Expression, purification, and characterization of the recombinant proform of eosinophil granule major basic protein.

The cDNA for the highly toxic eosinophil granule major basic protein (MBP) encodes a 25-kDa acidic precursor (proMBP) that is processed to form the 14-kDa mature MBP. To characterize the biochemical and biological properties of proMBP, and compare these to the known properties of MBP, we expressed recombinant proMBP in Chinese hamster ovary cells and purified the secreted form from supernatants. We developed a mAb specific for proMBP, J163-15E10, and by using a proMBP-specific RIA we found that recombinant proMBP was expressed quite efficiently at levels between 10 and 100 mg/l. By SDS-PAGE and immunoblotting analyses of bulk Chinese hamster ovary supernatants, recombinant proMBP was electrophoretically heterogeneous with an apparent molecular mass ranging from 3 x 10(4) to 1 x 10(5) daltons. Despite difficulties encountered because of the extreme molecular heterogeneity of the proform, two methods for purification of a predominant 33-kDa form of recombinant proMBP are presented. Glycosylation analysis of purified 33-kDa proMBP indicated that approximately 5 kDa is likely accounted for by the addition of one glycosaminoglycan group, three O-linked, and one N-linked complex type carbohydrate groups. Functional studies of purified recombinant proMBP were also conducted. Using amounts of proMBP determined to be optimal for MBP activity, it was shown that proMBP not only lacked the ability to inhibit protein synthesis in K562 cells, but it also lacked the ability to stimulate basophil histamine release or generate neutrophil superoxide anion release. Furthermore, proMBP inhibited in a dose-responsive manner the basophil histamine release and superoxide anion generation stimulated by MBP. The development of a mAb and RIA specific for proMBP will now make it possible to analyze biologic fluids for the presence of this protein, especially in pregnancy, when proMBP is increased.

Bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. LPS binding properties and effects on LPS-mediated cell activation.

We have previously shown that human bactericidal/permeability-increasing protein (BPI) is able to inhibit serum-dependent lipopolysaccharide (LPS)-mediated activation of human monocytes and neutrophils in vitro, and to counteract the lethal effects of LPS challenge in vivo. Lipopolysaccharide-binding protein (LBP) is a serum protein which participates in LPS-mediated activation of cells (Tobias, P. S., Mathison, J., Mintz, D., Lee, J. D., Kravchenko, V., Kato, K., Pugin, J., and Ulevitch, R. J. (1992) Am. J. Respir. Cell. Mol. Biol. 7, 239-245). We have proposed that BPI functions in a negative feedback loop which opposes this activation (Marra, M. N., Wilde, C. G., Collins, M. S., Snable, J. L., Thornton, M. B., and Scott, R. W. (1992) J. Immunol. 148, 532-537). We have now cloned and expressed recombinant forms of human BPI and LBP. Here we demonstrate that purified recombinant human LBP can replace the serum requirement for both LPS binding to human monocytes and LPS-mediated secretion of tumor necrosis factor alpha from these cells. These activities of LBP are inhibited by a neutralizing anti-CD14 monoclonal antibody. We further demonstrate that purified recombinant human BPI can inhibit LBP-mediated LPS binding to cells and their subsequent activation. Comparison of the LPS binding properties of BPI and LBP in enzyme-linked immunosorbent type assays and in the Limulus amebocyte lysate assay suggest that BPI has a stronger affinity for LPS than does LBP. Direct competition between BPI and LBP for LPS may explain the inhibition by BPI of the proinflammatory effects of LBP in the presence of LPS.

Endotoxin-binding and -neutralizing properties of recombinant bactericidal/permeability-increasing protein and monoclonal antibodies HA-1A and E5.

OBJECTIVE: To compare the endotoxin-binding and -neutralizing properties of bactericidal/permeability-increasing protein, the human monoclonal antiendotoxin antibody HA-1A, and the murine antiendotoxin antibody E5. DESIGN: Prospective, randomized, placebo-controlled laboratory study. SETTING: Biotechnology company research laboratory. SUBJECTS: Female CD-1 mice. INTERVENTIONS: Recombinant bactericidal/permeability-increasing protein, HA-1A, a human immunoglobulin M monoclonal antibody raised against Escherichia coli J5 (Rc) endotoxin, and E5, a murine immunoglobulin M monoclonal antibody raised against E. coli J5 endotoxin, were compared in the following assays: a) binding to rough lipopolysaccharide immobilized onto microtiter plates; b) inhibition of lipopolysaccharide activity in the limulus amebocyte lysate assay; c) inhibition of lipopolysaccharide-induced cytokine release in whole blood; and d) protection against lethal endotoxin challenge in CD-1 mice. MEASUREMENTS AND MAIN RESULTS: The binding affinity of bactericidal/permeability-increasing protein for immobilized lipopolysaccharide is apparently greater than the binding affinity of HA-1A or E5. Bactericidal/permeability-increasing protein neutralized lipopolysaccharide activity in the chromogenic limulus amebocyte lysate assay, while neither monoclonal antibody inhibited lipopolysaccharide activity. Similarly, bactericidal/permeability-increasing protein reduced lipopolysaccharide-mediated tumor necrosis factor production in human whole blood in vitro, whereas monoclonal antibodies had slight (HA-1A) or no (E5) effect on lipopolysaccharide activity in this system. Administration of bactericidal/permeability-increasing protein gave > 90% protection against an LD60 dose of endotoxin in CD-1 mice, while treatment with HA-1A or E5 did not improve survival rate. CONCLUSIONS: Neither monoclonal antibody was as effective as bactericidal/permeability-increasing protein at binding or neutralizing endotoxin in vitro or in vivo. The potent endotoxin-binding and -neutralizing properties of bactericidal/permeability-increasing protein indicate that it might be useful in the treatment of endotoxin-related disorders in humans.

The role of bactericidal/permeability-increasing protein as a natural inhibitor of bacterial endotoxin.

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.

Characterization of two azurphil granule proteases with active-site homology to neutrophil elastase.

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.

Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity.

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.

Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family.

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.