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Effective host defense against microbial invasion requires an innate immune system whose response is both rapid and independent of prior exposure (25). The neutrophil is a central cellular effector of the innate immune system whose importance to host defense is manifest in the increased frequency and severity of infections in patients who have defects in neutrophil quantity or quality. The mechanisms by which neutrophils exert their antimicrobial activity have been under investigation since the seminal work of Eli Metchnikoff, who demonstrated the phagocytic activity of these cells at the beginning of the 20th century. Activated neutrophils increase oxygen consumption during inflammatory responses in what has been termed the “respiratory burst,” reflecting assembly of a multicomponent neutrophil oxidase which transfers electrons to molecular oxygen, forming toxic radicals (6). Defects in the phagocyte oxidase proteins underlie chronic granulomatous disease (CGD), which is characterized by increased frequency of infections with certain bacterial and fungal pathogens (38). Nitric oxide is another small, readily diffusible antimicrobial mediator generated by activated neutrophils (54). Over the past two decades, there has been increasing recognition of oxygen-independent killing mechanisms relating to the following observations: (i) neutrophils from CGD patients are capable of killing a variety of microorganisms (52), (ii) normal neutrophils deprived of oxygen in vitro are also able to efficiently kill certain bacterial pathogens (52), (iii) crude acid extracts of neutrophils possess direct microbicidal activity which is oxygen independent (24), and (iv) cationic proteins and peptides isolated from such extracts are able to directly kill microorganisms in vitro (29). Encouraged by these observations, investigators have made use of protein chromatography and molecular cloning in order to isolate, sequence, and define a growing number of neutrophil granule-derived antimicrobial proteins and peptides (15, 22, 23). Neutrophil protein and peptide antibiotics are deployed by degranulation either extracellularly into inflammatory fluids or intracellularly into the phagolysosome, thereby exposing microorganisms to high concentrations of these agents. The antimicrobial proteins and peptides share in common a net positive charge which contributes to electrostatic interactions with negatively charged microbial surface components. However, despite similar charges, these agents vary markedly in size and structure as well as the mechanisms and selectivities of their cytotoxic actions. Here the focus is on a remarkably selective anti-infective component of human neutrophils known as the bactericidal/permeability-increasing protein (BPI).

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 (rBPI 21 ) 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 rBPI 21 's bactericidal action: E. coli K1/r was relatively susceptible (50% inhibitory concentration [IC 50 ], ∼10 nM), C. koseri was intermediate (IC 50 , ∼1,000 nM), Klebsiella pneumoniae was resistant (IC 50 , ∼10,000 nM), and Enterobacter cloacae and Serratia marcescens were highly resistant (IC 50 , >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, rBPI 21 consistently inhibited the TNF-inducing activity of all strains tested (IC 50 , 1 to 1,000 nM). The antibacterial effects of rBPI 21 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 rBPI 21 either alone or together with ampicillin and gentamicin profoundly inhibited release of this cytokine. Thus, supplementing newborn cord blood with rBPI 21 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 rBPI 21 may be of clinical benefit to neonates suffering from gram-negative bacterial infection and/or endotoxemia.

Vascular endothelial injury is responsible for many of the clinical manifestations of severe meningococcal disease. Binding and migration of activated host inflammatory cells is a central process in vascular damage. The expression and function of adhesion molecules regulate interactions between leukocytes and endothelial cells. Little is known about how meningococci directly influence these receptors. In this study we have explored the effect of Neisseria meningitidis on endothelial adhesion molecule expression and found this organism to be a potent inducer of the adhesion molecules CD62E, ICAM-1, and VCAM-1. Exposure of endothelium to a serogroup B strain of Neisseria meningitidis , B1940, and a range of isogenic mutants revealed that lipooligosaccharide (LOS) structure and capsulation influence the expression of adhesion molecules. Following only a brief exposure (15 min) to the bacteria, there were large differences in the capacity of the different mutants to induce vascular cell adhesion molecules, with the unencapsulated and truncated LOS strains being most potent ( P < 0.05). Furthermore, the pattern of cell adhesion molecule expression was different with purified endotoxin from that with intact bacteria. Meningococci were more potent stimuli of CD62E expression than was endotoxin, whereas endotoxin was at least as effective as meningococci in inducing ICAM-1 and VCAM-1. The effect of bactericidal/permeability increasing protein (rBPI 21 ), an antibacterial molecule with antiendotoxin properties, was also dependent on LOS structure. The strains which possessed a truncated or nonsialylated LOS, whether capsulated or not, were more sensitive to the inhibitory effects of rBPI 21 . These findings could have important implications for the use of antiendotoxin therapy in meningococcal disease.

The activity of recombinant bactericidal/permeability-increasing protein (rBPI 21 ), alone or in combination with sulfadiazine, on the intracellular replication of Toxoplasma gondii was assessed in vitro and in mice with acute toxoplasmosis. rBPI 21 markedly inhibited the intracellular growth of T. gondii in human foreskin fibroblasts (HFFs). Following 72 h of exposure, the 50% inhibitory concentration of rBPI 21 for T. gondii was 2.6 μg/ml, whereas only slight cytotoxicity for HFF cells was observed at the concentrations tested. Subsequent mathematical analyses revealed that the combination of rBPI 21 with sulfadiazine yielded slight to moderate synergistic effects against T. gondii in vitro. Infection of mice orally with C56 cysts or intraperitoneally (i.p.) with RH tachyzoites resulted in 100% mortality, whereas prolongation of the time to death or significant survival ( P = 0.002) was noted for those animals treated with 5 to 20 mg of rBPI 21 per kg of body weight per day. Treatment with rBPI 21 in combination with sulfadiazine resulted in significant ( P = 0.0001) survival of mice infected i.p. with tachyzoites but not of mice infected orally with T. gondii cysts. These results indicate that rBPI 21 is active in vitro and in vivo against T. gondii and that its activity is significantly enhanced when it is used in combination with sulfadiazine. To our knowledge, this is the first report of the activity of rBPI 21 against a protozoan parasite.

As a consequence of their bactericidal actions, many antibiotics cause the release of endotoxin, a primary mediator of gram-negative sepsis. Bactericidal/permeability-increasing protein (BPI) has bactericidal activity and neutralizes endotoxin in vitro and in vivo. We sought to examine the effect of a recombinant N-terminal fragment of BPI (rBPI21) in conjunction with cefamandole, a cephalosporin antibiotic, in the treatment of Escherichia coli bacteremia and septic shock in rabbits. Cefamandole (100 mg/kg of body weight) was injected intravenously. This was followed by simultaneous 10-min infusions of E. coli O7:K1 (9 x 10(9) CFU/kg) and rBPI21 (10 mg/kg). rBPI21 was continuously infused for an additional 110 min at 10 mg/kg/h. The administration of rBPI21 in conjunction with the administration of cefamandole prevented the cefamandole-induced increase of free endotoxin in plasma, accelerated bacterial clearance, ameliorated cardiopulmonary dysfunction, and thereby, prevented death, whereas neither agent alone was protective in this animal model. The efficacy of the combined treatment with rBPI21 and cefamandole suggests a synergistic interaction between the two agents. The data indicate that rBPI21 may be useful in conjunction with traditional antibiotic therapy.

Human bactericidal/permeability-increasing protein (BPI) from neutrophils and a recombinant amino-terminal fragment, rBPI23, bind to and are cytotoxic for gram-negative bacteria both in vitro and ex vivo in plasma or whole blood. To function in vivo as an extracellular bactericidal agent, rBPI23 must act in the presence of the lipopolysaccharide-binding protein (LBP), which also binds to but has no reported cytotoxicity for gram-negative bacteria. LBP, which is present at 5 to 10 micrograms/ml in healthy humans and at much higher levels in septic patients, mediates proinflammatory host responses to gram-negative infection. On the basis of these previous observations, we have examined the effect of recombinant LBP (rLBP) on the bactericidal activity of rBPI23 against Escherichia coli J5 in vitro. Physiological concentrations of rLBP (5 to 20 micrograms/ml) had little or no bactericidal activity but reduced by up to approximately 10,000-fold the concentration of BPI required for bactericidal or related activities in assays which measure (i) cell viability as CFUs on solid media or growth in broth culture and (ii) protein synthesis following treatment with BPI. LBP also potentiated BPI-mediated permeabilization of the E. coli outer membrane to actinomycin D by about 100-fold but had no permeabilizing activity of its own. Under optimal conditions for potentiation, fewer than 100 BPI molecules were required to kill a single E. coli J5 bacterium.

A recombinant 23-kDa amino-terminal fragment of human bactericidal/permeability-increasing protein (rBPI23), a potent lipopolysaccharide (LPS)-binding/neutralizing protein, was used as a probe to assess the role of endotoxin in the acute inflammatory responses elicited by gram-negative bacteria in rat subcutaneous air pouches. In initial experiments, rBPI23 prevented the Escherichia coli O111:B4 LPS-induced accumulation of polymorphonuclear leukocytes (PMN), tumor necrosis factor alpha (TNF-alpha), and nitrite (a stable end product of nitric oxide formation) in exudate fluids. Significant inhibition of TNF-alpha production was still evident when rBPI23 treatment was delayed for 30 min after LPS instillation. In subsequent experiments, rBPI23 also prevented the nitrite and early (2-h) TNF-alpha accumulation induced by three different strains of formaldehyde-killed gram-negative bacteria (E. coli O7:K1, E. coli O111:B4, and Pseudomonas aeruginosa 12.4.4) but did not inhibit the PMN or late (6-h) TNF-alpha accumulation induced by these bacteria. As with LPS challenge, a significant inhibition of early TNF-alpha production was still evident when rBPI23 treatment was delayed for 30 to 60 min after instillation of killed bacteria. The results indicate that in this experimental model the NO and early TNF-alpha responses to gram-negative bacterial challenge are mediated predominantly by endotoxin, whereas the PMN and late TNF-alpha responses may be mediated by other bacterial components. Moreover, the results indicate that rBPI23 can inhibit the bacterially induced production of certain potentially harmful mediators (TNF-alpha and NO) without entirely blocking the host defense, i.e., PMN response, against the bacteria.

The bactericidal/permeability-increasing protein (BPI) is an antibiotic- and endotoxin-neutralizing protein of granulocytes and epithelial cells. Constitutive expression of BPI, which increases upon interleukin 4 stimulation, by human dermal fibroblast was demonstrated, suggesting an important role of BPI in gram-negative bacterial clearance and a dampened response to endotoxin in the skin.

In this study, the efficacy of bactericidal/permeability-increasing protein (BPI) was assessed in a rat model of chronic otitis media with effusion. BPI injection prevented disturbance of the mucociliary clearance system of the middle ear. Hence, it is postulated that BPI can be a new therapy for chronic otitis media with effusion.

Bactericidal/permeability-increasing protein (BPI) inhibited growth of cell wall-deficient Acholeplasma laidlawii and L forms of certain strains of Staphylococcus aureus and Streptococcus pyogenes . However, the same strains of S. aureus and S. pyogenes with intact cell walls were not susceptible to the growth-inhibitory effects of BPI.

We have shown previously that the sensitivity of Escherichia coli to the neutrophil bactericidal/permeability-increasing protein (BPI) depends mainly on the polysaccharide chain length of outer membrane lipopolysaccharides (LPS) (J. Weiss, S. Beckerdite-Quagliata, and P. Elsbach, J. Clin. Invest. 65:619-628, 1980). Thus, rough strains of E. coli producing only short-chain LPS are more sensitive to BPI than smooth strains that produce LPS with varied chain lengths. We now show that changes in the bacterial growth environment can modify BPI sensitivity of smooth E. coli as much as 30-fold depending on the bacterial strain and the growth conditions examined. Changes in BPI sensitivity paralleled differences in binding affinity of E. coli for BPI and closely correlated with changes in the chain length of LPS produced under different growth conditions, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No concomitent changes in either the number of LPS molecules per cell or the bacterial protein profile were detected. Rough strains showed little or no growth-dependent variation in BPI sensitivity, further indicating that subtle alterations in bacterial constituents other than LPS do not significantly affect bacterial sensitivity to BPI. Thus, the BPI sensitivity of E. coli can be modulated not only by the genotypic conversion of the LPS phenotype, but also by environmental effects on LPS-polysaccharide formation in wild-type strains.