Partial inhibition of human neutrophil activation by FK-506 at supratherapeutic concentrations

Abstract

Summary. 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 °C, FK-506(fMet-LeuPhe)-or platelet-activating factor-induced superoxide production in neutrophils by about 30°7o. At therapeutic concentrations (0.1-1nM) FK-506 was ineffective. FK-506 did not inhibit exocytosis and rises in cytosolic Ca 2÷ concentration [Ca2+] i mediated by these stimuli, and it did not at all inhibit neutrophil activation induced by C5a, leukotriene B 4 and 4fl-phorbol 12-myristate 13-acetate. FK-506 (1 gM) inhibited A23187-induced exocytosis by about 35%, but A23187-induced superoxide production was unaffected. After preincubation for 5 min at 37 °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 nucleotidebinding 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 Hb60 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.

Tacrolimus: In Vitro Effects on Myelopoiesis, Apoptosis, and CD11b Expression

“…17 We observed no differences in upregulation of the CD11b surface adhesion molecule in response to chemotactic stimulation between neutrophils that had been "primed" with tacrolimus and untreated cells. This observation contrasts with studies showing tacrolimusinduced inhibition of other neutrophil functions induced by fMLP, including migration and the production of reactive oxygen intermediates, 18,19 but is consistent with a lack of a direct effect of tacrolimus on fMLPinduced chemotaxis. 20 Our present observations might be related to an intrinsically diminished responsiveness of cord blood neutrophils.…”

The Immunosuppressant Cyclosporin A Antagonizes Human Formyl Peptide Receptor through Inhibition of Cognate Ligand Binding

“…Therefore, the inhibition of fMLF-induced degranulation by CsA may be attributed to a mechanism irrelevant to FPR antagonism. In fact, it was reported that FK-506, a potent immunosuppressant, could inhibit several neutrophil functions induced by fMLF in a FPR-independent manner (31). A previous study conducted on human basophils showed that CsA failed to competitively displace receptor-bound [ 3 H]fMLF (32).…”

Evidence that inhibition of phorbol ester-induced superoxide anion formation by cyclosporin a in phagocytes is not mediated by direct inhibition of protein kinase C

“…These findings raise the question of whether CsA inhibits 02 formation through alterations in physical membrane properties. However, CsA did not affect limiting anisotropy (a static measure) [20] and rotational correlation time (a dynamic measure) [20] of a fluorescent probe in HL-60 membranes, nor did it affect the activity of NA+/K+-ATPase and MgZ+-ATPase in HE-60 membranes. These findings render it unlikely that CsA inhibits 02 formation through changes in physical membrane properties.…”

“…We also studied the effect of CsA on physical properties of HL-60 membranes using steadystate polarization and differential polarized phase fluorometry with 1,6-diphenylhexa-1,3,5-triene probe [20]. We assessed limiting anisotropy and rotational correlation time.…”

“…Steady-state and differential polarized phase fluorometry. Determination of steady-state and differential phase fluorescence of 1,6-diphenylhexa-l,3,5-triene-loaded HL-60 membranes was performed as described [20]. Membranes were incubated for 10 min at 25 ° in the presence of solvent (control) or CsA.…”