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Partial inhibition of human neutrophil activation by FK-506 at supratherapeutic concentrations
1Institut für Pharmakologie, Freie Universität Berlin, Germany.
Abstract:
The macrolide, FK-506, is a potent and effective inhibitor of lymphocyte activation. We studied the effects of FK-506 on human neutrophil activation induced by chemoattractants and by various substances which circumvent receptor stimulation. After preincubation for 5 min at 37 degrees C, FK-506 (1 microM) inhibited N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe)- or platelet-activating factor-induced superoxide production in neutrophils by about 30%. At therapeutic concentrations (0.1-1 nM) FK-506 was ineffective. FK-506 did not inhibit exocytosis and rises in cytosolic Ca2+ concentration [Ca2+]i mediated by these stimuli, and it did not at all inhibit neutrophil activation induced by C5a, leukotriene B4 and 4 beta-phorbol 12-myristate 13-acetate. FK-506 (1 microM) inhibited A23187-induced exocytosis by about 35%, but A23187-induced superoxide production was unaffected. After preincubation for 5 min at 37 degrees C, FK-506 inhibited fMet-Leu-Phe-induced superoxide production in dibutyryl cAMP-differentiated HL-60 cells by about 20%; preincubation with the drug for 24 h did not result in inhibition of superoxide production. FK-506 did not inhibit agonist-binding to formyl peptide receptors and fMet-Leu-Phe-stimulated GTP hydrolysis of heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) in membranes from dibutyryl cAMP-differentiated HL-60 cells. FK-506 did not change steady-state and differential polarized phase fluorescence in HL-60 membranes using 1,6-diphenylhexa-1,3,5-triene and 12-(9-anthroyloxy)-stearate as probes. Our results show that FK-506 at supratherapeutic concentrations partially inhibits neutrophil activation. Inhibition by FK-506 of fMet-Leu-Phe-induced superoxide production is rapid in onset and is not due to inhibition of agonist-binding to receptors, interference with G-proteins or protein kinase C, reduction of rises in [Ca2+]i or alteration in physical membrane state.
Insights
The macrolide FK-506, at high concentrations, partially inhibits human neutrophil activation, specifically superoxide production. This effect is rapid and not linked to receptor binding or G-protein signaling.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- FK-506 is a known inhibitor of lymphocyte activation.
- Neutrophil activation plays a critical role in immune responses.
Purpose of the Study:
- To investigate the effects of FK-506 on human neutrophil activation.
- To determine the mechanisms underlying FK-506's impact on neutrophils.
Main Methods:
- Human neutrophils and HL-60 cells were used to study activation.
- Superoxide production, exocytosis, and cytosolic Ca2+ concentration were measured.
- Receptor binding, G-protein activity, and membrane fluidity were assessed.
Main Results:
- FK-506 (1 microM) partially inhibited fMet-Leu-Phe- and PAF-induced superoxide production (~30%).
- Therapeutic concentrations of FK-506 were ineffective.
- FK-506 did not inhibit exocytosis, Ca2+ rises, or activation by other stimuli, except for partial inhibition of A23187-induced exocytosis.
Conclusions:
- FK-506 at supratherapeutic concentrations exhibits partial inhibition of neutrophil activation.
- The inhibitory effect on superoxide production is rapid and independent of receptor binding, G-protein signaling, or membrane alterations.
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