Secretory activity of equine polymorphonuclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide.
Neutrophil (PMN) contributions to the acute inflammatory process and host defense include generation of bioreactive oxygen metabolites and secretion of granule enzymes. We assessed equine PMN secretion using several PMN stimuli, singly and in combination with bacterial lipopolysaccharide (LPS). LPS avidly associated with equine PMN, as shown by strong PMN labeling with FITC-conjugated LPS. LPS alone (1 or 10 micrograms ml-1) was a weak stimulus for PMN superoxide anion (O2-) generation, but preincubation with LPS followed by phorbol ester (PMA, 10 ng ml-1) significantly augmented (P less than 0.01) secretion of O2- (19.38 nmol O2- per 2 x 10(6) PMN per 5 min) over the amount generated by PMA stimulation alone (13.75 nmol O2-). A qualitatively similar, but smaller O2(-)-generation response occurred when either opsonized zymosan or recombinant human C5a was used as the PMN stimulus. Arachidonic acid (ArA; 50-200 microM) was a potent stimulus, with secreted O2- levels similar to those from PMA-stimulated PMN. Preincubation of PMN with either the formyl peptide, fMLP, or platelet-activating factor before stimulation with ArA did not significantly increase O2- generation over levels obtained using ArA alone. Release of PMN granule enzymes was also quantitated. A small amount of lysozyme secretion resulted when PMN were exposed to LPS alone (8.20% of total cell content), and PMA stimulation caused marked release of PMN lysozyme (44.45%). Non-specific proteolytic activity in PMN supernatants, assessed by cleavage of a collagen-rich substrate, was minimal with LPS as a sole stimulus (5.08%). There was significant proteolytic activity (P less than 0.01) in supernatants from PMA-stimulated PMN (27.21%), and preincubation with LPS followed by PMA stimulation slightly enhanced (P less than 0.05) the release of PMN proteases (34.62%). The activities of beta-glucuronidase, acid phosphatase, and alkaline phosphatase were minimal in PMN supernatants when using LPS and PMA as stimuli. The activity of PMN granule enzymes was found to be sensitive to the presence of normal equine serum, and proteolytic activity was markedly reduced (80.13% reduction) in the presence of 10% pooled serum.
Bochsler
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Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville 37901-1071.
Slauson
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Neilsen
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D000818:Animals; D003594:Cytoplasmic Granules / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D006736:Horses; D006867:Hydrolases / Q000378:metabolism; D008070:Lipopolysaccharides; D009504:Neutrophils / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
D008070:Lipopolysaccharides; D013481:Superoxides; D015054:Zymosan; D006867:Hydrolases; D010447:Peptide Hydrolases; D016650:Fluorescein-5-isothiocyanate; D013755:Tetradecanoylphorbol Acetate