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Polymorphonuclear leukocyte opsonic receptor expression after hypoxia/reoxygenation
1Brown University School of Medicine, Department of Surgery, Providence, Rhode Island, USA.
Abstract:
We investigated the effects of hypoxia/reoxygenation (H/R) and subsequent stimulation of polymorphonuclear leukocytes (PMNs) with either formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA) on CD32, CD16, CD35, and CD11b/CD18 expression and on degranulation and superoxide anion production. H/R primed both adherent and fluid-phase PMNs for subsequent up-regulation of CD32 and CD16 (Fcgamma receptors) when stimulated with FMLP and primed both Fcgamma and complement (CD35, CD11b/CD18) receptors when stimulated with PMA. Kinetics assays demonstrated maximal up-regulation of CD32 and CD16 induced by H/R plus FMLP after 30 minutes of reoxygenation, whereas maximal receptor stimulation by H/R plus PMA occurred within 15 minutes of reoxygenation. Neither actinomycin D nor cycloheximide abrogated the effect of H/R with subsequent stimulation of PMNs on receptor expression; however, 10(-5) to 10(-8) mol/L concentrations of either taxol or phalloidin completely abrogated the effect of H/R plus FMLP or PMA on opsonic receptor expression. The effect of H/R plus FMLP on CD32 and CD16 expression was blocked by pertussis toxin, whereas staurosporine, H-7, H-9, and genistein had no effect. Conversely, the effect of H/R plus PMA on CD32, CD16, CD35, and CD11b/CD18 expression was blocked by staurosporine and H-7 but not by H-9, pertussis toxin, or genistein. The up-regulation of CD32, CD16, CD35, and CD11b/CD18 induced by H/R plus FMLP or PMA in the presence or absence of matrix proteins resulted in the increased rosetting of E-anti-CD32, E anti-CD16, E-Con A, EC3b, and EC3bi, respectively. Reduced nicotinamide adenine dinucleotide phosphate oxidase inhibition with diphenyleneiodonium blocked the effect of H/R on receptor expression, degranulation, and superoxide anion production. These results demonstrate that H/R primes PMNs for subsequent receptor up-regulation by divergent intracellular signal transduction pathways and that the receptors induced to the cell surface are biologically active.
Insights
Hypoxia/reoxygenation primes polymorphonuclear leukocytes (PMNs) for increased receptor expression. This priming enhances PMN function, impacting degranulation and superoxide production through distinct signaling pathways.
Area of Science:
- Immunology
- Cellular Biology
- Physiology
Background:
- Hypoxia/reoxygenation (H/R) is a critical condition affecting cellular function.
- Polymorphonuclear leukocytes (PMNs) play a key role in immune responses.
- Understanding PMN receptor modulation during H/R is crucial for immune research.
Purpose of the Study:
- To investigate the effects of H/R on PMN receptor expression (CD32, CD16, CD35, CD11b/CD18).
- To determine the impact of H/R on PMN degranulation and superoxide anion production.
- To elucidate the intracellular signaling pathways involved in H/R-induced PMN activation.
Main Methods:
- PMNs were subjected to hypoxia/reoxygenation (H/R) and stimulated with FMLP or PMA.
- Flow cytometry was used to assess receptor expression (CD32, CD16, CD35, CD11b/CD18).
- Degranulation and superoxide anion production were measured; signaling pathways were investigated using inhibitors.
Main Results:
- H/R primed PMNs for enhanced expression of Fcgamma (CD32, CD16) and complement receptors (CD35, CD11b/CD18) upon stimulation.
- Maximal receptor upregulation occurred at different time points for FMLP and PMA stimulation.
- H/R-induced receptor expression involved distinct intracellular pathways, modulated by cytoskeletal elements and protein kinases, and was dependent on NADPH oxidase activity.
Conclusions:
- H/R primes PMNs, leading to increased expression of functionally active opsonic receptors.
- Divergent intracellular signal transduction pathways mediate H/R-induced receptor upregulation.
- These findings highlight the complex role of H/R in modulating PMN immune function.