Interactions of Brucella abortus with bovine polymorphonuclear leukocytes: isolation and charcterization of neutrophil inhibitory components

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Canning, Peter
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Brucella abortus is an intracellular parasite capable of surviving within bovine polymorphonuclear leukocytes (PMN). The effects of B. abortus strain 2308 on bovine PMNs were examined to determine which PMN functions were impaired. Staphylococcus aureus ingestion and production of superoxide anion were not inhibited by heat-killed B. abortus. Iodination of proteins by PMNs (a measure of the myeloperoxidase-H(,2)O(,2)-halide antibacterial system) was markedly inhibited in the presence of live or heat-killed bacteria. The inhibitory activity could be associated with the supernatant from heat-killed cells and the components responsible for the suppression were characterized as heat and pH stable molecules with molecular weights of less than 1,000 daltons. Isolation of the inhibitory components by high performance liquid chromatography (HPLC) yielded two molecules, denoted fraction 3b and fraction 10, which decreased iodination activity in a concentration-dependent manner. Chemical characterization indicated both fractions 3b and 10 possessed properties of nucleotides or nucleotide-like molecules. Chemical characterization of B. abortus fractions 3b and 10 by HPLC and thin layer chromatography indicated that fraction 3b co-eluted and co-migrated with 5'-guanosine monophosphate (GMP) while fraction 10 co-eluted and co-migrated with adenine. The biological effects of GMP and adenine on PMN functions were similar to those obtained with fractions 3b and 10. Analysis of culture supernatants from viable B. abortus indicated that both GMP and adenine are produced by viable bacteria. These studies suggest that fractions 3b and 10 are GMP and adenine respectively and that these materials contribute to the inhibition of intracellular destruction of B. abortus by PMNs. Inhibition of anion fluxes across the plasma membranes of human PMNs has been shown to produce similar effects on PMN function as fractions 3b and 10. An assay for detecting and measuring fluxes of SO(,4)('2-) anions across the plasma membrane was adapted for use with bovine PMNs. Sulfate influx, but not efflux, was significantly inhibited by the anion channel blocker 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) and by 2'-deoxy-D-glucose. The ability of PMNs to migrate under agarose, ingest S. aureus, and iodinate protein was significantly depressed in the presence of DIDS. Superoxide anion production and antibody-dependent cell-mediated cytotoxicity by neutrophils were not impaired by DIDS.

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Tue Jan 01 00:00:00 UTC 1985