, whereas newborn PILIN increased by only 1.75-fold ( p < 0.005). This priming deficiency was not attributable to TNF-a receptors because phycoerythrin-conjugated TNF-a was associated with P h I N from adults and newborns equally. Thus, P h I N from newborns are not primed effectively in vitro with L P S or TNF-a. This defect may contribute to neonatal susceptibility to bacterial infection. (Pediatr Res 34: 243-248, 1993) Abbreviations PhIN, polymorphonuclear neutrophilic granulocyte IFN-y, interferon-y TNF-a, tumor necrosis factor-a

Previous research in our laboratory has shown that polymorphonuclear leukocytes (PMN) from neonates are not primed effectively in vitro with lipopolysaccharide (LPS) (from Escherichia coli O111:B4) compared with priming of adult PMN. This finding led us to speculate that differences between neonatal and adult LPS receptors may account for the lower response by neonatal PMN to LPS. In these experiments, we investigated if CD14 or other LPS receptors contribute to the priming activity of PMN by LPS. We found that unprimed neonatal and adult PMN expressed similar numbers of CD14 (11.6 ؎ 9.2 versus 18.6 ؎ 2.7 fluorescence units [FlU]; P > 0.05) and LPS-binding sites (2.94 ؎ 1.4 versus 4.94 ؎ 0.79 FlU; P > 0.05). Monoclonal antibody against CD14 (MY4) did not significantly change the binding of LPS to adult unprimed PMN, suggesting that LPS receptors other than CD14 receptors are predominant on PMN. However, when PMN were pretreated with LPS (10 ng/ml) for 45 min at 37؇C, expression of CD14 on adult PMN increased to 33.8 ؎ 4.9 FlU (P < 0.05 versus unprimed adult PMN) while that on neonatal PMN showed little change, increasing to 17.2 ؎ 10.3 FlU (P > 0.05 versus unprimed neonatal PMN; P < 0.05 versus primed adult PMN). Furthermore, MY4 specifically blocked oxidative-radical production from PMN primed with LPS (10 ng/ml) compared with that from control PMN (P < 0.01). These studies suggest that LPS primes PMN by activating CD14 expression. We conclude that lower expression of CD14 or failure to up-regulate CD14 after LPS pretreatment contributes to the inability of neonatal PMN to be primed by LPS.

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We examined the production of tumor necrosis factor (TNF) by mononuclear cells (MNC) after incubating adult or cord blood MNC with Listeria monocytogenes in vitro. With adult MNC cultures, we found that TNF activity reached a peak at 6 h (606 +/- 120 x 10(3) units/liter) and declined to the baseline by day 3. In contrast, using cord blood MNC, we found that TNF activity increased gradually reaching a peak at 24 h. In addition, the peak TNF activity using newborn MNC (189 +/- 26 x 10(3) U/liter) at 24 h was still lower than the peak using adult MNC at 6 h (p < 0.0002). In seeking an explanation for the decreased TNF secretion from newborn MNC, we examined the possibility that newborn cells produce TNF but failed to secrete it. However, lysates of newborn cells contained functionally and antigenically less TNF than adult cells. Based on these observations, we conclude that the overall TNF production by newborn cells incubated with L monocytogenes is decreased compared with similarly stimulated adult cells.

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