Enhancement of neonatal innate defense: Effects of adding an N-terminal recombinant fragment of bactericidal/permeability-increasing protein on growth and tumor necrosis factor-inducing activity of gram-negative bacteria tested in neonatal cord blood ex vivo

Innate defense against microbial infection requires the action of neutrophi ls, which have cytoplasmic granules replete with antibiotic proteins and pe ptides. Bactericidal/permeability-increasing protein (BPI) is found in the primary granules of adult neutrophils, has a high affinity for lipopolysacc harides (or "endotoxins"), and exerts selective cytotoxic, antiendotoxic, a nd opsonic activity against gram-negative bacteria. We have previously repo rted that neutrophils derived from newborn cord blood are deficient in BPI (O. Levy et al., Pediatrics 104:1327-1333, 1999). The relative deficiency i n BPI of newborns raised the possibility that supplementing the levels of B PI in plasma might enhance newborn antibacterial defense. Here we determine d the effects of addition of recombinant 21-kDa N-terminal BPI fragment (rB PI(21)) on the growth and tumor necrosis factor (TNF)-inducing activity of representative gram-negative clinical isolates. Bacteria were tested in cit rated newborn cord blood or adult peripheral blood. Bacterial viability was assessed by plating assay, and TNF-alpha release was measured by enzyme-li nked immunosorbent assay. Whereas adult blood limited the growth of all iso lates except Klebsiella pneumoniae, cord blood also allowed logarithmic gro wth of Escherichia coli K1/r and Citrobacter koseri. Bacteria varied in the ir susceptibility to rBPI(21)'s bactericidal action: E. coli K1/r was relat ively susceptible (50% inhibitory concentration [IC50], similar to 10 nM), C. koseri was intermediate (IC50, similar to 1,000 nM), Klebsiella pneumoni ae was resistant (IC50, similar to 10,000 nM), and Enterobacter cloacae and Serratia marcescens were highly resistant (IC50, >10,000 nM). All isolates were potent inducers of TNF-alpha activity in both adult and newborn cord blood. In contrast to its variable antibacterial activity, rBPI(21) consist ently inhibited the TNF-inducing activity of all strains tested (IC50, 1 to 1,000 nM). The antibacterial effects of rEPI(21) were additive with those of a combination of conventional antibiotics typically used to treat bacter emic newborns (ampicillin and gentamicin). Whereas ampicillin and gentamici n demonstrated little inhibition of bacterially induced TNF release, additi on of rBPI(21) either alone or together with ampicillin and gentamicin prof oundly inhibited release of this cytokine. Thus, supplementing newborn card blood with rBPI(21) potently inhibited the TNF-inducing activity of a vari ety of gram-negative bacterial clinical pathogens and, in some cases, enhan ced bactericidal activity. These results suggest that administration of rBP I(21) may be of clinical benefit to neonates suffering from gram-negative b acterial infection and/or endotoxemia.