Faculty Bibliography

The objective of this investigation was to examine the effects of low levels of Hg(II) on the respiratory burst of PMNs by monitoring O2 consumption, superoxide radical formation, and chemiluminescence. Hg(II) at concentrations of 10-100 ng/ml profoundly inhibited zymosan-stimulated human cells. This inhibition was immediate in onset and occurred with minimal loss of cell viability. Effects of Hg(II) on the PMN respiratory burst were compared with those of Sn, Pb, Se, Au, Ag and Cu. Only in the case of Ag and Cu did the inhibitory effects approach those of Hg. The results indicate that Hg(II) may serve as a specific inhibitor of components of the respiratory burst.

Human blood leukocytes generate intense luminol-dependent chemiluminescence (LDCL) following stimulation by streptococci and by Gram negative rods which had been preopsonized by cationic polyelectrolytes (histone, poly L-arginine-PARG, poly L-histidine-PHSTD). Streptococci but not Gram negative rods or hyaluronic acid-rich streptococci (group C) also induced intense LDCL following opsonization with the anionic polyelectrolytes-dextran sulfate or polyanethole sulfonate (liquoid) suggesting that the outer surfaces of different bacteria bound anionic polyelectrolytes to different extents. Both normal and immune serum, synovial fluids and pooled human saliva inhibited the LDCL responses induced by streptococci preopsonized with poly cations. On the other hand, bacteria which had been first preopsonized by the various body fluids and then subjected to a second opsonization by cationic ligands ("sandwiches"), induced a very intense LDCL response in leukocytes. Streptococci which had been preopsonized by PARG, histone or by PHSTD also triggered superoxide generation by blood leukocytes, which was markedly enhanced by a series of cytochalasins. PHSTD alone induced the formation of very large amounts of superoxide. Paradoxically, the same concentrations of cytochalasins B or C which markedly boosted the generation of superoxide following stimulation of leukocytes with soluble or particulate ligands, had a strong inhibitory effect on the generation of LDCL. On the other hand cycochalasins failed to inhibit LDCL which had been induced by phorbol myristate acetate (PMA). Peritoneal macrophages which had been harvested from C. parvum-stimulated mice, generated more LDCL and superoxide following stimulation by PARG than macrophages obtained from proteose peptone-stimulated mice. Macrophages which had been activated either by proteose peptone or by C. parvum and cultivated for 2 hours on teflon surfaces, generated much more LDCL than macrophages which had been cultivated for 24 hours on teflon surfaces. Both cationic and anionic polyelectrolytes mimic the effects of antibodies as activators of the oxygen burst in blood leukocytes and in macrophages. Such polyelectrolytes can serve as models to further study leukocyte-bacteria interactions in infectious and inflammatory sites.

Aggregation of bacteria by zinc and lysozyme was studied and compared with aggregation induced by a high-molecular-weight salivary agglutinin. Each ligand was found to exhibit a unique profile of properties when examined by both a microradiochemical centrifugation assay and a turbidimetric assay. Significant differences in rate of aggregation and bacterial species specificity were noted. Zinc- and lysozyme-mediated aggregations were shown to be calcium independent and to proceed rapidly at 0 degree C, in contrast to the salivary agglutinin. Zinc produced large, asymmetric aggregates, saliva produced intermediate-sized aggregates, and lysozyme produced the smallest aggregates. These size differences are consistent with many of the observed reaction properties.

Various cationic polyelectrolytes (poly-alpha-amino acids and histones), lectins, the chemotactic peptide, f-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187, and phorbol myristate acetate (PMA) were investigated regarding their capacity to induce luminol-dependent chemiluminescence (LDCL) and superoxide production by human blood leukocytes. Although when tested individually, poly-L-arginine (PARG), phytohemagglutinin (PHA), concanavalin A (Con A), or fMLP induced only a low to moderate LDCL response, very intense synergistic CL reactions were obtained by mixtures of PARG + PHA, PARG + Con A, PARG + PHA + fMLP, Ca2 + ionophore + PARG + PHA + fMLP, and PARG + PMA. The sequence of addition of the various agents to WBC in the presence of luminol absolutely determined the intensity of the LDCL signals obtained, the highest reactions being achieved when the WBC were preincubated for 2-3 min with A23187 followed by the sequential addition of fMLP, PARG, and PHA. These "multiple hits" induced CL reactions which were many times higher than those obtained by each factor alone. On the other hand, neither poly-L-lysine, poly-L-ornithine, poly-L-histidine, nor poly-L-asparagine, when employed at equimolar concentrations, cooperated efficiently with PHA and fMLP to trigger synergistic LDCL responses in leukocytes. Concomitantly with the induction of LDCL, certain ligand mixtures also triggered the production of superoxide. The LDCL which was induced by the "cocktail" of agents was markedly inhibited by sodium azide (93% inhibition), but to a lesser extent by catalase (10% inhibition) or by superoxide dismutase (20%-60% inhibition). On the other hand, scavengers of singlet oxygen and OH (sodium benzoate, histidine) did not affect the synergistic LDCL responses induced by these multiple ligands. Cytochalasin B also markedly inhibited the LDCL responses induced either by soluble stimuli or by streptococci preopsonized either with histone or with polyanethole sulfonate. The LDCL responses which were induced by mixtures of PARG and concanavalin A were also strongly inhibited by mannose, alpha-methyl mannoside, and poly-L-glutamic acid. The data suggest that the LDCL responses induced by the soluble ligands involved a myeloperoxidase-catalyzed reaction. The possible employment of "cocktails" of ligands to enhance the bactericidal effects of PMNs, macrophages, and natural killer cells on microbial cells and mammalian targets is discussed.

A leukotoxin from Actinobacillus actinomycetemcomitans was isolated by a procedure that includes polymyxin B extraction, ion-exchange chromatography, and gel filtration chromatography. The procedure resulted in the recovery of 48% of the toxin with a 99-fold increase in specific activity. The isolated toxin has a molecular mass of 180,000 daltons by gel filtration and 115,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It retains all the major biological characteristics previously documented for crude leukotoxin preparations, including susceptibility to heat and proteolytic enzymes and neutralization by sera from patients with juvenile periodontitis. The isolated leukotoxin destroys human but not rat or guinea pig polymorphonuclear leukocytes and has no apparent effect on human erythrocytes. The availability of the A. actinomycetemcomitans leukotoxin should facilitate studies on its chemistry and mode of action as well as its role in the pathogenesis of human periodontal disease.

Human and animal sera contain potent inhibitors of saliva-mediated aggregation of oral streptococci. The inhibitors consist of a high-molecular-weight heat-labile factor and a lower-molecular-weight heat-activated factor. The latter appears to be serum albumin. Analyses of purified blood-derived proteins indicated that several high-molecular-weight proteins (fibrinogen, fibronectin, and ferritin) were able to inhibit aggregation at low concentrations. These data suggest that high-molecular-weight proteins may modulate the aggregation process.

Human parotid saliva contains agglutinins which bind to the surface of streptococci and induce the formation of bacterial aggregates. Bacterial aggregation can be blocked by proteins released from viable PMNs and platelets or by sonic extracts prepared from these cells. PMN and platelet inhibitors display characteristic differences in molecular weight, protease, and temperature sensitivity. The mechanism of action of the inhibitors appears to involve a direct interaction with the salivary agglutinins rather than with the bacteria. It is thus possible that PMN and platelet-derived products might modulate saliva-mediated bacterial aggregation and thereby influence the course of infections in the oral cavity.