Arginine-rich proteins of polymorphonuclear leukocyte lysosomes. Antimicrobial specificity and biochemical heterogeneity.

The cationic antibacterial proteins of rabbit PMN lysosomes have been resolved into at least five subfractions. Each of these showed substantial selectivity in its antibacterial action against several pathogenic bacteria, including two smooth and two rough Escherichia coli strains, three Staphylococcus aureus strains, one S. albus, three proteus species and four different cultures of streptococcus. Each of the subfractions possesses a different electrophoretic mobility. Amino acid analyses of the three most cationic components revealed high contents of arginine consistent with their relative electrophoretic mobilities and very high arginine to lysine ratios. Aromatic amino acids were present in very low concentrations in these proteins and their light absorption at 2800 A was correspondingly weak. The evidence of antibacterial specificity, along with marked differences in the arginine-lysine ratios, shows that the cationic antibacterial components of rabbit PMN lysosomes are biologically and chemically heterogeneous.

Antimicrobial specificity of leukocyte lysosomal cationic proteins.

Diflerences in antimicrobial specificities against Staphylococcusaureus, Streptococcus faecalis (groupD),and Proteus vulgaris exist amongthe electrophoretically separable components of lysosomal cationic proteins of polymorphonuclear leukocytes.

Cationic protein-bearing granules of polymorphonuclear leukocytes: separation from enzyme-rich granules.

The cationic, antibacterial proteins of polymorphonuclear leukocytes are associated with a unique subcellular particle that is separable through zonal density gradient centrifugation from acid phosphatase-containing particles as well as from particles that contain alkaline phosphatase and lysozyme. Normal macrophages, macrophages stimulated by bacillus Calmette-Guérin, and liver cells lack this particle and the associated group of cationic proteins. Particles physically and biochemically similar to slower sedimenting enzyme-rich particles of polymorphonuclear leukocytes are shared by all the tlhree cell types.

Compartmentalization of cyclic AMP during phagocytosis by human neutrophilic granulocytes.

Immunocytochemistry shows that early during phagocytosis of zymosan, adenosine 3',5'-monophosphate (cyclic AMP) appears on the cell surface before the phagosome is internalized. The appearance of cyclic AMP on the cell surface is coincident with that of granule products and regulatory subunit of type I cyclic AMP-dependent protein kinase. Guanosine 3',5'-monophosphate is not associated with the initiation site of phagocytosis, but is observed throughout the granular cytoplasmic region. This sharply localized accumulation of cyclic AMP may serve as a signal for the initiation of phagocytosis.

Comparison of NADH and NADPH oxidase activities in granules isolated from human polymorphonuclear leukocytes with a fluorometric assay.

A fluormetric method for the determination of pyridine nucleotides has been adapted for use in studying the reduced pyridine nucleotide oxidases in human polymorphonuclear leukocytes. In the presence of strong base the oxidized forms of the pyridine nucleotides form a highly fluorescent product. The small amounts of NAD(P) formed by the oxidase reactions can be determined with great sensitivity. This method has been compared to the radioisotopic assay for NADPH oxidation. Both methods gave essentially the same results in terms of nanomoles NADP produced by control, resting, and phagocytizing samples. Both NADPH and NADH oxidase activities were insensitive to cyanide. NADPH oxidation had a pH optimum of 5.5, while that for NADH appeared to be 6.0. Granules isolated from phagocytizing cells routinely showed more activity toward both substrates (two to threefold) than granules from resting cells. Both activities were located primarily in a granule fraction prepared by differential centrifugation. Oxidation of NADPH was routinely four to five times that of NADH at all except very high substrate levels. Measurable NADH oxidation was rarely seen below 0.80 mM NADH, while NADPH oxidation was easily measurable at 0.20 mM. One patient with chronic granulomatous disease was studied. At low substrate levels, there was no activity toward either substrate in granules isolated from either resting or phagocytizing cells of this patient, while granules isolated from normal control cells showed substantial activity at these substrate levels. Purification of the activities had been initiated with linear sucrose gradients. Both activities co-sediment to a very dense region of the gradient, a region different from that in which membrane or azurophil granules usually equilibrate. The peak gradient fractions show a 10-30-fold increase in specific activity over comparable granule fractions. These data suggest that the oxidase activities are associated with one enzyme that has different affinities for the two substrates ans support the contention that the oxidation of NADPH is responsible for the metabolic burst accompanying phagocytosis in human PMNL.

CAP37, a human neutrophil-derived chemotactic factor with monocyte specific activity.

CAP37, an antimicrobial protein of human neutrophil granules, is a specific chemoattractant for monocytes. Purified to homogeneity by sequential chromatography over carboxymethyl Sephadex, G-75 Sephadex, and hydrophobic interaction HPLC, demonstratively endotoxin-free CAP37 was maximally chemotactic over a range of 1.3 X 10(-9)-10(-8) M. Thus it was active in the same molar concentrations as formyl-methionyl-leucyl-phenylalanine. CAP37 lacked chemotactic activity for neutrophils and lymphocytes. In checkerboard assays CAP37 had some chemokinetic activity as well. It was also chemotactic for rabbit mononuclear cells. Higher concentrations (2.7 X 10(-8) M) were required for activity with rabbit cells than with human. Sequence analysis of the first 42 NH2-terminal amino acid residues of CAP37 showed strong homologies with known serine proteases that mediate various functions in inflammation. However, a critical substitution of a serine for a histidine at position 41 suggested that CAP37 lacked serine protease action. This impression was supported by the failure of CAP37 to bind tritiated diisopropyl fluorophosphate. 89% of total CAP37 was released extracellularly from human neutrophils while they phagocytized Staphylococcus aureus. We propose that CAP37 released from neutrophils during phagocytosis and degranulation may mediate recruitment of monocytes in the second wave of inflammation.

Peritoneal macrophages of pathogen-free rats but not of conventional rats secrete elastolytic activity.

Elicited peritoneal macrophages from Sprague-Dawley rats conventionally bred and housed failed, as we have reported, to produce detectable elastolytic activity in culture. They did produce lysozyme and plasminogen activator. We now show that in contrast to these cells, macrophages from pathogen-free, barrier-sustained rats produced readily demonstrable elastolytic activity. Rats raised pathogen-free and subsequently housed conventionally for 2-4 wk appeared to lose the capacity to afford macrophages producing elastase. At the same time they acquired infections with several rat pathogens including Spironucleus muris, Kilham rat virus, sialodacryoadinitis virus, and mycoplasma pulmonis. The acquisition by the rats of one or more of these infections, conditions conducive to infection, or both factors may have suppressed their capacity to yield elastolytic activity.

Amino acid sequence of CAP37, a human neutrophil granule-derived antibacterial and monocyte-specific chemotactic glycoprotein structurally similar to neutrophil elastase.

We report the amino acid sequence of CAP37, a human neutrophil granule protein with antibacterial and monocyte-specific chemotactic activity. CAP37 is a single-chain protein consisting of 222 amino acid residues. It has three N-glycosylation sites, at Asn residues 100, 114 and 145. Some species of CAP37 are glycosylated at all three sites; some at Asn-114 alone, others at Asn-114 and Asn-110 or Asn-145. CAP37 has 45% sequence identity to human neutrophil elastase, and 30-37% identity to several other granule serine proteinases. Despite these similarities, CAP37 is not a serine proteinase because the active site residues serine and histidine are replaced.

Oxygen-independent antimicrobial action in sphingosine-treated neutrophils.

Sphingosine is reported to inhibit the oxidative burst and superoxide anion production of human polymorphonuclear neutrophils (PMN) phagocytosing in atmospheric oxygen (Wilson et al., 1986). We have confirmed its effect on superoxide production and examined the antimicrobial phagocytic capacity of PMN treated with sphingosine, comparing them with PMN, untreated but phagocytosing either under anaerobic conditions or in atmospheric oxygen. Sphingosine just like anaerobiosis partially inhibited, but did not eliminate, the bactericidal activity of PMN when compared to non-treated aerobic cells. In fact, sphingosine-treated PMN mimicked killing of Staphylococcus aureus (S. aureus) and Serratia marcescens (S. marcescens) due to anaerobic PMN. Moreover, our results with Salmonella typhimurium and sphingosine-treated cells duplicated results this laboratory published previously about comparative killing of Salmonella in aerobic versus anaerobic neutrophils. In these studies sphingosine-treated PMN took up bacteria as avidly as untreated PMN and retained their viability, as assessed by trypan blue exclusion. While sphingosine should not be completely substituted for anaerobic studies, it is a convenient screening reagent for the study of non-oxidative killing mechanisms of PMN. Results achieved with anaerobic and with sphingosine-treated cells suggest that O2-independent antimicrobial action is substantially more powerful than has been generally acknowledged.

An enzyme-linked immunoassay (ELISA) for measurement of lactoferrin.

An enzyme-linked immunosorbent assay has been developed for quantitation of lactoferrin (LF) in body fluids. An indirect double-sandwich method was used which allows a sensitivity of 3 ng LF/ml in samples of polymorphonuclear cell lysates and serum. Mean LF content of serum was 0.307 +/- 0.066 micrograms/ml (n = 18). Mean LF content of polymorphonuclear cells was 4.90 +/- 1.48 micrograms/10(6) PMN. Concentrations of LF were similar in serum and in plasma of EDTA anticoagulated blood. Advantages of this method include its rapidity, and radioactivity is not required.

Quantitation of a cationic antimicrobial granule protein of human polymorphonuclear leukocytes by ELISA.

The quantitation of CAP57, a highly hydrophobic, native cationic antigen of human polymorphonuclear leukocytes has been achieved using ELISA. An important feature determining the sensitivity and precision of the ELISA was the reduction of non-specific protein-protein binding, particularly in the inhibition assays, thus eliminating high backgrounds obtained with presently available methodology. Washing of the solid phase-bound antigen and blocking of the non-specific binding sites using a potassium phosphate buffer containing heparin largely contributed to this increased sensitivity. The inhibition assays were conducted using antigen concentrations over the range of 0.9-120 ng. The assay is highly specific and can be performed using monoclonal antibodies and polyclonal antibodies. Non-specific reactions were observed only when high concentrations of antigen (greater than 100 ng) were present in the inhibition mixture. The technique as described is extremely simple, highly reproducible and could be of value in the detection of cationic antimicrobial proteins in the clinical setting in the future.

Neutral proteases of human polymorphonuclear granulocytes: putative mediators of pulmonary damage.

Tissue proteolytic enzymes are currently believed to be critical to the pathogenesis of panacinar emphysema. Polymorphonuclear leukocytes (Polys) have several enzymes including elastase and cathepsin G in their azurophil granules. They have collagenase in their specific granules. We have found that this collagenase is doubly latent. It has the lysosomal type of latency that depends on the impermeability of the unit membrane that surrounds each specific granule. In addition it has a latency that is converted to activity by proteolytic enzymes. The cathepsin G of the azurophil granule is a potent activator of this latent collagenase once the collagenase is released from its membrane dependent latency. Thus latency of enzymes, the nature of the latency and accessibility of the latent enzymes to activating mechanisms must all be taken into account in any analysis of their contribution to pathogenesis of local lung disease. Equally important is that fact that polys are not a prominent cellular component of normal lung. Polys must be attracted to the lung by chemotactic peptides. These peptides must be released by the interaction of inflammatory stimuli, such as smoke particles, with complement components or they must be provided by other sources. The hypothesis that lung damage in panacinar emphysema is mediated by polys and their proteases is attractive and suggestive evidence supporting this is available. However, more evidence that takes into full account the cell biology of the proteases any poly turnover in the lung are needed to extend the hypothesis and to form a rational basis for therapeutic and prophylactic measures.

Origins and development of peptide antibiotic research. From extracts to abstracts to contracts.

That cationic proteins might be factors on the antimicrobial defenses of mammalian hosts and are apparently associated with the cytoplasmic granules of phagocytic leukocytes first became evident on the late nineteenth century. It remained, however, for development of sophisticated microanalytic techniques in microbiology, cell biology and protein biochemistry to place these hypotheses in the realm of established theory. This article is a brief summary of significant steps in the development of this theory. It also attempts to outline the firmly established scope and significance of these developments both for the theory of immunity to infection in the different phyla and for the now global quest for new antibiotics.

CAP 37, a 37 kD human neutrophil granule cationic protein shares homology with inflammatory proteinases.

We have previously shown that a major granule-associated cationic protein CAP 37 (Mr = 37 kD) derived from human PMN is a monocyte-specific chemoattractant. The N-terminal amino acid sequence of this novel chemotactic protein shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, a critical substitution of a serine for a histidine at position 41, results in its lack of serine protease activity.

A comparative analysis of human IgM rheumatoid factor degradation by purified elastase and total granule extracts from human polymorphonuclear leukocytes.

A modified digestion system using radiolabeled IgM rheumatoid factors (RF) and unlabeled IgG was used to examine IgM RF digestion by human polymorphonuclear leukocyte (PMN) elastase. Upon molecular sieve chromatography, the radioactive fragments coelute with fragments produced by elastase digestion of an IgM protein giving no RF activity. The fragments represent an Fab2-like fragment, an Fab-like fragment, and small peptides. Utilizing this same system, digests were performed at both acid and neutral pH to compare the proteolytic action of purified elastase on IgM RF (Ove) to the action of the total granule extract (TGE) from human PMN. At pH 4.5, purified elastase exhibits low-level protease activity, producing a slightly degraded IgM fragment with a molecular weight of about 800,000 daltons. In contrast, TGE at pH 4.5 completely degrades IgM RF to small peptides. At pH 7.5, the fragments produced by TGE digestion of IgM (Ove) coelute with fragments produced by elastase digestion under the same conditions. Thus elastase appears to be the major granule protease active in IgM RF degradation at the pH characterizing the inflammatory site.

Molecular approaches to leucotoxin as a virulence component in Actinobacillus actinomycetemcomitans.

A strategy has been developed to examine the hypothesis that leucotoxin is a critical virulence factor of Actinobacillus actinomycetemcomitans in a non-human primate (Macaca fascicularis). Firstly the leucotoxin gene from A. actinomycetemcomitans was cloned and sequenced. This DNA contained a functional leucotoxin gene, as protein extracts of Escherichia coli with the cloned sequences lysed appropriate human cell lines. The protein encoded by lktA shared at least 42% identity with P. haemolytica leucotoxin and with the alpha-haemolysins from E. coli and A. pleuropneumoniae. The lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be related to the LktC proteins from these other bacteria and with which it shared at least 49% amino acid identity. Despite the overall homology to the other leucotoxins/haemolysins, the LktA from A. actinomycetemcomitans has several unique properties including a very basic pI of 9.7, as compared to pIs approx. 6.2 for lktA proteins in other bacteria. Using the cloned genes as probes produced evidence that a TOX- strain contains the leucotoxin gene but fails to transcribe it at high levels. The second avenue of investigation was to develop methods for examining the humoral immune responses in the monkey to bacterial toxins such as lktA. A. actinomycetemcomitans was detected in subgingival plaque samples from approx. 40% of the animals. A. actinomycetemcomitans comprised less than 1% to 9% of the flora. Most A. actinomycetemcomitans isolates were serotype b and each of the monkeys had serum IgG antibody to A. actinomycetemcomitans serotype b (generally considered to be lktA-producing strains). An ELISA was developed to examine the isotype/subclass distribution, level and avidity of serum antibody in the monkey following parenteral immunization with a prototype bacterial exotoxin (tetanus toxoid). IgG1 and IgG3 antibody predominated over IgG2 and IgG4 after primary immunization. Secondary immunization elicited enriched IgG1 and IgG4 responses. Primary immunization increased avidity indices of IgG to tetanus toxoid from approx. 0.9 (baseline) to a mean of 1.72 and secondary immunization significantly increased the avidity index to 2.56.

Human neutrophil granule cationic protein CAP37 is a specific macrophage chemotaxin that shares homology with inflammatory proteinases.

Cationic antimicrobial protein CAP37 (Mr = 37 kD) is derived from the azurophilic granules of human PMN. In vitro and in vivo studies demonstrate that CAP37 is a novel monocyte-specific chemoattractant. The N-terminal amino acid sequence of CAP37 shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, substitutions in the catalytic triad (serine for a histidine at position 41 and glycine for a serine at position 175), may account for its lack of serine protease activity. A full length cDNA for CAP37 was identified in an HL60 cDNA library screened with oligonucleotide probes designed from the N-terminal amino acid sequence. Sequencing of the cDNA reveals a protein of 225 amino acids with significant nucleotide homology to cathepsin G and human neutrophil elastase.