Kenneth T. Miyasaki

Mechanism of extracellular release of human neutrophil calprotectin complex

Calprotectin is an abundant cytosolic protein complex of humanneutrophils with in vitro extracellular antimicrobial activity. Studiessuggest that calprotectin may be actively secreted from intact HL‐60cells and that it can be translocated to polymorphonuclear neutrophil(PMN) cell membranes. To examine whether calprotectin is secretedextracellularly, we incubated soluble and particulate stimuli,including live and heat‐inactivated Candida albicans, withwhole blood and measured calprotectin levels in the plasma. We comparedthe release of calprotectin to that of lactoferrin, a protein known tobe secreted by PMNs. Extracellular lactoferrin was detected afterincubation with any of the particulate stimuli. In contrast, asignificant increase in extracellular calprotectin was found only afterincubation with live C. albicans. Specifically, theincrease in extracellular calprotectin correlated directly with aproportional decrease in PMN viability. Our results indicate that humanPMN calprotectin is not secreted extracellularly except as a result ofcell disruption or death.

In vitro Antimicrobial Activity of the Human Neutrophil Cytosolic S-100 Protein Complex, Calprotectin, Against Capnocytophaga sputigena:

Calprotectin is a complex of two anionic proteins found in abundance in the cytosol of neutrophils, certain macrophages, and oral epithelial keratinocytes. Bacteria of the genus Capnocytophaga are pathogens of periodontal origin which can cause systemic infection in neutropenic subjects. Recently, it has been observed that Capnocytophaga may be internalized by neutrophils within the cytosol rather than within a membrane-delimited phagosome. The purpose of this study was to test the in vitro antibacterial effect of the cytosolic complex, calprotectin, against Capnocytophaga sputigena. Calprotectin was purified from the cytosol of human neutrophils by gel filtration and anion exchange FPLC, and it exerted potent in vitro antimicrobial effects against C. sputigena. Net bacteriostatic activity was exerted up to 18 h, after which bactericidal effects were observed. Both net bacteriostatic and bactericidal activity occurred at concentrations above 20 μg/mL and exhibited identical dose-response characteristics. Particle counts increased in the presence of calprotectin, despite net bacteriostasis as assessed by changes in colony-forming units (CFU). Dose-response characteristics and direct particle counts suggested that net bacteriostatic effects were the result of balanced cell division and death, rather than suspension of cell division. We conclude that calprotectin can be a significant contributor to host defense against infection by Capnocytophaga.

β-sheet antibiotic peptides as potential dental therapeutics1

Small, cysteine-rich, beta-sheet peptide antibiotics are found throughout the Animalia. Though broad spectrum in potential, they may exert selective antimicrobial effects under certain conditions. We have explored the antimicrobial properties of two families of beta-sheet peptide antibiotics, defensins and protegrins, against periodontopathic bacteria. The rabbit defensin NP-1 was active against facultative Gram-negative bacteria associated with early onset periodontitis, including Actinobacillus actinomycetemcomitans and the Capnocytophaga spp. Porcine protegrins showed even greater activity against those organisms, as well as against anaerobic bacteria associated with adult periodontitis, including Porphyromonas gingivalis Prevotella intermedia and Fusobacterium nucleatum. Based on these observations, we believe that protegrin-like beta-sheet peptide antibiotics may be useful dental therapeutics.

The distribution of the antimicrobial protein, calprotectin, in normal oral keratinocytes.

Calprotectin is a heterodimeric peptide isolated from neutrophil cytosol that exhibits profound antimicrobial effects. Using monoclonal antibody MAC 387, calprotectin was found to be expressed in oral keratinocytes from normal, non-inflamed oral mucosa. Orthokeratinized sites including the attached gingiva and hard palate expressed low levels of calprotectin with a restricted pattern; immunoreactants were identified only within subcorneal keratinocytes. Parakeratinized mucosa from the lips, soft palate, tongue and buccal mucosa expressed calprotectin in a more widespread, yet variable pattern, immunoreactants being detectable in only a portion of the spinous layer in some cases whereas in others the pattern of expression was more topographically diffuse. Antigen was not detected in basilar and lower strata cells. Both cytoplasmic and nuclear decoration could be identified. The results indicate that oral mucosa harbours an antimicrobial deterrent to micro-organisms that may enhance the physical epithelial barrier of host defence.

Sensitivity of Periodontal Pathogens to the Bactericidal Activity of Synthetic Protegrins, Antibiotic Peptides Derived from Porcine Leukocytes

Protegrins, small peptides (1900 to 2160 daltons) isolated from porcine leukocytes, are bactericidal against a broad range of medical pathogens in vitro under conditions which reflect the extracellular milieu. The purpose of this study was to determine whether Gram-negative, facultative periodontal pathogens were sensitive to the protegrins. Synthetic L- and D-enantiomers of protegrin 1 (PG-1 and D-PG-1, respectively), and L-enantiomers of protegrins 2, 3, and 5 (PG-2, PG-3, and PG-5) were tested against Actinobacillus actinomycetemcomitans (three strains) and Capnocytophaga spp. (three strains). Strains of both A. actinomycetemcomitans and Capnocytophaga spp. were sensitive to PG-1, and exhibited ED99 (dose at which 99% killing was observed after 1 hr at 37°C) of 0.5 to 3 ug/mL and 4 to 19 ug/mL, respectively. The D-form and the L-form were equally effective. Serum (above 5% v/v) inhibited the bactericidal effects of 10 ug/mL PG-1, but the inhibitory effect was overcome by concentrations of PG-1 at 100 ug/mL. Different patterns of sensitivity were observed when the effects of PG-1, D-PG-1, PG-2, PG-3, and PG-5 were compared against A. actinomycetemcomitans and the Capnocytophaga. We conclude that protegrins may be useful antimicrobial agents in therapy against periodontal infections.

Induction of Activated Lymphocyte Killing by Bacteria Associated with Periodontal Disease

Complex interactions occur among host defense cells during bacterial infection. Bacteria and bacterial products may enhance or inhibit the effector and regulatory activity of human lymphocytes. Accordingly, we tested the ability of human periodontal pathogens to activate peripheral blood lymphocytes using standard 51chromium-release assays to measure lymphocyte-mediated cytolysis. Human adherent-cell depleted peripheral blood lymphocytes (PBL) with the addition of glutaraldehyde-fixed bacteria at a 5:1 bacteria:lymphocyte ratio were incubated at 37°C for 24 hr in RPMI 1640 medium. Six of eight bacteria tested significantly augmented lymphocyte killing of the natural killer (NK) cell-sensitive human erythroleukemia cell line K562. E. corrodens, representing activating bacteria, was also able to induce the killing of NK-resistant targets (M14, Raji), comparable with induction by interleukin-2. Lipopolysaccharides extracted from A. actinomycetemcomitans strains, when incubated with PBL, were able to enhance cytotoxicity without the presence of whole bacteria. A majority of cytotoxicity was mediated by NK cells bearing Leu-11 and NKH-1 markers.

Secretion of myeloperoxidase isoforms by human neutrophils

The human neutrophil lysosomal enzyme, myeloperoxidase (MPO), exists in three major and chromatographically distinct forms, MPO I, MPO II, and MPO III. We used cation-exchange medium-pressure liquid chromatography and kinetic microenzyme assay (or spectrophotometric monitoring) to analyze the secretion of MPO isoforms by neutrophils exposed to N-formylmethionylleucylphenylalanine (FMLP), digitonin, the ionophore A23187, and serum-opsonized zymosan A (SOZ). All three MPO isomers were released into the fluid phase after neutrophils were exposed to these secretagogues. A significant proportional increase in MPO I was released when neutrophils were stimulated with SOZ. MPO I was released in higher proportions than found in the whole cell constituency when neutrophils were stimulated with FMLP + cytochalasin B, A23187, and digitonin, but this was not statistically significant.

Preferential Adsorption of Human Neutrophil Myeloperoxidase Isoform III by Oral Bacteria

Neutrophil myeloperoxidase (MPO) adsorbs to bacteria as a pre-requisite for killing by the MPO/hydrogen-peroxide/chloride system. Three chromatographically distinct isoforms of MPO (MPO I, MPO II, and MPO III) have been isolated from human neutrophils. The purpose of this study was to determine whether oral bacteria-including Actinobacillus actinomycetemcomitans, Capnocytophaga sputigena, Haemophilus aphrophilus, and Eikenella corrodens-differentially adsorb the three major isoforms of MPO from a mixture of MPO I, II, and III, and to assess the effect of pH and normal human serum (NHS) on MPO adsorption. MPO III adsorbed preferentially (i) at high bacterial concentrations, (ii) in the pH range of 6.0-8.0, and (iii) in the presence and absence of NHS. These results support the role of MPO III in the killing of oral bacteria.

Differential Modulation of Adherence of Oral Streptococci by Human Neutrophil Myeloperoxidase

In this study, the modulation of adherence of hydrogen peroxide (H2O2)-producing and non-H2O2-producing strains of oral streptococci by the host leukocyte enzyme myeloperoxidase (MPO) was examined. It was found that exposure to MPO decreased adherence of many strains of oral streptococci to saliva-coated hydroxyapatite beads in the presence of exogenous H2O 2 and chloride. The MPO-H2O2-Cl- system increased the adherence of one strain. In the absence of exogenous H 2O2, the MPO-H2O2-Cl- system decreased the adherence of H2O2producing strains only. Glucose increased streptococcal H2O2 production and also increased the anti-adhesive activity of MPO in the absence of exogenous H2O2. We conclude that: (1) host leukocytes can modulate the adherence of oral streptococci via MPO; (2) endogenous production of H 2O2 by the oral streptococci can provide sufficient substrate H2O2 to drive this system; and (3) MPO will exert differential modulatory effects on the adherence of oral streptococci, based in part upon the level of endogenous H2O2 production and in part upon the particular characteristics of the adhesins of the bacteria.