John K. Spitznagel

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.

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.

Nonoxidative Antimicrobial Reactions of Leukocytes

Increasingly abundant evidence supports the hypothesis that PMNs and perhaps alveolar macrophages have antimicrobial mechanisms independent of the presences of molecular oxygen for effective action against an array of bacteria and against some fungi. Eosinophils have mechanisms toxic for schistosomula and Trichinella larvae. In all instances the antimicrobial substances isolated have been cationic proteins and, in PMNs, associated with the azurophil cytoplasmic granules of the PMNs. Several of these substances have thus far demonstrated no enzymic function. Two of these substances are serine proteases but in one, chymotrypsin-like protein, the antimicrobial action depends on the cationic properties of the protein and is independent of the proteolytic action of the substance. In most instances, these proteins are cationic due to relatively large proportions of arginine. In two instances, a large proportion of lysine is present. All have high proportions (about 50%) of hydrophobic amino acid. Such proteins occur in the PMNs of man, rabbit, guinea pig, rat, cow, and chicken. The present view is that they are most active against gram-negative bacteria. At least two of them-37-kd and 57-kd proteins (Shafer and Spitznagel, 1983)-act on S. typhimurium in a manner analogous to that of polymyxin B through binding to lipid A. Currently available results shows that anaerobic PMNs have substantial antimicrobial capacity. Whether this capacity is due to the O2-independent mechanisms discussed in this chapter remains to be established with greater certainty.

Differential distribution of distinct forms of myeloperoxidase in different azurophilic granule subpopulations from human neutrophils

Myeloperoxidase (MPO), a characteristic enzyme of human polymorphonuclear neutrophils (PMN), is localized in specialized lysosomal or azurophilic granules, and can be resolved into three distinct forms (I, II, III) by ion-exchange chromatography. Granules were isolated from single donor PMN and fractionated with centrifugation into two different azurophilic subpopulations (high and low density) by banding in a continuous sucrose density gradient. Ion-exchange chromatography of granule extracts indicated that the lower density granules contained mainly MPO forms II and III while the higher density granules appeared to contain all three forms, but in much reduced amounts. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed that, the mobilities of the heavy subunits of MPO appeared to be inversely related to the density of the granule population from which they were extracted. These observations suggest that the different forms of MPO may have distinct functional roles and/or are a possible reflection of maturational differences among the granule subpopulations.

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.

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.

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.

Origins and development of peptide antibiotic research

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 singificant 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.

Oxygen independent microbicidal mechanisms of human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMN) have several antimicrobial systems that can be viewed as belonging to two groups. One group depends upon oxidative processes. These include the superoxide anion (O2-), the myeloperoxidase-chloride-hydrogen peroxide system (MPHCl), and free hydroxyl radicals (•OH) (1). The other group (Table 1) functions independently of oxidative processes and includes increased hydrogen ion concentrations, various cationic proteins, cathepsin G, lysozyme, and apolactoferrin (2). The oxidative bactericidal processes are complex and depend upon soluble enzymes or cofactors present in the cytosol and upon enzymes located on or in membranes. The nonoxidative processes appear to depend solely upon proteins found within the PMN granules (3). Although there is indirect evidence that favors the dominance under various conditions of one or the other of these antimicrobial systems, it is unclear, at present, which Table 1 Oxygen Independent Antibacterial Components Cationic Proteins Elastase Cathepsin G Other Azuorphil Granule Proteins Lysozyme Lactoferrin mechanisms are mainly responsible for, or crucial in, the bactericidal activity of PMN in the various environments found in the body. For example, accumulating evidence suggests that oxygen-independent antimicrobial systems operate in vivo in human PMN as well as in those of chickens and rabbits (2).Table mechanisms are mainly responsible for, or crucial in, the bactericidal activity of PMN in the various environments found in the body. Fo example, accumulating evidence suggests that oxygenindependent antimicrobial systems operate in vivo in human PMN as well as in those of chickens and rabbits (2).

Oxygen-Independent Antimicrobial Systems in Polymorphonuclear Leukocytes

It has long been recognized that neutrophil polymorphonuclear granulocytes (PMN) exercise antimicrobial action only at very short range, and that they must attach to and ingest their targets in order to kill them with maximum efficiency. Metchnikoff (1905) first recognized this. Kanthack and Hardy (1894) extended Metchnikoff’s ideas to show the importance of degranulation of cytoplasmic granules. They showed that bacilli stopped growing only when they came in contact with and degranulated guinea pig granulocytes. Bacilli in the same fluid untouched by granulocytes continued to grow logarithmically. Remarkably, Kanthack’s work was forgotten. But Hirsch (1962) and Zucker-Franklin (Zucker-Franklin and Hirsch, 1964) rediscovered and extended these concepts, emphasizing the importance of the phagolysosomes formed about microbes as they are endocytized by heterophil (or neutrophil) polymorphonuclear granulocytes. Their studies focused attention on the importance of substances carried by cytoplasmic granules and deposited in phagolysosomes of PMN. It is now generally agreed that there are two principal granule classes, the specific granules and the azurophil granules (Bainton and Farquhar, 1968; Ullyot et al, 1973). The granules are now known to comprise several antibacterial substances including cationic antibacterial protein (CAP), lysozyme (LYZ), lactoferrin (LF), and myeloperoxidase (MPO). The distribution of several of these substances in the granules is shown in Table 1.