Modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations.

We tested the in vitro susceptibility of Candida albicans to three defensins from human neutrophilic granulocytes (HNP-1, 2, and 3), a homologous defensin from rabbit leukocytes (NP-1), and four unrelated cationic peptides. Although the primary amino acid sequences of HNP-1, 2, and 3 are identical except for a single amino-terminal amino acid alteration, HNP-1 and HNP-2 killed C. albicans but HNP-3 did not. C. albicans blastoconidia were protected from HNP-1 when incubations were performed in the absence of oxygen or in the presence of inhibitors that blocked both of its mitochondrial respiratory pathways. Neither anaerobiosis nor mitochondrial inhibitors substantially protected C. albicans exposed to NP-1, poly-L-arginine, poly-L-lysine, or mellitin. Human neutrophilic granulocyte defensin-mediated candidacidal activity was inhibited by both Mg2+ and Ca2+, and was unaffected by Fe2+. In contrast, Fe2+ inhibited the candidacidal activity of NP-1 and all of the model cationic peptides, whereas Mg2+ inhibited none of them. These data demonstrate that susceptibility of C. albicans to human defensins depends both on the ionic environment and on the metabolic state of the target cell. The latter finding suggests that leukocyte-mediated microbicidal mechanisms may manifest oxygen dependence for reasons unrelated to the production of reactive oxygen intermediates by the leukocyte.

Defensins. Natural peptide antibiotics of human neutrophils.

We extracted a granule-rich sediment from normal human neutrophils and subjected it to chromatographic, electrophoretic, and functional analysis. The extract contained three small (molecular weight less than 3,500) antibiotic peptides that were named human neutrophil peptide (HNP)-1, HNP-2, and HNP-3, and which will be referred to as "defensins." HNP 1-3, a mixture of the three defensins, killed Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli effectively in vitro when tested in 10 mM phosphate buffer containing certain nutrients, but it had little or no bactericidal activity in nutrient-free buffer. In contrast, the nutrient-free buffer supported a high degree of activity by HNP 1-3 against Cryptococcus neoformans. In addition to its antibacterial and antifungal properties, HNP 1-3 directly inactivated herpes simplex virus, Type 1. Two of the individual purified defensins, HNP-1 and HNP-2, were as microbicidal as the mixture HNP 1-3. HNP-3 was less active than the other defensins against most but not all of the microbes tested. Immunoperoxidase stains revealed HNP 1-3 to have a granular localization in the neutrophil's cytoplasm by light microscopy. Frozen thin section immunogold transmission electron microscopy showed HNP 1-3 to be localized in azurophil granules. These studies define a broad-spectrum antimicrobial system in human neutrophils. The defensin system may operate in conjunction with or independently from oxygen-dependent microbicidal processes to enable human neutrophils to inactivate and destroy potential pathogens.

High- and low-affinity receptors regulate platelet responses to phorbol diesters and teleocidin.

We examined binding of 3H-phorbol dibutyrate (3H-PDBu) to gel filtered human platelets (GFP) and discovered that GFP possess two classes of receptors for phorbol diesters (PDE). High-affinity (HA) receptors, approximately 5000/GFP, bound 3H-PDBu with an apparent dissociation constant (KD) of approximately 12 nM. Low-affinity receptors were approximately 5 times more numerous (2.4 X 10(4)/GFP) and had a 10-fold lower affinity for 3H-PDBu (apparent KD = 115 nM). The potencies of phorbol myristate acetate (PMA) and PDBu paralleled their binding affinities to the PDE receptors. Teleocidin (Tel), although structurally distinct from PDE, competed with 3H-PDBu for its HA-receptors (KI Tel = 1.9 nM). Binding of PDE to HA- or LA- receptors was rapid, reversible, saturable and stereospecific. The HA- and LA-receptors modulated different platelet responses. HA-receptors regulated the secretion of beta-thromboglobulin from alpha-granules and the release of N-acetyl-beta-D-hexosaminidases from lysosomes. LA-receptors mediated both platelet aggregation and the release of serotonin from dense granules. This is the first demonstration of two physiologically active classes of PDE/Tel receptors in human platelets, and demonstrates that particular platelet responses may be directed by distinct classes of receptors for specific agonists.

Comparison of electrophoretic mobility and membrane sialic acid content of erythrocytes from adult and umbilical cord blood.

Determinations of cell electrophoretic mobility at low ionic strength and of ghost sialic acid content show that erythrocytes from umbilical cord blood and from adult donors are identical in these two glycoprotein-related properties. Using a streak deflection electrophoresis in 16.5 mM Tris-acetic acid buffer, pH 7.4, no increased streak width indicating electrophoretic heterogeneity could be detected when mixed suspensions of adult and umbilical cord blood erythrocytes were compared with suspensions of adult cells alone. Sialic acid content of 100 nmol/mg protein were obtained for both populations of cells.

Purification and antibacterial activity of antimicrobial peptides of rabbit granulocytes.

Six antimicrobial peptides, corresponding to the family of "lysosomal cationic proteins" described previously by Zeya and Spitznagel (H. I. Zeya and J. K. Spitznagel, J. Bacteriol. 91:750-754, 1966; H. I. Zeya and J. K. Spitznagel, J. Bacteriol. 91:755-762, 1966), were purified from rabbit peritoneal granulocytes by preparative acrylamide gel electrophoresis and reversed-phase high-pressure liquid chromatography. Each of the peptides was of low molecular weight (ca. 4,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two most cationic peptides, NP-1 and NP-2, were active against a broad spectrum of gram-positive and gram-negative bacteria. The remaining four peptides, NP-3A, NP-3B, NP-4, and NP-5, had more selective antibacterial activity. None of the peptides was active against Bordetella bronchiseptica, a common pathogen of domestic rabbits. Antibacterial activity was best expressed at near neutral pH under conditions of low ionic strength.

Activity of rabbit leukocyte peptides against Candida albicans.

Six related cysteine-rich, low-molecular-weight peptides were purified from rabbit peritoneal granulocytes and tested in vitro for fungicidal activity against Candida albicans. Two peptides (NP-1 and NP-2) were highly effective, one (NP-3a) was moderately active, and three (NP-3b greater than NP-4 much greater than NP-5) had substantially less potency. There was a general, but imperfect, correlation between the candidacidal potency of each peptide and its net cationic charge. Candidacidal activity by NP-1 was concentration and time dependent and occurred rapidly under optimal low-ionic-strength conditions. It was inhibited by increasing either the ionic strength or Ca2+ concentration of the incubation mixtures, but was relatively unaffected by Mg2+. Candidacidal activity was independent of H+ concentrations between pH 5 and 8, but decreased below pH 5. Candidacidal activity was temperature sensitive and was virtually abolished when NP-1 was incubated with C. albicans at 0 degrees C. Cysteine-rich antimicrobial peptides such as NP-1 and NP-2 may equip leukocytes to deal with infections caused by C. albicans and other fungi that are susceptible to their microbicidal effects.

Correlation of binding of rabbit granulocyte peptides to Candida albicans with candidacidal activity.

NP-1, a candidacidal peptide purified from rabbit granulocytes, bound extensively and with biphasic kinetics to Candida albicans. The primary phase of binding was temperature independent and occurred even at 0 degrees C. This primary binding was relatively specific, reversible, saturable, and of high capacity. It was inhibited by increased salt concentrations in the incubation medium, but was relatively unaffected by increasing the calcium ion concentration or by lowering the incubation temperature to 0 degrees C. The secondary phase of binding was only noted under conditions that supported candidacidal activity. Secondary binding was inhibited by millimolar concentrations of calcium, but not magnesium, ions and did not occur at 0 degrees C or when subtoxic concentrations of NP-1 were tested. NP-2 and NP-3a, other potent candidacidal peptides from rabbit granulocytes, also bound directly and extensively to C. albicans and competed for binding with NP-1. NP-4 and NP-5, less candidacidally active homologs of the aforementioned peptides, showed relatively little direct binding activity and competed poorly for binding with NP-1 or NP-2. NP-3b, another less candidacidal homolog, bound extensively to C. albicans, but did not compete effectively with NP-1 or NP-2. By comparing candidacidal and binding activity of the peptides, we conclude that the candidacidal activity of NP-1 involves primary binding to C. albicans followed by postbinding events that are temperature dependent and inhibitable by calcium ions.

Synergistic activity of rabbit granulocyte peptides against Candida albicans.

Rabbit granulocytes contain six antimicrobial peptides that are structurally homologous to the human neutrophil "defensins." NP-5, a rabbit defensin, lacks significant activity against Candida albicans. Nevertheless, its addition to submicromolar concentrations of rabbit NP-1, NP-2, or NP-3a potentiates their candidacidal effect. Thus, granulocyte defensins can act synergistically against potential pathogens.

Polymorphic expression of defensins in neutrophils from outbred rats.

We isolated and characterized a rat neutrophil defensin, RatNP-2, that differs from the previously described defensin RatNP-1 by containing Ser-7 in place of Arg-7. Although the resulting charge difference rendered RatNP-2 easily distinguishable from RatNP-1 on polyacrylamide gel electrophoresis gels, the two defensins exhibited very similar antimicrobial efficacies against Salmonella typhimurium, Staphylococcus aureus, and Candida albicans. The polymorphonuclear leukocytes of Sprague-Dawley rats obtained from one of two breeders also showed a marked polymorphism for defensin RatNP-4. This defensin was absent in two of seven animals and present in 1x or 2x relative amounts in the others. These observations indicate that a striking degree of defensin polymorphism exists in the polymorphonuclear leukocytes of outbred rodents.

Effects of proteases and neuraminidase on RBC surface charge and agglutination. A kinetic study.

Electrophoretic mobility, membrane sialic acid content and agglutinability by "incomplete" antisera against Rh-o, hr' and k antigens were determined for red blood cells in the course of treatment with trypsin, ficin and neuraminidase. Neuraminidase gradually produces a slight to moderate agglutinability as it reduced surface charge density in proportion to the amount of sialic acid removed. Proteases acted in two distinct steps. The first stage is characterized by the cells rapidly becoming highly agglutinable and by the unmasking of new negative charge as the first half of the sialic acid is removed. In the second stage the cells show a slight gain in agglutinability as surface charge is removed in proportion to sialic acid removal as in the case of neuraminidase. Neuraminidase-treated cells are considerably less agglutinable than cells reduced to the same zeta-potential by protease treatment. The greater efficacy of proteases compared to neuraminidase in making cells agglutinable could be because they not only reduce surface charge density but also increase antigen-antibody bond strength, render antigens more mobile in the membrane to allow clustering in regions of cell to cell antibody bridging and remove glycopeptide chains which may be causing steric hindrance to antigen-antibody binding or to cell-cell contact.

Cell electrophoretic, membrane sialic acid and quantitative hemagglutination studies on some MN variants.

The group of conditions exhibiting diminished MN antigenicity, increased saline agglutinability, decreased electrophoretic mobility and reduced membrane content of sialic acid includes enzyme-treated cells, the hereditary MNSs variants Mk and Mg, En(a-) and the acquired condition of persistent mixed-field polyagglutinability. Here we report our studies on the above serological, chemical and biophysical properties of Mg, Mk and EnaEn? CELLS AND ON TWO ADDITIONAL HEREDItary variants, Miltenberger III and V, (Mi-III and Mi-V). The latter clearly fits into this group of conditions. On the other hand, Mi-III shows its kinship to the broad group of abnormalities of membrane glycophorin but it deviates from normal in the opposite direction. That is we find evidence of decreased saline agglutinability, increased electrophoretic mobility and of increased sialic acid content. Moreover, in the rare MsMi-III Mk phenotype, the opposing effects evident in the heterozygotes tend to balance out their serologic and physicochemical expressions in the double heterozygote.

Antibacterial activity of microbicidal cationic proteins 1 and 2, natural peptide antibiotics of rabbit lung macrophages.

Microbicidal cationic proteins 1 and 2, peptides derived from rabbit lung macrophages, were tested for bactericidal activity against various bacterial species. Both were highly active against diverse gram-positive and gram-negative organisms under conditions of near-neutral pH (between 7 and 8) and relatively low ionic strength. Susceptible species included Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Escherichia coli, and Serratia marcescens. Streptococcus agalactiae, type 1A, was less susceptible than the aforementioned organisms or S. agalactiae, type 3. Bordetella bronchiseptica, a common commensal and pathogen of the rabbit respiratory tract, was completely resistant to both peptides.

Increased content of microbicidal cationic peptides in rabbit alveolar macrophages elicited by complete Freund adjuvant.

We measured the microbicidal peptides MCP-1 and MCP-2 in rabbit alveolar macrophages (AM), comparing rabbits pretreated with complete Freund adjuvant with untreated control animals. Levels of MCP-1 increased from 4.7 +/- 0.6 microgram/10(7) resident AM to 13.2 +/- 0.1 microgram/10(7) complete Freund adjuvant-elicited AM. MCP-2 levels rose from 1.8 +/- 0.1 microgram/10(7) resident AM to 7.3 +/- 0.4 microgram/10(7) complete Freund adjuvant-elicited AM. The activities of five lysosomal hydrolases (beta-D-glucuronidase, beta-D-galactosidase, acid phosphatase, N-acetyl-beta-D-galactosaminidase, and N-acetyl-beta-D-glucosaminidase) were 44 to 96% higher in complete Freund adjuvant-elicited AM, and lysozyme activity was three- to fourfold higher. As MCP-1 and MCP-2 are major constituents of rabbit AM and exhibit potent antibacterial and antifungal properties, they may contribute to the expression of microbicidal activity in both resident and activated states.

Microbicidal cationic proteins of rabbit alveolar macrophages: amino acid composition and functional attributes.

We purified two microbicidal cationic proteins, MCP-1 and MCP-2, from rabbit alveolar macrophages. MCP-1 was remarkably rich in arginine (25.5 mol%) and half cystine (18.7 mol%) residues and constituted approximately 1.5% of the total protein content of Freund adjuvant-elicited alveolar macrophages. MCP-2 was approximately half as abundant as MCP-1 and contained relatively less arginine (14.9 mol%) and half cystine (9.8 mol%). The amino acid compositions of MCP-1 and MCP-2 resembled those reported for the lysosomal cationic proteins of rabbit granulocytes, but were distinct from those of any known histone. MCP-1 (1 microgram/ml) killed 99.6% of Candida albicans in 20 min, whereas MCP-2 killed approximately 80% under similar conditions. Both proteins rapidly suppressed O2 consumption by C. albicans and induced a rapid loss of intracellular 86Rb+. Although more information is needed about the biological origin, distribution, and roles of macrophage microbicidal proteins, it seems likely that MCP-1 and MCP-2 contribute to the microbicidal efficacy of rabbit alveolar macrophages.

Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein.

We examined the bactericidal activity of two proteins that are abundant in the cytoplasmic granules of human eosinophils, major basic protein (MBP) and eosinophil cationic protein (ECP). Unlike the human neutrophil's peptide defensins, both MBP and ECP killed stationary phase Staphylococcus aureus 502A in a simple nutrient-free buffer solution. Although MBP also killed Escherichia coli ML-35 with considerable efficacy under these experimental conditions, the in vitro activity of ECP against E. coli was considerably enhanced if mid-logarithmic phase bacteria replaced stationary phase organisms or if the assay medium was enriched with trypticase soy broth. The antibacterial activity of both eosinophil proteins was modulated by incubation time, protein concentration, temperature and pH. A pBR322-transformed derivative of E. coli ML-35 was used to examine the effects of ECP and MBP on integrity of the bacterial inner membrane (IM) and outer membrane. Although both MBP and ECP caused outer and inner membrane permeabilization when nutrients were present, only MBP was effective under nutrient-free conditions. Two proton ionophores (DNP and carbonyl cyanide m-chlorophenyl hydrazone) protected E. coli from the bactericidal effects of ECP but not from MBP. These findings establish that MBP and ECP have bactericidal properties and suggest that these proteins kill E. coli by similar but nonidentical mechanisms marked by an attack on the target cell's membranes. In view of evidence that high concentrations of ECP and MBP exist in cytoplasmic granules whose contents are translocated to phagocytic vacuoles, we suggest that MBP and ECP contribute to the eosinophil's ability to kill ingested bacteria.

Purification and antimicrobial properties of three defensins from rat neutrophils.

Three cysteine-rich cationic peptides, designated RatNP-1, RatNP-3, and RatNP-4, were purified from an acid extract of rat polymorphonuclear neutrophils, sequenced, and tested for antimicrobial activity. The peptides ranged from 29 to 32 amino acids in length (Mr, 3,252 to 3,825), and each contained all eight invariantly conserved "framework" residues that are characteristic of defensins. Each of the peptides killed Escherichia coli ML-35, Acinetobacter calcoaceticus HON-1, Staphylococcus aureus 502A, and Candida albicans 820 in vitro. RatNP-1, the most cationic rat defensin, was also the most potent. With this report, a total of 13 distinct defensins have been characterized in the polymorphonuclear leukocytes of four mammalian species. The existence of the defensin system in rats should facilitate investigations of the in vivo role of defensins in experimental infections.