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Secretory activity of equine polymorphonuclear leukocytes: stimulus specificity and priming effects of bacterial
P N Bochsler1, D O Slauson, N R Neilsen
1Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville 37901-1071.
Abstract:
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.
Insights
Bacterial lipopolysaccharide (LPS) primes equine neutrophils (PMN) to enhance their inflammatory response to other stimuli like phorbol ester. This priming effect significantly boosts superoxide anion generation and protease release, crucial for host defense.
Area of Science:
- Immunology
- Veterinary Medicine
- Cellular Biology
Background:
- Neutrophils (PMN) are critical for acute inflammation and host defense, mediating their functions through reactive oxygen metabolites and granule enzyme secretion.
- Understanding equine PMN responses to various stimuli is essential for comprehending inflammatory processes and host defense mechanisms in horses.
Purpose of the Study:
- To assess equine PMN secretion in response to different stimuli, including bacterial lipopolysaccharide (LPS) alone and in combination with other activators.
- To investigate the priming effect of LPS on equine PMN's ability to generate superoxide anion (O2-) and release granule enzymes.
Main Methods:
- Equine PMN were exposed to various stimuli: bacterial LPS, phorbol ester (PMA), opsonized zymosan, recombinant human C5a, arachidonic acid (ArA), fMLP, and platelet-activating factor.
- PMN association with FITC-conjugated LPS was assessed via labeling.
- Superoxide anion (O2-) generation and release of granule enzymes (lysozyme, non-specific proteases, beta-glucuronidase, acid phosphatase, alkaline phosphatase) were quantified.
Main Results:
- Bacterial LPS avidly associated with equine PMN but was a weak stimulus for O2- generation alone.
- Preincubation with LPS significantly augmented O2- generation and protease release when followed by PMA stimulation, indicating a priming effect.
- Arachidonic acid (ArA) was a potent stimulus for O2- generation, but fMLP or platelet-activating factor did not further enhance this response.
- PMN granule enzyme release (lysozyme, proteases) was significantly increased by PMA, with a slight enhancement when LPS pre-incubation preceded PMA.
- Proteolytic activity was markedly reduced in the presence of normal equine serum.
Conclusions:
- Bacterial LPS primes equine neutrophils, enhancing their responsiveness to subsequent stimuli like PMA, which is critical for effective host defense.
- The study elucidates the complex secretory responses of equine PMN to various stimuli, highlighting the synergistic effects of LPS and PMA.
- Equine PMN enzyme activity is sensitive to serum components, suggesting a regulatory role for serum in modulating inflammatory responses.