KOZO YASUI,l* MIDORI MASUDA,2 TAKAFUMI MATSUOKA,1 MUNEHIRO YAMAZAKI,1. ATSUSHII KOMIYAMA,1 TARO AKABANE,' AND KENJIRO MURATA2.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1988, p. 1864-1868 0066-4804/88/121864-05$02.00/0 Copyright X 1988, American Society for Microbiology
Vol. 32, No. 12
Miconazole and Amphotericin B Alter Polymorphonuclear Leukocyte Functions and Membrane Fluidity in Similar Fashions KOZO YASUI,l* MIDORI MASUDA,2 TAKAFUMI MATSUOKA,1 MUNEHIRO YAMAZAKI,1 ATSUSHII KOMIYAMA,1 TARO AKABANE,' AND KENJIRO MURATA2
Department of Pediatrics, Shinshu University School of Medicine, Matsumoto 390,1 and Department of Clinico-laboratory Medicine, Kansai Medical University, Moriguchi, Osaka 570,2 Japan Received 22 April 1988/Accepted 21 September 1988
The influence of miconazole on polymorphonuclear leukocytes (PMN) was investigated and compared with that of amphotericim B (AmB). Human PMN were preincubated in vitro with miconazole or AmB at therapeutically attainable concentrations in plasma, and their chemotactic functions were assessed with the synthetic chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP). Changes in membrane fluidity of PMN were examined by an excimer-forming lipid technique. Adherence of PMN was measured by a nylon fiber column method. Miconazole and AmB pretreatment irreversibly depressed PMN random migration and chemotaxis under agarose but did not influence superoxide anion production. Both miconazole and AmB increased PMN adherence. Miconazole and AmB lowered the binding affinity of FMLP receptors on PMN and decreased the membrane fluidity in a similar manner. These results demonstrate that miconazole and AmB alter selected in vitro membrane properties of human PMN.
Amphotericin B (AmB) remains the drug of choice for
([3H]FMLP) with a specific activity of 50.4 Ci/mmol was purchased from Dupont, NEN Research Products (Boston, Mass.). Unlabeled FMLP, ferricytochrome c (type III), and superoxide dismutase were obtained from Sigma Chemical Co. (St. Louis, Mo.). Deoxycholate-free miconazole powder (Florid; molecular weight, 416.13; Mochida Seiyaku Co., Tokyo, Japan) and deoxycholate-free AmB powder (Fungizone; molecular weight, 924.09; Japan Squibb & Sons, Tokyo, Japan) were gifts. These antifungal agents were dissolved to a concentration of 10 mM in dimethyl sulfoxide (Fisher Scientific Co., Fair Lawn, N.J.) and were diluted for use in Dulbecco buffer containing 0.1% gelatin. Treatment of PMN with antifungal agents. PMN were preincubated at 37°C for 30 min with various concentrations of miconazole or AmB. After the incubation, the cells were washed twice and assayed for their motility, binding of a chemoattractant, superoxide anion release, adherence, and membrane fluidity. PMN motility. PMN motility was measured by the agarose plate method (15) with 10 nM FMLP, this concentration being effective for maximal directed migration of PMN. The center well on the plate received a 10-pdl volume of cell suspension containing 108 PMN per ml either untreated or treated with miconazole or AmB, and the outer and inner wells received an equal volume of a chemoattractant and a nonchemotactic medium, respectively. After incubation for 3 h at 37°C, migration was evaluated by measurement of linear distance moved in the direction of the chemoa,ttractant (chemotaxis) compared with that for control medium (random migration). The values are shown as migration distance in millimeters. Each experiment was done in dupli-
most systemic fungal infections. The interaction of AmB
with ergosterol in fungal cell membranes has been thoroughly studied (10, 14, 161. There are fewer studies of the interaction of AmB with human cell membranes. Most reported studies of the effect of AmB on human polymorphonuclear leukocytes (PMN) have been in vitro investigations (1, 2, 11, 12). The clinical significance of the reported in vitro AmB-induced changes in PMN function is unclear. However, clinical reports of lethal pulmonary reactions after simultaneous administration of AmB and leukocyte transfusions in patients with profound neutropenia (22, 23) are consistent with drug-induced alteration of PMN functions. A possible explanation for these pulmonary reactions is drug-induced alteration of the PMN membrane with subsequent PMN aggregation in the lungs leading to tissue damage (2, 11). Miconazole, an imidazole derivative, is an intravenous antifungal drug that is often used as an alternative to AmB. Miconazole and AmB exert their antifungal effect by alteration of fungal cell membrane permeability (4, 8, 20). Hence, miconazole might also influence human PMN membranes and alter measurable parameters of in vitro PMN functions. In this study, we analyzed the effects of miconazole on PMN functions and PMN membrane fluidity and compared them with those of AmB. MATERIALS AND METHODS PMN preparation. Heparinized (20 U/ml) peripheral blood was obtained from healthy volunteers. PMN were isolated from the blood samples by a modification of the method of Boyum (3). After lysis of erythrocytes with 0.83% NH4C1, PMN were washed twice with Dulbecco phosphate-buffered
cate.
Superoxide anion release assay. Superoxide anion (02-) measured spectrophotometrically at a wavelength of 550 nm by the superoxide dismutase-inhibitable reduction of ferricytochrome c (24). The total volume of all reaction mixtures was 1 ml; they contained 100 pAl of PMN suspension (107/ml), 50 ,ul of 2 mM ferricytochrome c, and 25 ,ul of buffer (sample tube) or superoxide dismutase (reference tube). After the reaction mixture was temperature equilibrated
saline (pH 7.4; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2PO4,
was
1.5 mM KH2PO4, 0.9 mM CaCl2, 0.5 mM MgCl2) and resuspended in the buffer until use. The purity of PMN was greater than 95% as judged by Giemsa-stained smears. Chemicals. [3H]N-formylmethionyl-leucyl-phenylalanine *
Corresponding author. 1864
EFFECTS OF MICONAZOLE AND AmB ON HUMAN PMN
VOL. 32, 1988
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