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

O Levy

R Sisson

J Kenyon

E Eichenwald

A Macone

D Goldmann

Departments of Medicine,^{General Clinical Research Center,}^{Laboratory Medicine,}^{and Infectious Disease,}^{Children's Hospital of Boston, Harvard Medical School,}^{and Division of Newborn Medicine, Brigham & Woman's Hospital,}^{Boston Massachusetts}

^{Corresponding author. Mailing address: Department of Medicine, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Phone: (617) 355-6369, ext. 1701. Fax: (617) 734-6152. E-mail:}ude.dravrah.hct.1a@o_yvel.

Received 2000 Feb 1; Revisions requested 2000 Apr 14; Accepted 2000 Jun 20.

Abstract

Innate defense against microbial infection requires the action of neutrophils, which have cytoplasmic granules replete with antibiotic proteins and peptides. Bactericidal/permeability-increasing protein (BPI) is found in the primary granules of adult neutrophils, has a high affinity for lipopolysaccharides (or “endotoxins”), and exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria. We have previously reported that neutrophils derived from newborn cord blood are deficient in BPI (O. Levy et al., Pediatrics 104:1327–1333, 1999). The relative deficiency in BPI of newborns raised the possibility that supplementing the levels of BPI in plasma might enhance newborn antibacterial defense. Here we determined the effects of addition of recombinant 21-kDa N-terminal BPI fragment (rBPI₂₁) on the growth and tumor necrosis factor (TNF)-inducing activity of representative gram-negative clinical isolates. Bacteria were tested in citrated newborn cord blood or adult peripheral blood. Bacterial viability was assessed by plating assay, and TNF-α release was measured by enzyme-linked immunosorbent assay. Whereas adult blood limited the growth of all isolates except Klebsiella pneumoniae, cord blood also allowed logarithmic growth of Escherichia coli K1/r and Citrobacter koseri. Bacteria varied in their susceptibility to rBPI₂₁'s bactericidal action: E. coli K1/r was relatively susceptible (50% inhibitory concentration [IC₅₀], ∼10 nM), C. koseri was intermediate (IC₅₀, ∼1,000 nM), Klebsiella pneumoniae was resistant (IC₅₀, ∼10,000 nM), and Enterobacter cloacae and Serratia marcescens were highly resistant (IC₅₀, >10,000 nM). All isolates were potent inducers of TNF-α activity in both adult and newborn cord blood. In contrast to its variable antibacterial activity, rBPI₂₁ consistently inhibited the TNF-inducing activity of all strains tested (IC₅₀, 1 to 1,000 nM). The antibacterial effects of rBPI₂₁ were additive with those of a combination of conventional antibiotics typically used to treat bacteremic newborns (ampicillin and gentamicin). Whereas ampicillin and gentamicin demonstrated little inhibition of bacterially induced TNF release, addition of rBPI₂₁ either alone or together with ampicillin and gentamicin profoundly inhibited release of this cytokine. Thus, supplementing newborn cord blood with rBPI₂₁ potently inhibited the TNF-inducing activity of a variety of gram-negative bacterial clinical pathogens and, in some cases, enhanced bactericidal activity. These results suggest that administration of rBPI₂₁ may be of clinical benefit to neonates suffering from gram-negative bacterial infection and/or endotoxemia.

Abstract

Host defense against bacterial invasion requires an innate immune system with the ability to respond to infection independent of prior exposure to the pathogen (16). As evidenced by the increased frequency and severity of infections in patients who are neutropenic, neutrophils are important cellular effectors of the innate immune system. Neutrophils exert microbicidal activity by at least three mechanisms: (i) generation of oxygen radicals by the phagocyte oxidase (1), (ii) generation of nitric oxide (36), and (iii) mobilization of antibiotic proteins and peptides which are stored in neutrophil cytosolic granules (14, 20).

Neutrophil microbicidal activity is diminished in a variety of clinical conditions. Chronic granulomatous disease is caused by mutations in the genes encoding components of the leukocyte oxidase enzyme (22). Specific granule deficiency is associated with decreased defensin content, but this disorder is associated with pleiotropic neutrophil abnormalities, leaving it uncertain as to whether an increased rate of infection is solely related to deficiency of these broadly cytotoxic peptides (13, 26).

The neutrophils of newborns, who are particularly susceptible to invasive bacterial infections, have also been found to function suboptimally (6, 29, 38). Although gram-positive bacteria (particularly group B streptococcus) cause the majority of bacterial infections in newborns, gram-negative bacteria account for up to 20 to 40% of newborn bacterial infection (2), are associated with a relatively high mortality rate (∼40% [31]), and have been increasing in incidence at some medical centers (30).

We have recently demonstrated that newborns are selectively deficient in a neutrophil antibiotic protein with selective activity against gram-negative bacteria: bactericidal/permeability-increasing protein (BPI). BPI is a 55-kDa protein found in primary (azurophilic) granules with high affinity for the lipid A moiety of gram-negative bacterial lipopolysaccharides (LPSs) (21). BPI exerts selective cytotoxic, antiendotoxic, and opsonic activities against gram-negative bacteria (12). As predicted by their lower mean BPI content, newborn neutrophils have relatively low bactericidal activity against the encapsulated serum-resistant pathogen Escherichia coli K1/r (21). This result suggested that the relatively low BPI content of newborn neutrophils may contribute to the increased risk to newborns of gram-negative sepsis. Moreover, this study raised the possibility that supplementing newborns with exogenous BPI might enhance their antibacterial and antiendotoxic activities. Importantly, BPI's action in blood is greatly enhanced by synergy with the membrane attack complex of the complement system (33), whose function is impaired in newborns by virtue of markedly lower levels of C3 as well as C8 and C9 (37). Thus, it is unknown whether the effects of BPI that have been demonstrated in adult whole blood (33) would also be manifested in newborn blood.

With these considerations in mind, we undertook the current study to determine the effect of addition of exogenous BPI on the survival and cytokine-inducing activity of gram-negative bacteria isolated from newborns with bacteremia clinically associated with sepsis syndrome. We decided to measure tumor necrosis factor alpha (TNF-α) as a marker of endotoxin-induced cytokine release, because this cytokine is known to be elevated in newborns with bacterial sepsis (3) and is believed to contribute to the pathophysiology of septic shock by damaging neonatal tissues (4, 10, 23). Exogenous BPI was provided in the form of rBPI₂₁, a recombinant modified N-terminal fragment which carries the antibacterial and antiendotoxic activities of the holoprotein (17, 24) and is currently being evaluated for potential clinical utility in meningococcemia (15) and other applications (12).

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health/National Center for Research Resources/General Clinical Research Center grant M01RR02172, an American Academy of Pediatrics Resident Research Award, and a grant from XOMA (US) LLC.

We acknowledge the support and help of the following groups and individuals: from Children's Hospital, Philip Pizzo, Frederick Lovejoy, and Joseph Majzoub for advice and encouragement; Dixon Yun for assistance with computer graphics; Cheryl Sweeney and Eileen Gorss for administrative support; and Irena Clark, Pamela Sale, and the technical staff of the Bacteriology Lab; from The Brigham & Woman's Hospital, the nursing, midwife, and obstetrical staff for assistance with cord blood collection; and from XOMA (US) LLC, Stephen Carroll and Patrick Scannon for advice and encouragement.

ACKNOWLEDGMENTS

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