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Endotoxin initiates the systemic inflammatory response, haemodynamic changes, and multi-organ failure that may occur as a consequence of systemic gram-negative bacterial infection. The serum protein lipopolysaccharide-binding protein (LBP) binds to the lipid A component of bacterial endotoxin and facilitates its delivery to the CD14 antigen on the macrophage, where inflammatory cytokines are released and a cascade of host mediators is initiated. The neutrophil granular protein bactericidal/permeability-increasing protein (BPI) competes with LBP for endotoxin binding and functions as a molecular antagonist of LBP-endotoxin interactions. We have measured concentrations of both proteins in body fluids from 49 consecutive patients. In 16 of 17 samples of fluid from closed-space infections, BPI was present in greater concentration than LBP (median BPI/LBP ratio 7.6 [95% CI 2.32-22.1]). The ratio of BPI and LBP was not significantly different from 1.0 in abdominal fluid from 10 patients with peritonitis (ratio 0.235 [0.18-0.47]), whereas the BPI/LBP ratio was low in 22 non-infected body fluids (0.01 [0.001-0.04]) and concentrations of both proteins approached those in normal human plasma. BPI concentrations were directly correlated with the quantity of neutrophils within clinical samples (rs = 0.81, p < 0.0001). Thus, within abscess cavities BPI is available in sufficient quantities for effective competition with LBP for endotoxin. BPI may attenuate the local inflammatory response and the systemic toxicity of endotoxin release during gram-negative infections.

Objectives: To study the toxicology and pharmacology of the endotoxin-neutralizing agent, bactericidal/permeability-increasing protein., Design: Prospective, randomized, placebo-controlled laboratory study., Setting: Academic research laboratory., Subjects: CD-1 mice (n = 259); Sprague Dawley rats (n = 26); New Zealand White rabbits (n = 19)., Interventions: Pharmacokinetics of intravenously injected bactericidal/permeability-increasing protein was assessed in mice. Toxicology was tested in mice and rats. Efficacy of intravenously administered bactericidal/permeability-increasing protein as an endotoxin-neutralizing agent was tested in mice, rats, and rabbits., Measurements and Main Results: Administration of a single 10-mg/kg bolus injection of bactericidal/permeability-increasing protein resulted in no alterations in hematologic, renal, or hepatic function, activity level, or weight gain in animals observed over a 7-day study period. A single bolus injection (10 mg/kg) of bactericidal/permeability-increasing protein protected 15 of 16 mice from a lethal endotoxin challenge (mortality rate 1/16 [6.25%]) compared with a 100% (16/16) mortality rate in the saline-treated controls (p < .001). Bactericidal/permeability-increasing protein administered up to 1 hr after endotoxin provided significant protection against lethal endotoxin challenge. Furthermore, bactericidal/permeability-increasing protein reduced the induration and dermal necrosis observed in the localized dermal Shwartzman reaction., Conclusions: Bactericidal/permeability-increasing protein is a potent antiendotoxin that neutralizes endotoxin in vivo and prevents mortality in animal models of lethal endotoxemia.

Objective: To evaluate changes in levels of polymorphonuclear leukocyte surface bactericidal/permeability-increasing protein (BPI), plasma BPI, and plasma lipopolysaccharide (LPS) binding protein (LBP) in normal human volunteers administered Escherichia coli LPS and in patients with sepsis and gram-negative infections., Design: Survey; case series., Setting: Clinical research center and surgical intensive care unit of a medical school and an associated tertiary care hospital., Patients or Other Participants: Volunteers (n = 10) screened prior to study by history and physical examination to exclude those with underlying diseases or hematologic abnormalities. Consecutive sample of surgical intensive care unit patients (n = 10) meeting criteria for sepsis syndrome with gram-negative infection. An additional patient with systemic inflammatory response syndrome but no gram-negative infection. All patients were studied on meeting the criteria. Three of the patients with sepsis syndrome and the patient with systemic inflammatory response syndrome were evaluated on recovery (approximately 25 days after initial study). Because these studies in volunteers and patients overlapped temporally, the control values were those of volunteers evaluated prior to LPS administration. No matching was employed., Measurements and Results: Compared with controls, LPS-challenged volunteers and patients with sepsis both exhibited significant granulocytosis (P < .01) and increased concentrations of polymorphonuclear leukocyte surface BPI (P < .01) and of plasma LBP (P < .01). Plasma BPI concentrations were increased (P < .01) in volunteers following LPS administration. There was a trend toward increased concentrations of plasma BPI in patients, but this was not significant relative to controls. Maximum concentrations of plasma LBP were approximately 250- and 3000-fold higher than plasma BPI concentrations in endotoxemic volunteers and in patients, respectively., Conclusions: Circulating polymorphonuclear leukocytes increase expression of BPI in response to LPS or gram-negative sepsis. Subsequently, concentrations of plasma BPI and LBP increase. Because both LBP and BPI bind to LPS, it is suggested that endogenously derived plasma levels of BPI are likely to be inadequate to compete for LPS binding to the much more abundant LBP in the circulation.

Bactericidal/permeability-increasing protein (BPI) is a natural constituent of human neutrophils. Recombinant BPI has been shown to bind to bacterial lipopolysaccharide (LPS), and to neutralize the ability of LPS to stimulate inflammatory cells in vitro and in vivo. BPI shares sequence homology and immunocrossreactivity with another endogenous LPS binding protein, lipopolysaccharide binding protein (LBP). Despite the homology, these proteins have opposite effects on LPS. LBP mediates cell activation by low, otherwise nonstimulatory concentrations, while BPI neutralizes LPS bioactivity. Exogenous LPS binding proteins in the form of monoclonal antibodies have been developed with the goal of generating antiendotoxin therapeutics to treat gram-negative sepsis and related syndromes. Here we show that LPS-binding and neutralizing properties of BPI compare favorably with two monoclonal antibodies tested, HA-1A and XMMEN-OE5. BPI also competes effectively with LBP for LPS. Thus, BPI may represent an endogenous LPS-regulatory molecule suitable for use as a potent antiendotoxin therapeutic.

Systemic release of endotoxin (LPS) after Gram-negative infection initiates a cascade of host cytokines that are thought to be the direct cause of shock, multisystem organ failure, and death. Endogenous LPS-binding proteins may play a role in regulating LPS toxicity in vivo. The human neutrophil granule protein bactericidal/permeability-increasing protein (BPI) shares sequence homology and immunocrossreactivity with an acute phase lipopolysaccharide binding protein (LBP) which has been shown to bind to LPS and accelerate LPS activation of neutrophils and macrophages. Although structurally similar, LBP and BPI are apparently functionally antagonistic. We previously showed that BPI inhibits LPS-mediated neutrophil activation in vitro. Here we demonstrate that BPI binds to LPS near the lipid A domain, and formation of the LPS-BPI complex abrogates detrimental host responses to LPS. For example, BPI blocks LPS-stimulated TNF release in vitro and in vivo, and LPS complexed to BPI is not pyrogenic in rabbits. Results demonstrating that BPI is released by stimulated human neutrophils further support the idea that BPI functions extracellularly in vivo to neutralize endotoxin. Taken together, these data argue that BPI neutralizes the toxic effects of LPS in vivo, and that BPI may represent a new therapeutic approach to the treatment of endotoxic shock.

The ontogeny of a 57-Kd cationic antimicrobial protein (CAP57) that has substantial similarities to bactericidal permeability increasing protein (BPI) has been determined immunocytochemically. CAP57 was detected in the granules of mature peripheral blood neutrophils. However, it was absent from other cells of the peripheral blood: eosinophils, red blood cells (RBCs), and mononuclear cells. In human bone marrow, CAP57 was confined to the neutrophilic series. The earliest stage of development of the myeloid cells at which CAP57 was demonstrated was the promyelocyte. Double immunofluorescent labeling showed that CAP57 was detected in cells positive for myeloperoxidase. The absence of lactoferrin in certain cells (promyelocytes) containing CAP57 indicated that CAP57 was synthesized and packaged in a population of granules prior to the development of granules that contain lactoferrin. CAP57 could not be demonstrated in HL60 cells either by enzyme-linked immunosorbent assay (ELISA) or by immunocytochemistry. However, the presence of another granule-associated cationic antimicrobial protein of molecular weight 37 Kd (CAP37) was readily detected in undifferentiated HL60 cells. Amino acid sequence analysis showed that CAP57 and BPI were identical. Further indication of the identity between CAP57 and BPI was that monoclonal anti-CAP57 antibodies cross reacted with BPI. Sucrose density-gradient centrifugations showed CAP57 was confined to a granule population that exhibited a buoyant density intermediate of the previously described light and heavy azurophil granules. Further resolution of the individual azurophil granule populations by Percoll density-gradient centrifugation revealed that CAP57 was most concentrated in the density range of 1.093 to 1.100 g/cc. These results strongly suggest the unique finding that CAP57 may be associated with a heretofore unreported granule type.

Much of the tissue damage associated with emphysema and other inflammatory diseases has been attributed to the proteolytic activity of neutrophil elastase, a major component of the azurophil granule. Recently, two additional azurophil granule proteins with NH2-terminal sequence homology to elastase were isolated (Gabay, J. E., Scott, R. W., Campanelli, D., Griffith, J., Wilde, C., Marra, M. N., Seeger, M., and Nathan, C. F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5610-5614) and designated azurophil granule protein 7 (AGP7) and azurocidin. Azurocidin and AGP7 represent significant protein components of the azurophil granule, together comprising approximately 15% of the acid-extractable protein as judged by reverse-phase high performance liquid chromatography analysis. AGP7 migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as four distinct glycoforms of molecular mass 28-34 kDa, whereas azurocidin exhibits three predominant bands with molecular mass of 28-30 kDa. Treatment of intact azurophil granules with [3H]diisopropyl fluorophosphate resulted in labeling of elastase, cathepsin G, and AGP7, whereas azurocidin was not labeled. Tryptic mapping of 3H-labeled AGP7 allowed us to identify and sequence the active-site polypeptide that has 70% identity to elastase over 20 residues. The active site peptide of azurocidin was also identified by sequence analysis of tryptic fragments and showed 65% identity to the active site of elastase. Surprisingly, the catalytic serine of azurocidin is replaced by glycine, explaining its inability to label with [3H]diisopropyl fluorophosphate. Thus, we have identified two azurophil proteins closely related to neutrophil elastase, one of which has apparently lost its proteolytic activity due to mutation of the catalytic serine.

Neutrophil granules contain proteins important in host defense against bacterial pathogens. Granule proteins released from activated neutrophils facilitate opsonization, phagocytosis, tissue digestion, and antimicrobial activity. Three similar, if not identical, neutrophil proteins, bactericidal/permeability-increasing protein (BPI), 57,000 m.w. cationic antimicrobial protein, and bactericidal protein have been described that specifically kill gram negative bacteria. Since LPS is a structure common to all gram-negative bacteria, we investigated whether the microbicidal protein BPI affects biologic activity of LPS in vitro. Human neutrophils can be activated both in vitro and in vivo by LPS. Upon stimulation, surface expression of CR1 and CR3 increases markedly. Using flow microfluorimetry, we analyzed surface expression of CR1 and CR3 as a measure of neutrophil stimulation in response to LPS. CR up-regulation on neutrophils was TNF independent, suggesting direct LPS stimulation of neutrophils in this system. Purified BPI completely inhibited CR up-regulation on neutrophils stimulated with both rough and smooth LPS chemotypes at 1.8 to 3.6 nM (100 to 200 ng/ml). By comparison, the polypeptide antibiotic polymyxin B completely inhibited the same dose of LPS at 0.4 nM. The inhibitory activity of BPI appeared to be specific for LPS because neutrophil stimulation by formylated peptide or TNF was unaffected. The specificity of BPI for LPS was further demonstrated by inhibition of LPS activity in the limulus amebocyte lysate assay. Therefore, the role of BPI in infection may not be limited to its microbicidal activity, but it may also regulate the neutrophil response to LPS.

Nine polypeptide peaks with antibiotic activity were resolved from human polymorphonuclear leukocyte azurophil granule membranes. All but 1 of the 12 constituent polypeptides were identified by N-terminal sequence analysis. Near quantitative recovery of protein and activity permitted an assessment of the contribution of each species to the overall respiratory-burst-independent antimicrobial capacity of the cell. Three uncharacterized polypeptides were discovered, including two broad-spectrum antibiotics. One of these, a defensin that we have designated human neutrophil antimicrobial peptide 4, was more potent than previously described defensins but represented less than 1% of the total protein. The other, named azurocidin, was abundant and comparable to bactericidal permeability-increasing factor in its contribution to the killing of Escherichia coli.

Primary (azurophil) granules of neutrophils contain proteins which play a major role in the killing and digestion of bacteria in the phagolysosome. We have isolated and characterized a novel antimicrobial peptide from the azurophil granule fraction of discontinuous Percoll gradients. We have named this peptide human neutrophil peptide 4 (HNP-4) based on its structural similarity to a group of antimicrobial polypeptides known as defensins (HNP 1-3). Using size exclusion and reverse-phase high performance liquid chromatography, HNP-4 was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. The amino acid sequence determined from isolated HNP-4 and from tryptic fragments of reduced and alkylated peptide is: NH2-Val-Cys-Ser-Cys-Arg-Leu-Val-Phe-Cys-Arg-Arg-Thr-Glu- Leu-Arg-Val-Gly-Asn-Cys-Leu-Ile-Gly-Gly-Val-Ser-Phe-Thr-Tyr-Cys-Cys-Thr- Arg-Val - COOH. Based on this sequence, HNP-4 has a calculated molecular weight of 3715 and a theoretical pI of 8.61. HNP-4 shows structural similarity to the family of three human defensins. HNP-4 and the defensins have identical cysteine backbones and, like the defensins, HNP-4 is rich in arginine (15.2 mol %). However, the amino acids at 22 of the 33 positions differ between HNP-4 and human defensins. Further, HNP-4 is significantly more hydrophobic than the defensins, as determined by its retention time on reverse-phase high performance liquid chromatography. In vitro, purified HNP-4 was shown to kill Escherichia coli, Streptococcus faecalis, and Candida albicans. Compared to a mixture of the other human defensins, HNP-4 was found to be approximately 100 times more potent against E. coli and four times more potent against both S. faecalis and C. albicans.