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In cystic fibrosis the bronchiectatic conducting airways have large numbers of neutrophils in their walls and in their luminal contents. The neutrophil's primary granule enzyme activities of elastase and peroxidase are increased in the sputum of these patients. It has been postulated that these enzymes--together or individually--act to damage the airway epithelium. However, only peroxidase activity has consistently correlated with the degree of structural and functional airway disease in these patients with leakage of plasma protein into the airway lumen (Regelmann et al., Pediatr Pulmonol, 1995; 19:1-9). The present study was designed to test whether human neutrophil-derived myeloperoxidase can independently produce bronchial epithelial damage without the presence of proteases, as measured by increased permeability of the airway epithelium. Human peripheral blood neutrophils were purified, their primary granules isolated, and their peroxidase purified using affinity and ion exchange column chromatography. Activity of the proteinase-free peroxidase was measured using a chromogenic substrate. The effect of this peroxidase on the permeability of excised rat tracheas was measured using radioactive and fluorescent-labeled non-ionic molecules of varying molecular weight. Rat tracheas exposed to 15 minute treatments with either 130 U of peroxidase or hydrogen peroxide (10(-5) M) did not show a significant increase in the permeability of the epithelium to [3H]inulin, [14C]sucrose, and fluorescein isothiocyanate dextran 20 compared with control tracheas. However, those tracheas exposed to 130 U peroxidase followed by 10(-5) M hydrogen peroxide showed an increased permeability to each of the three test solutes. We conclude that proteinase-free myeloperoxidase, in the presence of non-toxic concentrations of its substrates, hydrogen peroxide and halide, produced increases in permeability to non-ionic molecules in the rat trachea within 15 minutes.

Patients with cystic fibrosis (CF) of the same age differ significantly in their degree of pulmonary disease. Based on preliminary observations, we postulated that the activity of my-eloperoxidase would be significantly increased in patients with greater structural lung damage than in those with less lung damage. Acid extracts of weighed sputum samples were assayed for lactoferrin concentrations by ELISA. Activities of peroxidase, cathepsin G, and elastase (with and without proteinase 3) were determined by kinetic analysis using chromogenic substrates. The patients were divided into quartiles based on their Brasfield chest-radiograph score. Patients in the first quartile (least amount of structural lung abnormality) were compared to those in the fourth quartile. The concentration of lactoferrin, a specific (secondary) granule protein of neutrophils, did not differ between the two patient groups. However, the activities of the neutrophil primary granule proteins, peroxidase, elastase, and elastase plus proteinase 3, were significantly elevated in the group with the most structural lung abnormality. Sputum albumin concentration was used to estimate leakages of plasma proteins into the airways. Peroxidase activity, but not the activity of cathepsin G, of elastase, or of elastase plus proteinase 3, correlated significantly with albuminig sputum in both quartile groups. To confirm the association of sputum peroxidase activity with differences in lung structure and to test its correlation with lung function, spirometry was performed in a second group of patients during the week prior to the time of sputum sampling. In this second group, increased sputum peroxidase activity was associated with worse Brasfield scores and with decreased percent-predicted forced expiratory volume in 1 sec. Lactoferrin concentration again did not differ between the groups. Bacterial factors also did not differ. The results indicate that the degree of lung damage in patients with CF was more closely related to sputum peroxidase activity than to age, gender, CFTR genotype, or sputum bacterial parameters. Further study is required to determine whether or not peroxidase activity in the airway causes this lung damage. © 1995 Wiley-Liss, Inc.

Short (10- to 11-mer) hydrophilic peptides based on the structure of the 55-kDa bactericidal protein (BP55, B/PI, and CAP57) from human neutrophil granules were identified from the hydropathy plot of the 456-amino-acid sequence predicted from the nucleotide sequences of cDNA clones for BP55 and B/PI. Peptides corresponding to amino acid residues 90 to 99 (peptide #90-99), 86 to 99, or 90 to 102 of BP55 were bactericidal toward 5 x 10(6) Pseudomonas aeruginosa cells at 0.6 x 10(-5) to 1.5 x 10(-5) M and killed an Escherichia coli rough strain at 3 x 10(-5) M. The #90-99 peptide with a cysteine added at the amino terminus (C#90-99) was approximately 10 times more active than #90-99, killing P. aeruginosa at 1.5 x 10(-6) M. Peptides representing amino acid residues 27 to 37, 118 to 127, and 160 to 170 and the first 10 amino acids of the signal sequence for BP55 were not bactericidal. When coupled to either keyhole limpet hemocyanin or ovalbumin protein carriers through the thiol group, the C#90-99 peptide was not diminished on a molar basis in its capacity for killing of P. aeruginosa. Two other relatively hydrophilic peptides with an added amino-terminal cysteine, peptides C#227-236 and C#418-427, were not bactericidal at 1.2 x 10(-4) M or at 100 times the effective bactericidal concentration of C#90-99. The C#90-99 peptide killed E. coli at 1.5 x 10(-5) M, or at 10 times the concentration required to kill an equal number of P. aeruginosa cells. Although Pseudomonas cepacia and Staphylococcus aureus were resistent to killing by the parent BP55 molecule, they were susceptible to the C#90-99 and #90-99 peptides in the same concentration range as was E. coli. When all peptides were compared for the ability to neutralize E. coli O55:B5 endotoxin in a Limulus amoebocyte lysate assay, the C#227-236, C#418-427, and #160-170 peptides completely inhibited gelation at a 10(-4) M concentration. All other synthetic peptides, including bactericidal peptide #90-99 and its congeners, lacked endotoxin-neutralizing activity at the highest concentration tested (4.5 x 10(-4) M). A hybrid of the C#227-236 and #90-99 peptides (CHybrid) was identical to the C#227-236 peptide component in effectiveness for carrying out endotoxin neutralization and was fivefold better than the #90-99 peptide in its capacity for killing P. aeruginosa.

Killing of Pseudomonas aeruginosa by a 55-kDa bactericidal protein (BP 55), a 30-kDa protein (BP 30), cathepsin G, elastase, and proteinase 3 has been compared. P. aeruginosa was resistant to killing by elastase and proteinase 3. BP 55 at a 50% lethal dose (LD50) of 0.23 micrograms of protein per 5 x 10(6) bacteria per ml killed P. aeruginosa and was far more active than BP 30 and cathepsin G. The LD50s of BP 30 and cathepsin G were 16.9 and 28.3 micrograms of protein per 5 x 10(6) bacteria per ml, respectively. Preincubation of BP 55 or BP 30 with lipopolysaccharide (LPS) from P. aeruginosa inhibited bactericidal activity. The N-terminal amino acid sequence of BP 55 and BP 30 revealed no relationship between the two proteins. However, a monoclonal antibody (AHN-15) reacted with both proteins by Western immunoblot. The bactericidal activity of cathepsin G toward P. aeruginosa appeared to be dependent on the availability of the active site of the enzyme; bactericidal activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and by the specific cathepsin G inhibitor, Z-Gly-Leu-Phe-CH2Cl. The enzyme and bactericidal activities of cathepsin G were also inhibited by LPS from P. aeruginosa. LPS from P. aeruginosa was shown to be a competitive inhibitor of the enzyme activity of cathepsin G. Elastase enzyme activity was also inhibited noncompetitively by LPS, but the enzyme was not bactericidal. We have concluded that all three bactericidal proteins (BP 55, BP 30, and cathepsin G) may bind to the LPS of the outer membrane of P. aeruginosa. It appears that the enzyme active site must be available for cathepsin G to kill P. aeruginosa and that the active site may be involved in the binding of cathepsin G to P. aeruginosa.

Proteinase 3 (PR-3) is a human polymorphonuclear leukocyte (PMNL) serine proteinase that degrades elastin in vitro and causes emphysema when administered by tracheal insufflation to hamsters (Kao, R. C., Wehner, N. G., Skubitz, K. M., Gray, B. H., and Hoidal, J. R. (1988) J. Clin. Invest. 82, 1963-1973). We have determined the primary structure of several PR-3 peptides and have analyzed catalytic properties of the enzyme. The enzyme has considerable amino acid sequence homology with two other well characterized PMNL neutral serine proteinases, elastase and cathepsin G. Furthermore, the NH2-terminal amino acid sequence of PR-3 is identical to that of the target antigen of the anti-neutrophil cytoplasmic autoantibodies associated with Wegener's granulomatosis. PR-3 degrades a variety of matrix proteins including fibronectin, laminin, vitronectin, and collagen type IV. It shows no or minimal activity against interstitial collagens types I and III, respectively. The analysis of peptides generated by PR-3 digestion of insulin chains and the activity profile against a panel of chromogenic synthetic peptide substrates show that PR-3 prefers small aliphatic amino acids (alanine, serine, and valine) at the P1 site. The elastase-like specificity of PR-3 is consistent with its striking sequence homology to elastase at substrate binding sites. PR-3 is inhibited by alpha 1-proteinase inhibitor (ka = 8.1 x 10(6) M-1 S-1; delay time = 25 ms) and alpha 2-macroglobulin (ka = 1.1 x 10(7) M-1 S-1; delay time = 114 ms) but not by alpha 1-anti-chymotrypsin. In contrast to elastase and cathepsin G, PR-3 is not inhibited by secretory leukoprotease inhibitor and is weakly inhibited by eglin c. Thus, PR-3 is distinct from the other PMNL proteinases.

Novel peptide 33mers have been designed by incorporating beta-conformation stabilizing residues from the beta-sheet domains of alpha-chemokines and functionally important residues from the beta-sheet domain of human neutrophil bactericidal protein (B/PI). B/PI is known for its ability to kill bacteria and to neutralize the action of bacterial endotoxin (lipopolysaccharide, LPS) which can induce septic shock leading to eventual death. Here, the goal was to make short linear peptides which demonstrate good beta-sheet folding and maintain bioactivity as in native B/PI. A library of 24 peptide 33mers (betapep-1 to betapep-24) were synthesized with various amino acid substitutions. CD and NMR data acquired in aqueous solution indicate that betapep peptides form beta-sheet structure to varying degrees and self-associate as dimers and tetramers like the alpha-chemokines. Bactericidal activity toward Gram-negative Pseudomonas aeruginosa was tested, and betapep-19 was found to be only about 5-fold less potent (62% kill at 1.2 x 10(-7) M) than native B/PI (80% kill at 2.9 x 10(-8) M). At LPS neutralization, betapep-2 and -23 were found to be most active (66-78% effective at 1.2 x 10(-6) M), being only about 50-100-fold less active than B/PI (50% at 1.5 x 10(-8) M). In terms of structure-activity relations, beta-sheet structural stability correlates with the capacity to neutralize LPS, but not with bactericidal activity. Although a net positive charge is necessary for activity, it is not sufficient for optimal activity. Hydrophobic residues tend to influence activities indirectly by affecting structural stability. Furthermore, results show that sequentially and spatially related residues from the beta-sheet domain of native B/PI can be designed into short linear peptides which show good beta-sheet folding and retain much of the native activity. This research contributes to the development of solutions to the problem of multiple drug-resistant, opportunistic microorganisms like P. aeruginosa and of agents effective at neutralizing bacterial endotoxin.

Background. Bactericidal permeability increasing protein (BPI) binds to and neutralizes lipopolysaccharide (LPS, endotoxin). Small synthetic peptides based on the amino add sequence of the LPS binding domain of BPI neutralize LPS, albeit inefficiently. Although the LPS binding domain of native BPI possesses a β-turn secondary structure, this structure is not present in small derivative peptides. The purpose of this study was to determine whether the addition of a β-turn to a BPI-derived peptide is associated with more potent endotoxin antagonism. Methods. We generated a hybrid peptide (BU3) on the basis of (1) a portion of the LPS binding domain from BPI and (2) amino acids known to initiate a β-turn. BU3 folds with a β-turn, and we tested its effects on LPS neutralization and LPS-induced tumor necrosis factor-α secretion, comparing it with BPI-derived peptide BG22 that lacks a β-turn and to an irrelevant peptide (BG16). Results. Compared with BG22, BU3 demonstrated enhanced LPS neutralization and inhibition of LPS-induced tumor necrosis factor-α secretion in vitro and a similar diminution of endotoxemia and tumor necrosis factor-α secretion in a murine model of endotoxemia. Conclusions. These data demonstrate the potential for enhancing the biologic activity of a BPI-derived peptide endotoxin antagonist via manipulation of its conformational structure.

Objectives: To generate a recombinant fusion protein (FP) based on the endotoxin-binding domain of bactericidal/permeability-increasing protein (BPI) and the constant domain of IgG and to test its ability to inhibit lipopolysaccharide (LPS)-induced macrophage tumor necrosis factor α (TNF-α) secretion. Design: A murine macrophage cell line, RAW 264.7, was transfected with a BPI-IgG FP before incubation with LPS. The amount of LPS-induced TNF-α protein secreted was measured and compared with that secreted by cells transfected with a control construct. Setting: Basic science research laboratory. Main Outcome Measure: Secreted TNF-α protein concentration. Results: After transfection, RAW 264.7–cell FP expression was detected in cell lysates and supernatants. At each LPS dose tested, cells transfected with the FP gene secreted less TNF-α than did cells transfected with a control construct. Conclusions: The FP possesses substantial antiendotoxin activity, as delineated by inhibition of LPS-induced TNF-α secretion by murine macrophages transfected with the fusion gene construct. In the future, such FP may be used as a clinical reagent to reduce the morbidity and mortality associated with serious gram-negative bacterial infections in surgical patients. Arch Surg. 1996;131:1173-1178

Twenty-seven amino acid peptides with sequences corresponding to a proposed endotoxin binding region of bactericidal permeability increasing protein (BPI):1) inhibit lipopolysaccharide induced macrophage tumor necrosis factor-alpha (TNF-alpha) secretion, 2) have bactericidal activity against gram-negative bacteria, and 3) protect mice from a lethal lipopolysaccharide (LPS) challenge. Unfortunately, peptides have a short halflife in vivo. Therefore, we have chemically conjugated the BPI based peptide, BG38, to a larger carrier protein, keyhole limpet hemocyanin (KLH), and characterized its ability: 1) to inhibit LPS induced macrophage TNF-alpha secretion and 2) to decrease plasma endotoxin and TNF-alpha levels following an i.v. injection of E. coli 0111:B4 LPS. BG38-KLH inhibited cultured macrophage TNF-alpha secretion in response to LPS derived from four pathogenic strains of gram-negative bacteria in a dose dependent manner (90% inhibition at 50 microgram/ml, P0.05 Student's t test). BG38-KLH also decreased serum endotoxin (90%, P0.05 Student's t test) and peak TNF-alpha levels (30% inhibition, P0.05 Student's t test) following E. coli LPS challenge in a murine gram-negative bacterial sepsis model. Novel endotoxin antagonists based upon a small domain of BPI represent promising reagents for the treatment of serious gram-negative bacterial infections.

Background. Bactericidal permeability increasing protein (BPI), Limulus anti-lipopolysaccharide factor (LALF), and lipopolysaccharide binding protein (LBP) are three distinct proteins that bind to lipopolysaccharide (LPS). Intriguingly, binding of BPI and LALF to LPS results in neutralization of LPS activity, whereas the binding of LBP to LPS creates a complex that results in augmentation of LPS activity. Despite their different effector functions, we hypothesized that peptides based on the sequences of the proposed LPS-binding motif from each protein would neutralize LPS in vitro. Methods. Three peptide sequences, each 27 amino acids in length, of the proposed LPS-binding motif of BPI (BG38), LALF (BG42), and LBP (BG43) were synthesized. These peptides were then tested for their: (1) ability to inhibit macrophage secretion of TNF-α after stimulation by LPS derived from Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens; and (2) bactericidal activity against these same four gram-negative bacteria in vitro. Results. Synthetic peptides BG38 (BPI-derived), BG42 (LALF-derived), and BG43 (LBP-derived) but not control peptide significantly inhibited LPS-induced tumor necrosis factor-α secretion by macrophages and mediated the lysis of gram-negative bacteria in vitro. In addition, preincubation of LPS with peptide BG38 mediated complete protection subsequent to lethal endotoxin challenge. Conclusions. These data demonstrate that small peptides derived from BPI, LALF, and LBP retained significant endotoxin-netralizing and bactericidal activity against many different gram-negative bacteria in vitro. Identification of this conserved LPS-binding region within each protein may aid in the development of new immunomodulatory reagents for use as adjuvant therapy in the treatment of gram-negative bacterial sepsis.

Human neutrophil bactericidal protein (B/PI) is known for its ability to kill bacteria and to neutralize the action of endotoxin. Short linear peptides derived from residues 80-109 have been synthesized and their bactericidal and endotoxin neutralizing activities have been assayed. A series of 'walk-through' decapeptides, overlapping 3 to 4 residues, indicates that endotoxin neutralizing and partial bactericidal activities can be localized within the N- and C-terminal portions, respectively, of the 80-109 sequence. Bactericidal activity toward Pseudomonas aeruginosa was localized in central peptides of the walk-through series and greatest in peptide 90-99. By using longer peptides, residues 86-104 and 82-108, both bactericidal and endotoxin neutralizing activities are significantly enhanced. Bactericidal activity of peptide 82-108 is now only 6-fold less than that of parent B/PI and 9-fold more potent than peptide 86-104. The 82-108 peptide was 7-fold more active at endotoxin neutralization than 86-104 but showed less enhanced activity, being approx. 470-times less active than B/PI. Cyclized 82-108 peptide retained bactericidal activity but did not improve in capacity to neutralize endotoxin.

Publisher Summary This chapter describes assay, purification, and properties of myeloblastin. Myeloblastin is a neutral serine endopeptidase produced during myeloid differentiation and stored in the azurophilic granules of polymorphonuclear leukocytes (PMNs). Assays for myeloblastin are based on its preference for small aliphatic amino acids at the P1 site of susceptible peptide bonds. Although myeloblastin degrades a wide variety of extracellular matrix molecules, including elastin, fibronectin, vitronectin, collagen type IV, and the core protein of proteoglycans, the esterolytic or amidolytic activity of the enzyme against selected blocked amino acid or oligopeptide substrates has proved to be most useful in assessing enzyme activity. t-butyloxy carbonyl p-nitrophenylester is a nonspecific and sensitive substrate that is suitable to work with myeloblastin particularly in studies evaluating inhibitors. Release of p-nitrophenol is measured by the increase in absorbance at 347.5 nm. Myeloblastin is a basic protein and appears as three isoforms in polyacrylamide gel electrophoresis at neutral pH. It is inhibited by typical inhibitors of serine peptidases, such as phenylmethylsulfonyl fluoride and diisopropyl fluorophosphates.

Secretory leukocyte protease inhibitor (SLPI) is a 12 kD nonglycosylated serine antiproteinase secreted by cells of mucosal surfaces. In human lung, SLPI is present in the respiratory epithelium. It is the major barrier to tissue destruction mediated by the polymorphonuclear leukocyte (PMN) serine proteinases, elastase and cathepsin G, within the upper respiratory tract. We have recently described a third PMN serine proteinase, proteinase-3, that like elastase causes lung matrix destruction and experimental emphysema. The current studies examine interactions between SLPI and proteinase-3. The results show that: (1) SLPI and its reactive-site variants have no or minimal inhibitory activity against proteinase-3; (2) native SLPI does not complex with proteinase-3; (3) proteinase-3 selectively degrades both native and oxidized SLPI; (4) the cleavage of SLPI by proteinase-3 occurs at the peptide bond COOH-terminal to Ala-16 in the NH2-terminal domain of SLPI.

The susceptibility of paired mucoid and nonmucoid variants of Pseudomonas aeruginosa isolated from 13 patients with cystic fibrosis (CF) to killing by a 55,000-Da bactericidal protein (BP55) from human polymorphonuclear leukocytes was studied. Mucoid and nonmucoid variants were equally sensitive to killing by BP55 at both pH 5.6 and pH 7.2. Eleven of the isolates were resistant to the bactericidal activity of 10% normal human serum but were as sensitive as the serum-sensitive isolates to BP55. Similarly, the 15 isolates with lipopolysaccharides (LPS) containing O-polysaccharide side chains (smooth LPS) were as sensitive to BP55 as those isolates with rough LPS.P. aeruginosa isolates from patients in poor clinical condition were more likely to have LPS of the smooth type and to be resistant to killing by 10% human serum than the isolates from patients in good clinical condition. We have concluded that the susceptibility of the P. aeruginosa isolates from patients with CF to killing by BP55 does not correlate with mucoid or nonmucoid variations, with the presence or absence of smooth LPS, or with the sensitivity or resistance to killing by normal human serum.

Studies were designed to explore the possibility that human polymorphonuclear leukocyte granule constituents in addition to elastase (HLE) had the potential to cause emphysema. A two-step purification of three serine proteinases was developed. Granule extract proteins were initially separated by dye-ligand affinity chromatography. Fractions eluted were divided into four pools. Hamsters were given a single intratracheal instillation of saline +/- 0.1 mg protein of each pool. While pool 2 contained HLE and cathepsin G, the most dramatic bullous emphysema developed in animals treated with pool 4. The esterase from pool 4, designated proteinase 3 (PR-3) was purified, characterized in vitro, and tested for its ability to cause emphysema. PR-3 is a neutral serine proteinase with isoenzyme forms. Its ability to degrade elastin at pH 6.5 is slightly greater than that of HLE, but it is less active than HLE at pH 7.4 or 8.9. PR-3 has weak activity against azocasein. Its ability to degrade hemoglobin is intermediate to that of HLE and cathepsin G at pH 7.4. PR-3 has no activity against chromogenic substrates specific for HLE or cathepsin G. Its pI is substantially less than HLE or cathepsin G. It is also immunologically distinct from HLE. It induces emphysema in hamsters commensurate with that of HLE. We conclude that PR-3 may be important in the pathogenesis of human emphysema.

Neutrophil proteases are believed to play a role in the pathogenesis of a variety of human diseases. While many studies of proteases in models of disease have focused on elastase, neutrophils contain several proteases some of which share a high degree of homology. This report describes the production and characterization of an lgG1 murine monoclonal antibody (AHN-10) that reacts with human neutrophil elastase but not with the other major neutrophil neutral proteases: cathepsin G, proteinase 3, collagenase, or the newly purified neutral protease, esterase N. AHN-10 inhibited the elastinolytic activity of purified human neutrophil elastase and could detect elastase in alcohol-fixed cytospin preparations. The epitope recognized by AHN-10 was resistant to treatment with NaIO4, suggesting that the epitope is not a carbohydrate. AHN-10 should be useful for the immunolocalization of neutrophil elastase in tissue specimens and as a stable source of characterized antibody for quantitative identification of neutrophil elastase.

The specific activity of aspartokinase (ATP: l -aspartate 4-phosphotransferase, EC 2.7.2.4) decreases rapidly to a 10 to 20-fold lower level at the end of the growth phase in Bacillus licheniformis grown on a minimal glucose-salts medium. Concurrent with this decrease in specific activity is a loss of sensitivity to feedback inhibition by l -lysine and a slower decrease in the concerted inhibition caused by l -lysine and l -threonine. Growth of the cells in the minimal medium plus l -lysine and l -threonine caused the production of aspartokinase with characteristics similar to those of enzyme from cells harvested 4 h after the end of growth. Growth in the presence of either l -lysine or l -threonine did not affect the specific activity of the enzyme but did alter its inhibition sensitivity. The addition of l -lysine to the medium during growth on the minimal medium caused a rapid loss of sensitivity to lysine inhibition, 50% desensitization occurring in 15 min. These latter conditions did not produce an alteration in the specific activity of the enzyme or in the concerted inhibition by lysine and threonine. Despite the marked alteration in feedback-inhibition properties, changes in physical or kinetic properties of purified aspartokinase were not observed. Throughout the purification steps there was no evidence that more than one aspartokinase was present in B. licheniformis cells. It is concluded that this enzyme is unusually plastic and assumes alternate forms depending on the physiology of the cell.