Human Polymorphonuclear Leukocyte Granules Research Articles

Proteinase-3 as the major autoantigen of c-ANCA is strongly expressed in lung tissue of patients with Wegener's granulomatosis.

Proteinase-3 (PR-3) is a neutral serine proteinase present in azurophil granules of human polymorphonuclear leukocytes and serves as the major target antigen of antineutrophil cytoplasmic antibodies with a cytoplasmic staining pattern (c-ANCA) in Wegener's granulomatosis (WG). The WG disease appears as severe vasculitis in different organs (e.g. kidney, nose and lung). Little is known about the expression and distribution of PR-3 in the lung. We found that PR-3 is expressed in normal lung tissue and is upregulated in lung tissue of patients with WG. Interestingly, the parenchymal cells (pneumocytes type I and II) and macrophages, and not the neutrophils, express PR-3 most strongly and may contribute to lung damage in patients with WG via direct interaction with antineutrophil cytoplasmic antobodies (ANCA). These findings suggest that the PR-3 expression in parenchymal cells of lung tissue could be at least one missing link in the etiopathogenesis of pulmonary pathology in ANCA-associated disease.

Molecular Analysis of Myeloperoxidase Deficiency Shows Heterogeneous Patterns of the Complete Deficiency State Manifested at the Genomic, mRNA, and Protein Levels

Myeloperoxidase (MPO) is a glycosylated hemoprotein contained in the azurophil granules of human polymorphonuclear leukocytes (PMNs). MPO is thought to play a role in the oxidative antimicrobial activity of neutrophils by catalyzing the formation of hypochlorous acid, a potent microbicide, from hydrogen peroxide and chloride anions. Seven unrelated individuals with complete MPO deficiency, a relatively common heritable defect of neutrophils, were identified during routine blood tests. Molecular analyses were conducted to determine the level of the abnormality in these individuals. Western blot analysis showed that 6 of the 7 donors were devoid of immunoreactive MPO protein, while neutrophils from one individual contained only the 55-Kd subunit. Northern analysis of bone marrow RNA from one MPO-deficient donor showed the presence of the normal-sized 3.3-kb transcript indicating that the defect in MPO biosynthesis in this case was posttranscriptional. Southern analysis of four MPO-deficient donors showed a normal Bgl II digestion pattern, whereas an abnormal restriction pattern was observed in a fifth individual. Although the Bgl II pattern was similar to that observed in an unrelated subject described by Nauseef (Blood 73:290, 1989), our study strongly suggests that the point mutation does not reflect a polymorphism. Taken together, these analyses show the existence of diverse abnormalities associated with MPO-deficiency that may be detected at the level of the MPO polypeptide, mRNA, and gene.

Molecular analysis of myeloperoxidase deficiency shows heterogeneous patterns of the complete deficiency state manifested at the genomic, mRNA, and protein levels

Myeloperoxidase (MPO) is a glycosylated hemoprotein contained in the azurophil granules of human polymorphonuclear leukocytes (PMNs). MPO is thought to play a role in the oxidative antimicrobial activity of neutrophils by catalyzing the formation of hypochlorous acid, a potent microbicide, from hydrogen peroxide and chloride anions. Seven unrelated individuals with complete MPO deficiency, a relatively common heritable defect of neutrophils, were identified during routine blood tests. Molecular analyses were conducted to determine the level of the abnormality in these individuals. Western blot analysis showed that 6 of the 7 donors were devoid of immunoreactive MPO protein, while neutrophils from one individual contained only the 55-Kd subunit. Northern analysis of bone marrow RNA from one MPO-deficient donor showed the presence of the normal-sized 3.3-kb transcript indicating that the defect in MPO biosynthesis in this case was posttranscriptional. Southern analysis of four MPO-deficient donors showed a normal Bgl II digestion pattern, whereas an abnormal restriction pattern was observed in a fifth individual. Although the Bgl II pattern was similar to that observed in an unrelated subject described by Nauseef (Blood 73:290, 1989), our study strongly suggests that the point mutation does not reflect a polymorphism. Taken together, these analyses show the existence of diverse abnormalities associated with MPO-deficiency that may be detected at the level of the MPO polypeptide, mRNA, and gene.

Structure, chromosomal assignment, and expression of the gene for proteinase-3. The Wegener's granulomatosis autoantigen.

Proteinase-3 (PR-3) is a neutral serine proteinase present in the azurophil granules of human polymorphonuclear leukocytes. It degrades a variety of extracellular matrix proteins including elastin in vitro and causes emphysema when administered by tracheal insufflation to hamsters. It is identical to the target autoantigen (c-ANCA) associated with Wegener's granulomatosis and to myeloblastin, a serine proteinase first identified in HL-60 leukemia cells. In this study, the gene encoding PR-3 was cloned and sequenced. The gene spans approximately 6.5 kilobase pairs and consists of five exons and four introns. The genomic organization of PR-3 is similar to that of the other serine proteinases expressed in hemopoietic cells. Each residue of the catalytic triad of PR-3 is located on a separate exon, and the positions of the residues within the exons are similar to those in human leukocyte elastase and cathepsin G. The phase and placement of the introns in the PR-3 gene are also similar to those in human leukocyte elastase and cathepsin G. The 400-base pair (bp) 5'-flanking sequence of the PR-3 gene contains a TATA box at position 379. There is no CAAT box promoter element. The 3'-untranslated region is 200 bp, extending from a TGA stop codon to the site of polyadenylation 10 bp after the canonical AATAAA signal. Amplification of PR-3 from a human/hamster hybrid cell line localizes the gene to human chromosome 19. Evidence from Northern analysis suggests that PR-3 expression is primarily confined to the promyelocytic/myelocytic stage of bone marrow development.

Uptake and utilization of human polymorphonuclear leukocyte granule myeloperoxidase by mouse peritoneal macrophages

Functional myeloperoxidase contained in granules of polymorphonuclear neutrophil leukocytes or in fixed whole cells can be endocytosed by mouse peritoneal macrophages. Acquired myeloperoxidase was distributed in what we considered to be the secondary lysosomal system and, following a phagocytic stimulation, was delivered to newly formed phagosomes containing the targets.

Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters.

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.

Nonoxidative antimicrobial effects of human polymorphonuclear leukocyte granule proteins on Chlamydia spp. in vitro.

Proteins from isolated granules of human polymorphonuclear leukocytes were assessed for their nonoxidative microbicidal effect on chlamydiae by two different methods: a radioisotope assay for elementary body integrity and a biological assay for inclusion development. Crude granule extract, which consisted of a mixture of all granule proteins, caused a 20 to 30% decrease in infectivity and a 52% decrease in infectivity when incubated with Chlamydia psittaci CAL-10 and Chlamydia trachomatis serovar E, respectively. To define more specifically the components that were damaging to chlamydiae, crude granule extract was subjected to Sephadex G-75 column chromatography and isolated granule fractions were obtained. Only fractions containing lysozyme as the major component consistently caused reductions in infectivity of C. trachomatis elementary bodies. In contrast, fractions collected after the lysozyme fraction, containing proteins with molecular masses of 13,000 daltons or less, had detrimental effects on C. psittaci infectivity. Additional experiments using highly purified human polymorphonuclear leukocyte lysozyme confirmed its infectivity-reducing action upon C. trachomatis but not upon C. psittaci.

A monoclonal antibody that inhibits the antimicrobial action of a 57 KD cationic protein of human polymorphonuclear leukocytes.

Two monoclonal antibodies (mAb) specific for epitopes of a 57,000 m.w., cationic antimicrobial protein (CAP57) purified from granules of human polymorphonuclear leukocytes (PMN) have been produced. Both were IgG1 mouse antibodies with typical heavy and light chain structure. The mAb reactive with CAP57 failed to react specifically with other heretofore defined PMN or serum proteins as shown by ELISA. Both mAb showed specific reactivity in Western blots with CAP57. One of these mAb (P1G8) inhibited the antimicrobial action of CAP57 by 50% at a ratio of 62.5 micrograms antibody per microgram CAP57. The other mAb, P2A5, had no inhibitory capacity for CAP57. Binding constants of the two mAb for the antigen were determined and were found to be virtually identical. Thus, the greater inhibitory capacity of P1G8 for bacterial killing by CAP57 was not directly related to binding strength of the mAb. Competition experiments showed that unlabeled P1G8 could compete as well against radiolabeled P2A5 as could unlabeled P2A5. In the reverse experiment, it was seen that P1G8 competed with radiolabeled P1G8 for CAP57 better than unlabeled P2A5. These findings could be due to two antibodies that recognize different but adjacent epitopes on CAP57, one of the epitopes (P1G8) being closer to structure(s) of the protein essential to its antimicrobial action. Immunocytochemical studies showed positive staining with both mAb. The reaction was restricted to the cytoplasm of peripheral blood PMN and was of a granular pattern. Other peripheral blood cells (which included red blood cells, eosinophils, monocytes, and lymphocytes) failed to bind either mAb.

Cephalosporin antibiotics can be modified to inhibit human leukocyte elastase.

Several laboratories, including our own have reported the synthesis and activity of certain low relative molecular mass inhibitors of mammalian serine proteases, especially human leukocyte elastase (HLE, EC 3.4.21.37), an enzyme whose degradative activity on lung elastin has been implicated as a major causative factor in the induction of pulmonary emphysema, and which is present in the azurophil granules of human polymorphonuclear leukocytes (PMN). Normally, these granules fuse with phagosomes containing engulfed foreign material (such as bacteria), and HLE, in combination with other lysosomal enzymes, catabolizes the particles. Under certain pathological conditions, however, PMN become attached to host protein (elastin fibres, basement membrane, connective tissue, immune complexes), and in response to this adherence, the granules may fuse with the PMN outer membrane and release their contents, including HLE, directly onto the tissue. Besides emphysema, HLE may also contribute to the pathogenesis of disease states such as adult respiratory distress syndrome, and its potential involvement in rheumatoid arthritis makes HLE inhibitors of considerable interest. It is known that cephalosporin antibiotics (for example, cephalothin (compound I, Table 2)) are acylating inhibitors of bacterial serine proteases which help synthesize the cell wall by performing a transpeptidation reaction on a peptidyl substrate bearing a D-Ala-D-Ala terminus. We now report that neutral cephalosporins (that is, compounds not bearing a free carboxyl at position C-4) can be modified to become potent time-dependent inhibitors of HLE.

Myeloperoxidase Biosynthesis by a Human Promyelocytic Leukemia Cell Line: Insight Into Myeloperoxidase Deficiency

The biosynthesis and processing of myeloperoxidase (MPO), a cationic enzyme present in the azurophilic granules of human polymorphonuclear leukocytes (PMNs), were studied in the human promyelocytic leukemia cell line, HL-60. HL-60 cells produce large quantities of enzymatically active MPO that has the same electrophoretic behavior as MPO isolated from normal PMNs. Mature MPO is a glycoprotein of approximately 150,000 molecular weight (mol wt) composed of two heavy-light protomers (alpha 2 beta 2) with subunits of 59,000 and 13,500 mol wt, respectively, under reducing conditions. The primary translation product of MPO messenger RNA (mRNA) isolated from HL-60 cells was a single polypeptide of mol wt 80,000. In HL-60 cells labeled with [35S]-methionine, the labeled MPO isolated by immunoprecipitation had a mol wt of 89,000. Treatment of this 89-kilodalton (kDa) species with endoglycosidase H produced a 79-kDa peptide, suggesting that the 89-kDa protein contained high-mannose side chains. The 89-kDa species had no detectable peroxidase activity. During chase experiments some of the 89-kDa peptide was processed to smaller species of mol wt 39,000, 59,000, and 13,500, although a fraction of the 89-kDa peptide remained unprocessed after a chase of 100 hours. In addition, a small amount of the 89-kDa peptide appeared in the medium without any of the processed smaller peptides. These studies suggest that the primary translation product in MPO biosynthesis is an 80-kDa peptide that undergoes cotranslational cleavage of the signal peptide and glycosylation to produce an 89-kDa pro-MPO, that pro-MPO is a single polypeptide containing the alpha and beta subunits of MPO and contains endoglycosidase H-susceptible high-mannose side chains, and that posttranslational modification of pro-MPO results in targeting to the lysosome and proteolytic maturation of pro-MPO to active enzyme. In light of the previous observation that MPO-deficient and normal PMNs contain an 89-kDa protein immunochemically related to MPO, these studies on MPO biosynthesis indirectly support the hypothesis that defective posttranslation processing by pro-MPO may underlie hereditary MPO deficiency.

Myeloperoxidase biosynthesis by a human promyelocytic leukemia cell line: insight into myeloperoxidase deficiency

The biosynthesis and processing of myeloperoxidase (MPO), a cationic enzyme present in the azurophilic granules of human polymorphonuclear leukocytes (PMNs), were studied in the human promyelocytic leukemia cell line, HL-60. HL-60 cells produce large quantities of enzymatically active MPO that has the same electrophoretic behavior as MPO isolated from normal PMNs. Mature MPO is a glycoprotein of approximately 150,000 molecular weight (mol wt) composed of two heavy-light protomers (alpha 2 beta 2) with subunits of 59,000 and 13,500 mol wt, respectively, under reducing conditions. The primary translation product of MPO messenger RNA (mRNA) isolated from HL-60 cells was a single polypeptide of mol wt 80,000. In HL-60 cells labeled with [35S]-methionine, the labeled MPO isolated by immunoprecipitation had a mol wt of 89,000. Treatment of this 89-kilodalton (kDa) species with endoglycosidase H produced a 79-kDa peptide, suggesting that the 89-kDa protein contained high-mannose side chains. The 89-kDa species had no detectable peroxidase activity. During chase experiments some of the 89-kDa peptide was processed to smaller species of mol wt 39,000, 59,000, and 13,500, although a fraction of the 89-kDa peptide remained unprocessed after a chase of 100 hours. In addition, a small amount of the 89-kDa peptide appeared in the medium without any of the processed smaller peptides. These studies suggest that the primary translation product in MPO biosynthesis is an 80-kDa peptide that undergoes cotranslational cleavage of the signal peptide and glycosylation to produce an 89-kDa pro-MPO, that pro-MPO is a single polypeptide containing the alpha and beta subunits of MPO and contains endoglycosidase H-susceptible high- mannose side chains, and that posttranslational modification of pro-MPO results in targeting to the lysosome and proteolytic maturation of pro- MPO to active enzyme. In light of the previous observation that MPO- deficient and normal PMNs contain an 89-kDa protein immunochemically related to MPO, these studies on MPO biosynthesis indirectly support the hypothesis that defective posttranslation processing by pro-MPO may underlie hereditary MPO deficiency.

Physiological effects of a bactericidal protein from human polymorphonuclear leukocytes on Pseudomonas aeruginosa.

The physiological changes seen in Pseudomonas aeruginosa after exposure to a bactericidal protein (BP) from the granules of human polymorphonuclear leukocytes were studied. It was demonstrated, using radiolabeled proline or leucine, that both the rate of cellular uptake and amino acid incorporation into trichloroacetic acid-insoluble material were markedly decreased immediately after exposure to BP. The rate of O2 consumption by P. aeruginosa was decreased immediately after exposure to BP and continued to decline exponentially until it ceased completely 30 min after exposure to BP. In the presence of 30 mM CaCl2 or MgCl2, bacteria were protected from death due to BP and respiration rates were unaffected. The cellular ATP pool of P. aeruginosa remained constant for up to 2 h after exposure to BP. Membrane depolarization was measured by the influx of the lipophilic anion thiocyanate. It was shown that the cytoplasmic membrane of P. aeruginosa was partially depolarized after exposure to BP. Purified BP killed 95% of 5 X 10(6) CFU of P. aeruginosa at a concentration of 60 to 100 ng of protein per ml. Although the concentration of bacteria and BP varied with each type of experiment, the BP/bacteria ratio required to cause a 95 to 99% loss in viability remained constant. We propose that cytoplasmic membrane depolarization is the biochemical lesion responsible for the other physiological changes seen and ultimately for the death of P. aeruginosa induced by BP.

Effect of plant SH-protease on human polymorphonuclear leukocyte granule elastase.

ABSTRACTIn the present study, attempts were made to examine the effects of plant SH‐protease on human polymorphonuclear leukocyte granule elastase. S‐2484 amidolytic activity in extracted solution using 0.1% Triton X‐100 was remarkably higher than in the solution without Triton X‐100. The authors confirmed the possibility that the presence of solvents such as Triton X‐100 might be required for extraction of elastase from the granule membrane. It was also confirmed that ficin and papain inhibited extraction of elastase from the granule membrane by Triton X‐100, and that SH‐protease can inhibit S‐2484 amidolytic and Congo red‐elastinolytic activities of the released elastase. However, it is not clear whether SH‐protease inhibits the release of proelastase from the granule membrane or inhibits activation of released proelastase to elastase. The authors assume that there may be competitive inhibition between SH‐protease and elastase against the substrate.

Bactericidal activity of a granule extract from human polymorphonuclear leukocytes against Bacteroides species.

The microbicidal activity of an acetate extract of human polymorphonuclear leukocyte granules was tested against Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis and Bacteroides thetaiotaomicron. All strains tested were killed by the extract, and there were no significant differences between the different Bacteroides species.

Gonococci with mutations to low-level penicillin resistance exhibit increased sensitivity to the oxygen-independent bactericidal activity of human polymorphonuclear leukocyte granule extracts.

Gonococci which cause disseminated gonococcal infection are nearly always highly penicillin sensitive, in contrast to many isolates causing uncomplicated gonorrhea. We questioned whether any of the known chromosomal mutations to low-level penicillin resistance might adversely affect virulence. The penA2 locus is known to result in low-level resistance to penicillins, whereas mtr-2 results in nonspecific resistance to a variety of antimicrobial agents. We found that the penA2 and mtr-2 mutations each markedly increased sensitivity of strain FA19 to oxygen-independent killing by human polymorphonuclear leukocyte mixed or isolated azurophilic granule extracts. The penA2 and mtr-2 mutations had no effect on sensitivity to serum antibody and complement. Isogenic opaque or transparent variants of several strains of Neisseria gonorrhoeae were equally resistant to human polymorphonuclear leukocyte mixed granule extract bactericidal systems. There were also no differences in susceptibility of piliated type 1 and nonpiliated type 4 variants to human polymorphonuclear leukocyte mixed granule extracts. Since the penA2 and mtr-2 loci are known to increase the degree of cross-linking of cell wall peptidoglycan, the structure of peptidoglycan apparently affects sensitivity to killing by one or more polymorphonuclear leukocyte azurophilic granule extract bactericidal systems. These observations might explain why gonococci with mutations similar to penA2 and mtr-2 are almost never isolated from patients with disseminated gonococcal infection.

Gonococci with mutations to low-level penicillin resistance exhibit increased sensitivity to the oxygen-independent bactericidal activity of human polymorphonuclear leukocyte granule extracts.

Gonococci with mutations to low-level penicillin resistance exhibit increased sensitivity to the oxygen-independent bactericidal activity of human polymorphonuclear leukocyte granule extracts.

Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules.

A serine esterase with potent proteolytic activity against native bovine lens capsule type IV collagen was isolated and purified from extract of human polymorphonuclear leukocytes (PMN). The type IV collagenolytic activity co-purified with N-t-benzyloxycarbonyl-L-alanine nitroanilidase, and was inhibited by phenylmethanesulfonyl fluoride and N-acetyl-Ala-Ala-Ala-Ala chloromethyl ketone. In addition, the purified enzyme had elastolytic activity, reacted with a specific antibody to PMN elastase, and, therefore, appeared to be identical with this enzyme. A simple, reproducible assay for the detection of type IV collagenase activity using insoluble bovine anterior lens capsule collagen was defined. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the enzyme released large molecular weight peptides (greater than 30,000) from the insoluble substrate. The enzyme was also active against native, pepsin-solubilized type IV collagen; five reaction products could be identified. These data suggest that PMN elastase may be involved in the degradation of basement membrane collagen in physiologic and pathologic states.

Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes

Proteins from human polymorphonuclear leukocyte granules were extracted with 0.2 M acetate, pH 4.0, and fractionated by Sephadex G-100 column chromatography. The fractions demonstrated selective bactericidal action against a deep rough cell wall mutant of Escherichia coli O111:B4 with rough lipopolysacharide and cell wall mutants of Salmonella typhimurium LT-2 with lipoplysacharide of Ra, Rc, Rd1, Rd2, and Re types. Smooth parent strains were most resistant to the bactericidal action. Fractions with greatest activity for the mutants were from valley regions (regions of low protein concentration) between three high protein peaks comprising myeloperoxidase, protease, and lysozyme, respectively. Susceptibility of the mutants to bactericidal action increased as sugar residues decreased in lipopolysaccharide. Gram-positive bacteria were susceptible to different fractions than were the gram-negative bacteria.

Purification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes.

Purification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes.

Susceptibility of Lipopolysaccharide Mutants to the Bactericidal Action of Human Neutrophil Lysosomal Fractions

Acetate extracts of purified human neutrophil granules (a mixed population containing specific and azurophil granules) were dialyzed against phosphate-buffered saline (pH 7.0) and tested for bactericidal activity against smooth parent and rough mutant, gram-negative bacteria. Rough (Re) mutants of Escherichia coli, Salmonella typhimurium, and Salmonella minnesota were exquisitely more sensitive to extracts of human polymorphonuclear leukocyte granules than were their smooth (S) parents. The mean lethal dose (LD(50)) for the parent strains was 25 to 50 mug of granule protein per ml. As much as 500 mug of extract per ml failed to kill 100% of the S parents. The LD(50) for the rough mutants was 1.5 to 2.0 mug of the same granule extract per ml; 100% killing occurred with 5 to 10 mug of lysosomal protein per ml. Conditions affecting the growth of the bacteria greatly affected their sensitivity to the granule extracts. Granule extract killed bacteria grown with aeration to log phase 10 to 15 times more efficiently than the same bacteria grown to stationary phase under static conditions. The bactericidal incubation mixture also influenced results, in that greater killing occurred with tryptone than with phosphate or N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid-buffered saline. Bactericidal activity depended on lysosomal protein concentration, time, and temperature. Boiled lysosomal fractions failed to kill the S parents but retained 20 to 50% of their ability to kill the Re mutants. Parents (smooth) were killed more efficiently at pH 5 to 6, whereas their Re mutants were killed more efficiently at pH 7 to 8.