Biomedical subjects

P Elsbach

Publications and source records attributed to P Elsbach.

At least 19 recordsLinked to original sources

PubMed1998

The bactericidal/permeability-increasing protein (BPI) in antibacterial host defense.

The bactericidal/permeability-increasing protein (BPI) is a 456-residue cationic protein produced only by precursors of polymorphonuclear leukocytes (PMN) and is stored in the primary granules of these cells. The potent (nM) cytotoxicity of BPI is limited to gram-negative bacteria (GNB), reflecting the high affinity (<10 nM) of BPI for bacterial lipopolysaccharides (LPS). The biological effects of isolated BPI are linked to complex formation with LPS. Binding of BPI to live bacteria via LPS causes immediate growth arrest. Actual killing coincides with later damage to the inner membrane. Complex formation of BPI with cell-associated or cell-free LPS inhibits all LPS-induced host cell responses. BPI-blocking antibodies abolish the potent activity of whole PMN lysates and inflammatory fluids against BPI-sensitive GNB. The antibacterial and the anti-endotoxin activities of BPI are fully expressed by the amino terminal half of the molecule. These properties of BPI have prompted preclinical and subsequent clinical testing of recombinant amino-terminal fragments of BPI. In animals, human BPI protein products protect against lethal injections of isolated LPS and inocula of GNB. Phase I trials in healthy human volunteers and multiple Phase I/II clinical trials have been completed or are in progress (severe pediatric meningococcemia, hemorrhagic trauma, partial hepatectomy, severe peritoneal infections, and cystic fibrosis) and two phase III trials (meningococcemia and hemorrhagic trauma) have been initiated. In none of >900 normal and severely ill individuals have issues of safety or immunogenicity been encountered. Preliminary evidence points to overall benefit in BPI-treated patients. These results suggest that BPI may have a place in the treatment of life-threatening infections and conditions associated with bacteremia and endotoxemia.

Animals↗

PubMed1998

Role of the bactericidal/permeability-increasing protein in host defence.

Much has been learned recently about the structure and function of 55 kDa bactericidal/permeability-increasing protein (BPI), a member of a genomically conserved lipid-interactive protein family. Analysis of BPI fragments and the crystal structure of human BPI have established that BPI consists of two functionally distinct domains: a potently antibacterial and anti-endotoxin amino-terminal domain (approximately 20 kDa) and a carboxy-terminal portion that imparts opsonic activity to BPI. A recombinant amino-terminal fragment (rBPI21) protects animals against the effects of Gram-negative bacteria and endotoxin. In man, rBPI21 is nontoxic and non-immunogenic and is in Phase II/III clinical trials with apparent therapeutic benefit.

Animals↗

PubMed1997

An opsonic function of the neutrophil bactericidal/permeability-increasing protein depends on both its N- and C-terminal domains.

The host response to Gram-negative bacterial infection is influenced by two homologous lipopolysaccharide (LPS)-interactive proteins, LPS-binding protein (LBP) and the bacteridical/permeability-increasing protein (BPI). Both proteins bind LPS via their N-terminal domains but produce profoundly different effects: BPI and a bioactive N-terminal fragment BPI-21 exert a selective and potent antibacterial effect upon Gram-negative bacteria and suppress LPS bioactivity whereas LBP is not toxic toward Gram-negative bacteria and potentiates LPS bioactivity. The latter effect of LBP requires the C-terminal domain for delivery of LPS to CD14, so we postulated that the C-terminal region of BPI may serve a similar delivery function but to distinct targets. LBP, holoBPI, BPI-21, and LBP/BPI chimeras were compared for their ability to promote uptake by human phagocytes of an encapsulated, phagocytosis-resistant strain of Escherichia coli. We show that only bacteria preincubated with holoBPI are ingested by neutrophils and monocytes. These findings suggest that, when extracellular holoBPI is bound via its N-terminal domain to Gram-negative bacteria, the C-terminal domain promotes bacterial attachment to neutrophils and monocytes, leading to phagocytosis. Therefore, analogous to the role of the C-terminal domain of LBP in delivery of LPS to CD14, the C-terminal domain of BPI may fulfill a similar function in BPI-specific disposal pathways for Gram-negative bacteria.

Antimicrobial Cationic Peptides↗

PubMed1997

Lipopolysaccharide (LPS)-binding proteins BPI and LBP form different types of complexes with LPS.

Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are closely related LPS-binding proteins whose binding to LPS has markedly different functional consequences. To gain better insight into the possible basis of these functional differences, the physical properties of LBP-LPS and BPI-LPS complexes have been compared in this study by sedimentation, light scattering, and fluorescence analyses. These studies reveal dramatic differences in the physical properties of LPS complexed to LBP versus BPI. They suggest that of the two proteins, only LBP can disperse LPS aggegates. However, BPI can enhance both the sedimentation velocity and apparent size of LPS aggregates while inhibiting LPS-LBP binding even at very low (1:40 to 1:20) BPI:LPS molar ratios.

Acute-Phase Proteins↗

PubMed1997

p15s (15-kD antimicrobial proteins) are stored in the secondary granules of Rabbit granulocytes: implications for antibacterial synergy with the bactericidal/permeability-increasing protein in inflammatory fluids.

The bactericidal potency toward complement-resistant Escherichia coli of bactericidal/permeability-increasing protein (BPI) released from polymorphonuclear leukocytes (PMNs) in glycogen-induced inflammatory peritoneal exudates of rabbits is dependent on synergy with extracellular p15s. This synergy depends on the high molar ratio of p15s to BPI in the extracellular fluid (approximately 50:1), which greatly exceeds the intracellular ratio (approximately 5:1). To explore the possible basis of the greater accumulation of p15s in inflammatory fluid, we examined the subcellular localization of BPI and p15 in PMNs. Immunogold electron microscopy confirmed the storage of BPI in primary granules and showed that p15s are stored in secondary granules. Reverse-transcription polymerase chain reaction of density-fractionated rabbit bone marrow cells verified that p15s are expressed later than BPI during myeloid differentiation. As the inflammatory response evolves, p15 mRNA appears earlier in blood and exudate cells than mRNA for BPI, consistent with release of progressively less mature precursors from bone marrow. Finally, Ca(2+)-ionophore-mediated exocytosis of p15s occurs more readily than release of BPI. We therefore propose that localization of a synergistic partner of BPI (p15s) in more readily released secondary granules allows the neutrophil to mobilize potent BPI-dependent antibacterial activity extracellularly without significant depletion of intracellular BPI stores.

Animals↗

PubMed1996

The potent anti-Staphylococcus aureus activity of a sterile rabbit inflammatory fluid is due to a 14-kD phospholipase A2.

The cell-free fluid (ascitic fluid, AF) of a sterile inflammatory peritoneal exudate elicited in rabbits is potently bactericidal for complement-resistant gram-negative as well as gram-positive bacterial species. This activity is absent in plasma. We now show that essentially all activity in AF against Staphylococcus aureus is attributable to a group II 14-kD phospholipase A2 (PLA2), previously purified from AF in this laboratory. Antistaphylococcal activity of purified PLA2 and of whole AF containing a corresponding amount of PLA2 was comparable and blocked by anti-AF-PLA2 serum. At concentrations present in AF (approximately 10 nM), AF PLA2 kills > 2 logs of 10(6) S. aureus/ml, including methicillin-resistant clinical isolates, and other species of gram-positive bacteria. Human group II PLA2 displays similar bactericidal activity toward S. aureus (LD90 approximately 1-5 nM), whereas 14-kD PLA2 from pig pancreas and snake venom are inactive even at micromolar doses. Bacterial killing by PLA2 requires Ca2+ and catalytic activity and is accompanied by bacterial phospholipolysis and disruption of the bacterial cell membrane and cell wall. These findings reveal that group II extracellular PLA2, the function of which at inflammatory sites has been unclear, is an extraordinarily potent endogenous antibiotic against S. aureus and other gram-positive bacteria.

Animals↗

PubMed1996

Potent CD14-mediated signalling of human leukocytes by Escherichia coli can be mediated by interaction of whole bacteria and host cells without extensive prior release of endotoxin.

How invading microorganisms are detected by the host has not been well defined. We have compared the abilities of Escherichia coli and lipopolysaccharides (LPS) purified from these bacteria to prime isolated neutrophils for phorbol myristate acetate-stimulated arachidonate release, to trigger respiratory burst in 1% blood, and to increase steady-state levels of tumor necrosis factor alpha mRNA in whole blood. In all three assays, bacteria were > or = 10-fold more potent than equivalent amounts of LPS and could trigger maximal cellular responses at ratios as low as one bacterium per 20 to 200 leukocytes. Both E. coli and LPS-triggered responses were enhanced by LPS-binding protein and inhibited by an anti-CD14 monoclonal antibody and the bactericidal/permeability-increasing protein (BPI). However, whereas O polysaccharide did not affect the potency of isolated LPS, intact E. coli carrying long-chain LPS (O111:B4) was less potent than rough E. coli (J5). Furthermore, material collected by filtration or centrifugation of bacteria incubated under conditions used to trigger arachidonate release or chemiluminescence was 5- or 30-fold less active, respectively, than whole bacterial suspensions. Extracellular BPI (not bound to bacteria) inhibited bacterial signalling, but BPI bound to bacteria was much more potent. Taken together, these findings indicate that E. coli cells can strongly signal their presence to human leukocytes not only by shedding LPS into surrounding fluids but also by exposing endotoxin at or near their surface during direct interaction with host cells.

Acute-Phase Proteins↗

PubMed1995

Antibacterial proteins of granulocytes differ in interaction with endotoxin. Comparison of bactericidal/permeability-increasing protein, p15s, and defensins.

Bactericidal/permeability-increasing protein (BPI), antibacterial 15-kDa protein isoforms (p15s), and defensins (neutrophil peptides or NPs) are granule-associated antibacterial proteins of polymorphonuclear leukocytes (PMN) that have both direct and synergistic growth inhibitory activity against Gram-negative bacteria. In this study, we have compared in vitro the abilities of these antibacterial proteins, alone and in combination, to inhibit the endotoxic activity of isolated LPS and whole bacteria. All three proteins blocked endotoxin activity in: 1) the Limulus amoebocyte lysate assay, 2) priming of PMN for enhanced arachidonate release, and 3) stimulating leukocyte oxidase activity in 1% blood. However, the proteins differ markedly in both relative potency (BPI >> p15s = NP1) in the presence of the plasma LPS-binding protein and in the range of LPS chemotypes that can be inhibited. BPI potently neutralizes LPS of any chemotype, but p15s and defensins are less active against long-chain (S-type) LPS. In whole blood ex vivo, the p15s and NP1 are approximately 1000-fold less potent than BPI, but at subinhibitory doses act in synergy with BPI to inhibit the TNF-inducing activity of a serum-resistant encapsulated strain of Escherichia coli (K1/r). The anti-endotoxic effects of p15 and NP1 against E. coli K1/r in whole blood appear secondary to growth arrest, because, in marked contrast to BPI, they are not evident against nonviable bacteria (pretreated with antibiotic) nor isolated LPS. Thus, BPI stands out for its ability to inhibit isolated or bacterial LPS under physiologic conditions. However, p15s and defensins may also contribute to suppression of endotoxic signaling by Gram-negative bacteria via synergistic (with BPI) growth inhibition upon extracellular release of these proteins from PMN during inflammation.

Animals↗

PubMed1995

Extracellular accumulation of potently microbicidal bactericidal/permeability-increasing protein and p15s in an evolving sterile rabbit peritoneal inflammatory exudate.

To what extent the host defense role of granule-associated antibacterial proteins and peptides of PMN includes extracellular action has not been established. To address this question, we have analyzed the antibacterial activity of cell-free (ascitic) fluid (AF) obtained from glycogen-induced sterile inflammatory rabbit peritoneal exudates in which > 95% of the accumulating cells are PMN. AF, but not plasma collected in parallel, exhibits potent activity toward serum-resistant Gram-negative and Gram-positive bacteria. Total and specific antibacterial activity of AF increases during the first 12 h after injection of glycogen in parallel with the influx of PMN. At maximum, > 99% of 10(7) encapsulated Escherichia coli and Staphylococcus aureus are killed in 30 min/ml of AF. Neutralizing antibodies against the bactericidal/permeability-increasing protein (BPI) of PMN abolishes activity of AF toward encapsulated E. coli but has no effect on activity vs staphylococci. However, BPI alone (approximately 1 microgram/ml in AF) can only account for < or = 20% of AF activity toward E. coli. AF also contains 15 kD PMN proteins (p15s) that act in synergy with BPI. Purified BPI and p15s, in amounts present in AF, reconstitute the growth-inhibitory activity of AF toward encapsulated E. coli. These findings show for the first time an extracellular function of endogenous BPI, providing, together with the p15s, a potent microbicidal system toward Gram-negative bacteria resistant to plasma-derived proteins and phagocytes in inflammatory exudates.

Animals↗

PubMed1995

Prospects for use of recombinant BPI in the treatment of gram-negative bacterial infections.

The bactericidal/permeability-increasing protein (BPI), a potent cytotoxin specific for Gram-negative bacteria and an endotoxin-neutralizing agent, is a major component of the antimicrobial arsenal of mammalian polymorphonuclear leukocytes. The antibacterial and endotoxin-neutralizing activities of the N-terminal portion (approximately 25 kDa) of BPI are at least equal to those of the holoprotein (approximately 50 kDa). Recombinant N-terminal fragments of BPI are antibacterial and inhibit host cell responses to endotoxin in whole blood ex vivo and in animal experiments. BPI administered to both animals and man is apparently nontoxic and nonimmunogenic and acts synergistically with some antibiotics. Thus, the prospects for the therapeutic use of bioactive BPI fragments in serious Gram-negative bacterial infections are highly encouraging.

Animals↗

PubMed1994

Structural determinants of the action against Escherichia coli of a human inflammatory fluid phospholipase A2 in concert with polymorphonuclear leukocytes.

Extracellular 14-kDa phospholipases A2 (PLA2) in inflammatory exudates can contribute to bacterial phospholipid (PL) degradation during phagocytosis of Escherichia coli by polymorphonuclear leukocytes (PMN) and are highly active toward E. coli treated with the bactericidal/permeability-increasing protein (BPI) purified from PMN. PLA2 activity toward BPI-treated E. coli varies greatly among members of this conserved family of enzymes and apparently depends on a cluster of basic residues in a variable surface region near the NH2 terminus for recognition of this biological target (Weiss, J., Wright, G.W., Bekkers, A.C.A.P.A., van den Bergh, C.J., and Verheij, H.M. (1991) J. Biol. Chem. 266, 4162-4167). We have examined by site-specific mutagenesis of a recombinant PLA2 that is identical to an enzyme in human synovial fluid (containing His-6, Arg-7, Lys-10, and Lys-15 and a global net charge of +15) the role of basic residues in this region in PLA2 action against PLA-deficient (pldA-) E. coli. Substitution of Ser for Arg-7 +/- Gln for Lys-15 caused, respectively, about a 10- and 25-fold reduction in BPI-dependent PLA2 binding and activity to E. coli, but had no effect on hydrolysis of PL of autoclaved E. coli or dispersions of purified PL. PL degradation during phagocytosis was increased after pretreatment of E. coli (or PMN) with wild-type PLA2 followed by removal of unbound PLA2. Thus, the PLA2 binds to cells before phagocytosis followed by internalization of the enzyme along with E. coli and intracellular action. Mutant (e.g. R7S +/- K15Q) PLA2 show the same BPI-independent binding to E. coli as the wild-type enzyme but 10-30-fold reduced activity during phagocytosis, reflecting lower intracellular activity of these enzymes. Thus, structural determinants first implicated in PLA2 action toward E. coli treated with purified BPI apparently are also important in the intracellular action of PLA2 during phagocytosis by PMN.

Animals↗

PubMed1994

Bactericidal permeability-increasing protein in host defence against gram-negative bacteria and endotoxin.

The bactericidal permeability-increasing protein (BPI) is a highly conserved host-defence molecule produced and stored by myeloid cells only and a major constituent of the primary granules of human and rabbit polymorphonuclear leukocytes. The c. 50 kDa BPI and a c. 23 kDa bioactive N-terminal fragment are cytotoxic only for Gram-negative bacteria. This target-cell specificity reflects the high affinity (apparent Kd: 1-10 nM) of BPI for the lipid A portion of lipopolysaccharide (LPS or endotoxin). Native and recombinant (r) holo-BPI and the N-terminal fragment (rBPI-23) bind with equal affinity to all forms of isolated LPS examined and inhibit the numerous biological effects of LPS in vitro (including in whole blood ex vivo) as well as in animals. Under the same conditions the antibacterial potencies of holo-BPI and rBPI-23 against Gram-negative bacteria with rough chemotype LPS (whether encapsulated or not) are also the same, but against more resistant smooth chemotype Gram-negative bacteria rBPI-23 is up to 30-fold more potent than holo-BPI. Holo-BPI and rBPI-23 protect a broad range of animals against lethal cytotoxic effects of LPS and in some cases against lethal inoculations with live Gram-negative bacteria.

Animals↗

PubMed1994

Integration of antimicrobial host defenses: role of the bactericidal/permeability-increasing protein.

Our understanding of the complex and integrated host-defense systems against microbial infection has progressed rapidly with the characterization of individual components. However, the various factors must be studied not only in isolation, but also in a closer approximation to the in vivo situation, where these factors interact. This is well illustrated in recent studies of the role of the bactericidal/permeability-increasing protein.

Animals↗

PubMed1994

Effect of lipopolysaccharide (LPS) chain length on interactions of bactericidal/permeability-increasing protein and its bioactive 23-kilodalton NH2-terminal fragment with isolated LPS and intact Proteus mirabilis and Escherichia coli.

The target-specific cytotoxicity for gram-negative bacteria and the endotoxin-neutralizing activity of the 55-kDa bactericidal/Permeability-increasing protein (BPI) and its bioactive 23-kDa NH2-terminal fragment depend on the strong attraction of BPI for the lipid A region of lipopolysaccharides (LPS). We have shown before that smooth gram-negative bacteria with long-chain LPS are more resistant to BPI (especially holo-BPI) than are rough strains. It has been suggested that the high BPI resistance of some gram-negative bacteria, such as Proteus mirabilis, might also reflect the structural diversity of lipid A. To explore this possibility, we compared the antibacterial activity and binding of natural and recombinant holo-BPI and a recombinant NH2-terminal fragment (rBPI-23) to an isogenic rough (Re-LPS chemotype) and a smooth (S-LPS chemotype) strain of P. mirabilis and to LPS isolated from the two strains. Holo-BPI and rBPI-23 were both potently active against the Re strain of P. mirabilis (90% lethal dose, 20 nM). In contrast, the smooth strain was > or = 100 times more resistant to holo-BPI but only 10 times more resistant to rBPI-23. rBPI-23 was also more potent against several Escherichia coli strains from clinical bacteremia isolates. Differences in the antibacterial potency of BPI toward the Re and S strains of P. mirabilis correlated with differences in the binding of holo-BPI and rBPI-23 to these bacteria. In contrast, the binding of biosynthetically (in vitro transcribed and translated) 35S-labeled holo-BPI and NH2-terminal fragment to isolated Re- and S-LPS from P. mirabilis in solution was similar. Moreover, in the Limulus amebocyte lysate assay, holo-BPI and rBPI-23 potently neutralized both forms of LPS with equal effectiveness. Together, these results strongly suggest that BPI recognizes Proteus lipid A and that the relative resistance of (smooth) P. mirabilis to holo-BPI is due to the inhibitory effect of long polysaccharide chains of tightly packed LPS in the envelope.

Antimicrobial Cationic Peptides↗

PubMed1994

Bacterial lipopolysaccharide primes human neutrophils for enhanced release of arachidonic acid and causes phosphorylation of an 85-kD cytosolic phospholipase A2.

Production of leukotriene B4 (LTB4) by human neutrophils (PMN) in response to different stimuli is increased after pretreatment with lipopolysaccharides (LPS). We have analyzed the steps in arachidonic acid (AA) metabolism affected by LPS by examining release of AA and its metabolites from [3H]AA prelabeled PMN. Pretreatment of PMN for 60 min with up to 1 microgram/ml of LPS alone had no effect, but release of [3H]AA was stimulated up to fivefold during subsequent stimulation with a second agent. In the absence of LPS-binding protein (LBP), priming was maximal after pretreatment of PMN with 10 ng of LPS/ml for 60 min; in the presence of LBP maximal priming occurred within 45 min at 0.1 ng of LPS/ml and within 15 min at 100 ng of LPS/ml. Treatment of PMN with 10 ng of LPS/ml also increased uptake of opsonized zymosan by up to 60%. Phospholipids are the source of released [3H]AA. No release was observed from [14C]oleic acid (OA)-labeled PMN suggesting that phospholipolysis may be specific for [3H]AA-labeled phospholipid pools. Cytosol from PMN primed with LPS contains two to three times the phospholipase A2 (PLA2) activity of control PMN, against 1-palmitoyl-[2-14C]arachidonoyl-phosphatidylcholine. This activity is Ca2+ dependent and dithiothreitol resistant. LPS priming is accompanied by reduced migration during SDS-PAGE of an 85-kD protein, identified as a cytosolic PLA2. The extent and kinetics of this effect of LPS on cPLA2 parallel the priming of [3H]AA release, both depending on LPS concentration either with or without LBP. These findings suggest that priming by LPS of AA metabolism by PMN includes phosphorylation of an AA-phospholipid-selective cytosolic PLA2 that is dissociated from activation until a second stimulus is applied.

Acute-Phase Proteins↗

PubMed1994

Individual and synergistic effects of rabbit granulocyte proteins on Escherichia coli.

Affinity purification of crude acid extracts of rabbit polymorphonuclear leukocytes using Escherichia coli (J5) as adsorbent yields the bactericidal/permeability-increasing protein (BPI), two 15-kD species (p15s), and the two most potent (cationic) defensin species (neutrophil peptides [NP] -1 and -2). Tested in buffered isotonic medium, the relative antibacterial potency of these proteins against E. coli J5 is BPI (IC50 0.2 nM) > p15A (10 nM) > NP -1 (400 nM). Sublethal doses of p15A or NP-1 can synergize with BPI to decrease the dose required to inhibit the growth of E. coli by up to 50-fold. BPI and p15A display similar features of antibacterial action distinct from defensin NP-1, but NP-1 acts synergistically only with BPI and not with p15A. All aspects of the combined action of BPI and NP-1 resemble those observed with higher concentrations of BPI alone, implying that NP-1 enhances BPI potency. Neither NP-1 nor p15A alter the amount of BPI binding to E. coli but BPI enhances binding of p15A to E. coli, raising the possibility that synergy between these two proteins may occur at least partially at the level of binding. The potent synergistic actions of these proteins can also be demonstrated against serum-resistant clinical isolates of encapsulated E. coli tested in whole blood and plasma ex vivo, suggesting that such combined action may contribute to host defense in vivo.

Animals↗

PubMed1994

The bactericidal/permeability increasing protein of neutrophils is a potent antibacterial and anti-endotoxin agent in vitro and in vivo.

The Bactericidal/Permeability Increasing protein (BPI) is a major constituent of the azurophilic granules of human and rabbit polymorphonuclear leukocytes (PMN). The cDNA of the highly conserved protein has been isolated from man, rabbit and cow. The ca. 50 kDa BPI and a ca. 25 kDa bioactive N-terminal fragment are cytotoxic only for Gram-negative bacteria (GNB). This target-cell specificity reflects the strong attraction of the highly cationic protein for the negatively charged lipopolysaccharides (LPS) in the bacterial envelope. Native and recombinant (r) holo-BPI and the N-terminal fragment (rBPI-23) bind with high affinity (apparent Kd 1-10 nM) to all forms of isolated LPS so far examined, and inhibit the numerous biological effects of LPS in vitro (including in whole blood ex vivo) as well as in animals. Under the same conditions the antibacterial activities of holo-BPI and rBPI-23 against GNB with rough chemotype LPS are the same, but against serum-resistant and smooth chemotype GNB rBPI-23 is up to 30-fold more potent than holo-BPI. Holo-BPI and rBPI-23 protect mice, rats and rabbits against lethal cytotoxic effects of LPS and in some cases against lethal inoculations with live GNB.

Animals↗

PubMed1993

Antibacterial 15-kDa protein isoforms (p15s) are members of a novel family of leukocyte proteins.

We have previously described the isolation and initial characterization of functionally distinct 15-kDa protein isoforms (p15s) from rabbit polymorphonuclear leukocytes (PMN) that bind with high affinity to Escherichia coli and modulate the antibacterial actions of other leukocyte proteins on this Gram-negative bacterium. We now report the cloning and sequencing of two distinct cDNAs from a rabbit bone marrow library that encode p15s differing at only 2 residues (His-3, Arg-88 versus Arg-3, Trp-88). Tryptophan-directed chemical cleavage of two isoforms purified from a single rabbit confirms the existence of multiple isoforms with distinct function and primary structure in a single rabbit. The p15 cDNAs encode putative signal sequences and studies of cellular and subcellular localization indicate that the p15s are granule-associated proteins of PMN. Both purified isoforms bind avidly to lipopolysaccharide (LPS), the major component of the Gram-negative bacterial outer membrane. Analysis of the deduced primary structures of the p15s reveals homology to three other leukocyte proteins: CAP-18, an 18-kDa LPS-binding protein from rabbit PMN, pro-indolicidin, a 16-kDa precursor of an antibacterial peptide of bovine PMN, and cathelin, an 11-kDa cysteine protease inhibitor from porcine leukocytes, suggesting the existence of a novel family of leukocyte proteins with LPS-binding, antimicrobial, and protease-inhibitory activities.

Amino Acid Sequence↗