Vol. 14, No. 1 Printed in U.S.A.
INFECTION AND IMMUNITY, July 1976, p. 315-317 Copyright © 1976 American Society for Microbiology
Inhibition of Human Neutrophil Chemotaxis and Chemiluminescence by Amphotericin B BENGT BJORKST£N,I* CASANN RAY, AND PAUL G. QUIE Department of Pediatrics, UniversitY of Minnesota School of Medicine, Minneapolis, Minnesota 55455 Received for publication 23 February 1976
The effect of the antifungal drugs amphotericin B, fluocytosine, miconazole, griseofulvin, and nystatin on the chemotactic responsiveness of human neutrophils was studied. Amphotericin B in a concentration of 2 ,ug/ml inhibited chemotactic responsiveness, and in a concentration of 5 ,ug/ml it also inhibited chemiluminescence. The inhibition of chemotaxis could be reversed by washing the cells. The other antifungal drugs did not inhibit chemotaxis even in concentrations much higher than those obtained in human serum during treatment. Recent reports suggest that certain antimicrobial drugs in vitro interfere with granulocyte function; e.g., tetracycline inhibits neutrophil chemotaxis (8) and phagocytosis of Candida (3), certain sulfonamides interfere with intracellular killing (7), and gentamicin has an adverse effect on chemotaxis (A. J. Khan, H. E. Evans, L. Glass, and P. Khan, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 15th, Washington, D.C., Abstr. 65, 1975). These investigations raised the question of the effect of antifungal drugs on chemotaxis. Depression of neutrophil function by these antibiotics may adversely effect response to antimicrobial treatment. We report here the inhibitory effect of the antifungal drug amphotericin B on neutrophil chemotaxis and lack of suppression by fluocytosine, miconazole, griseofulvin, and Mycostatin (nystatin). Chemotaxis was assessed by the leading front technique (11). Suspensions of leukocytes were obtained by sedimentation of the erythrocytes of freshly drawn heparinized blood from healthy persons. The white cell-rich buffy coat was diluted with Hanks balanced salt solution (HBSS) containing 3 ml of 1 M HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.3) per 100 ml to a final concentration of 7 x 10' to 8 x 10"' polymorphonuclear leukocytes per ml. Samples of the cell suspensions were incubated for 30 min in a 37 C water bath with equal amounts of amphotericin B (Squibb & Sons, Princeton, N.J.), fluocytosine (Roche Laboratories, Nutley, N.J.), miconazole (Janssen R & D, New Brunswick, N.J.), griseofulvin (Schering Corp., Bloomfield, N.Y.), or Present address: Department of Pediatrics, University of UmeA, S. 901 85, Sweden.
Mycostatin (nystatin) (Lederle Laboratories, Pearl River, N.Y.) of different concentrations. All drugs were diluted in buffered HBSS. In the upper compartment of a modified Boyden chamber (Neuroprobe Corp., Bethesda, Md.) was deposited 0.4 ml of the leukocyte drug suspension, and this compartment was separated from the lower by a 5-,um membrane filter (Millipore Corp., Bedford, Mass.). Leukocytes from the same donors incubated with buffered HBSS served as controls. An Escherichia coli culture filtrate was used as the chemotactic agent (6). The chemotaxis of cells incubated with drugs was expressed as a percentage value of the chemotaxis of simultaneously run control leukocytes. In two experiments, the leading front had passed through the filter and the results of these assays were evaluated by counting the number of neutrophils on the bottom surface of the filters in 10 randomly selected fields, using a magnification of x360 and a reticule (5 by 5 ,um). Random migration was assessed by the agarose technique as described by Nelson et al. (9), using buffy-coat suspensions of leukocytes. Chemiluminescence during phagocytosis was measured by the method described by Allen et al. (1), using opsonized zymosan to stimulate neutrophil light production. The peak of chemiluminescence of neutrophils incubated with amphotericin B was expressed as a percentage of the peak chemiluminescence produced by control leukocytes from the same donor, incubated with opsonized zymosan without added amphotericin B. To study the effect of amphotericin B on neutrophil phagocytosis, the uptake of preopsonized, radiolabeled streptococci, group B, was measured at a bacteria/polymorphonuclear leukocyte ratio of 5 to 10:1, using a method re315
316
INFECT. IMMUN.
NOTES
cently developed in our laboratory (Peterson et al., submitted for publication). As shown in Table 1, incubation of leukocytes with amphotericin B inhibited chemotaxis. The concentrations used to demonstrate this effect of amphotericin B were within the range found in the plasma of patients treated with this drug. There was no effect of amphotericin B on random migration under agarose. No effect could be demonstrated by fluocytosine (1 to 100 ,ug/ml), miconazole (0.4 to 50 ,ug/ml), griseofulvin (3.1 to 50 ug/ml), or nystatin (0.8 to 100 ug/ ml), not even in concentrations much above those found in the plasma of patients treated with these drugs. Amphotericin B also inhibited neutrophil chemiluminescence, 5 ,ug/ml being moderately inhibitory (Fig. 1). Therefore, amphotericin B inhibits the oxidative metabolic response of granulocytes as well as chemotaxis. A 5-gg/ml concentration of amphotericin B did not affect phagocytosis, whereas 20 gg/ml was inhibitory. Because this concentration is higher than would be found in the plasma of patients treated with amphotericin B, the clinical relevance of this effect on phagocytosis is uncertain. Amphotericin B and nystatin, like the other polyene antibiotics, are fungicidal and have an affinity for sterols in the cell membrane, which becomes more permeable to water and solutes (3). The other three antifungal drugs are fungistatic; fluocytosine is incorporated into and modifies fungal ribonucleic acid (10) and miconazole has an effect primarily on the plasmalemma and the cell wall (10), whereas the mode of action for griseofulvin is not known in detail (4). The affinity for cell membranes may explain the effect of aniphotericin B on chemotaxis. Although nystatin has a similar mode of TABLE 1. Effect of exposure to amphotericin B on neutrophil responsiveness to chemotactic stimulus Concn of drug in which cells were incubated
Amphotericin B 5 ,ug/ml 2
gg/ml
Expt la
21,
3a
4b
5b
34 55 74 111 100
81 71 91 100 100
86
7 9 21 31 34
6
100
1.1
90
80 75
0 0 0'
70
60 50
40 F
30F 20F l0 1.
SX
0.5
2
5
20
Concentration of Amphotericin B (jig/ml) 1. FIG. Effect of exposure to amphotericitn B on neutrophil chemiluminescence (mean and range of five experiments). Results are expressed as a percentage of chemiluminescence by simultaneously run unincubated neutrophils from the same person.
action to that of amphotericin B, neutrophil cell membranes may differ in sensitivity to the two drugs. Future studies of the in vivo effect of amphotericin B on chemotaxis and chemiluminescence should be done to evaluate the clinical relevance of the in vitro findings. Such studies are complicated by the fact that patients receiving amphotericin B frequently have severe generalized mycotic infections secondary to an underlying disease or to immunosuppressive treatment. Finding abnormal chemotaxis in the granulocytes of these patients would be difficult to interpret. This work was supported in part by Public Health Service research grants AI06931, AI08821, and AI12402 from the National Institute of Allergy and Infectious Diseases, and by the J. C. Kempe Foundation, Sweden.
LITERATURE CITED
26 95 1,ug/ml 90 0.2 ,ug/ml 98 55 100 Hanks BSS The distance traveled by the leading front leukocytes expressed as a percentage of the simultaneously run control. Numbers indicate mean triplicate assays on three separate dates. b Number of leukocytes on bottom of filter. Results are expressed as number of cells in 10 highpower fields. 11
lb0
1. Allen, R. C., R. L. Stjernholm, and R. H. Steele. 1972.
Evidence for the generation of electronic excitation states in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem.
Biophys. Res. Commun. 47:679-684. 2. De Nollin, S., and M. Borgers. 1974. The ultrastructure of Candida albicans after in vitro treatment with miconazole. Sabouraudia 12:341-351. 3. Forsgren, A., D. Schmeling, and P. G. Quie. 1974. Effects of tetracycline on the phagocytosis function of human leukocytes. J. Infect. Dis. 130:412-415. 4. Franklin, T. J., and G. A. Snow. 1971. Biochemistry of antimicrobial action, p. 56-58. Academic Press Inc., New York.
VOL. 14, 1976 5. Goodman, L. S., and A. Gilman. 1975. The pharmacological basis of therapeutics, 5th ed., p. 1239-1241. Macmillan, New York. 6. Hill, H. R., R. D. Estenson, P. G. Quie, N. A. Hogan, and N. D. Goldberg. 1975. Modulation of human neutrophil chemotactic responses by cyclic 3'5'-guanosine monophosphate and cyclic 3'5'-adenosine monophosphate. Metabolism 24:447-456. 7. Lehrer, R. I. 1971. Inhibition by sulfonamides of the candidicidal activity of human neutrophils. J. Clin. Invest. 50:2498-2505. 8. Martin, R. R., G. A. Wau, R. B. Couch, H. Yeager, and
NOTES
317
V. Knight. 1974. Effects of tetracycline on leukotaxis. J. Infect. Dis. 129:110-116. 9. Nelson, R. D., P. G. Quie, and R. L. Simmons. 1975. Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J. Immunol. 115:1650-1656. 10. Polak, A. 1974. Effects of 5-fluorocytosine on protein synthesis and amino acid pool in Candida albicans. Sabouraudia 12:309-319. 11. Wilkinson, P. C. 1974. Chemotaxis and inflammation, p. 168-172. Churchill Livingston, London.
INFECTION AND IMMUNITY, July 1976, p. 315-317 Copyright © 1976 American Society for Microbiology
Inhibition of Human Neutrophil Chemotaxis and Chemiluminescence by Amphotericin B BENGT BJORKST£N,I* CASANN RAY, AND PAUL G. QUIE Department of Pediatrics, UniversitY of Minnesota School of Medicine, Minneapolis, Minnesota 55455 Received for publication 23 February 1976
The effect of the antifungal drugs amphotericin B, fluocytosine, miconazole, griseofulvin, and nystatin on the chemotactic responsiveness of human neutrophils was studied. Amphotericin B in a concentration of 2 ,ug/ml inhibited chemotactic responsiveness, and in a concentration of 5 ,ug/ml it also inhibited chemiluminescence. The inhibition of chemotaxis could be reversed by washing the cells. The other antifungal drugs did not inhibit chemotaxis even in concentrations much higher than those obtained in human serum during treatment. Recent reports suggest that certain antimicrobial drugs in vitro interfere with granulocyte function; e.g., tetracycline inhibits neutrophil chemotaxis (8) and phagocytosis of Candida (3), certain sulfonamides interfere with intracellular killing (7), and gentamicin has an adverse effect on chemotaxis (A. J. Khan, H. E. Evans, L. Glass, and P. Khan, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 15th, Washington, D.C., Abstr. 65, 1975). These investigations raised the question of the effect of antifungal drugs on chemotaxis. Depression of neutrophil function by these antibiotics may adversely effect response to antimicrobial treatment. We report here the inhibitory effect of the antifungal drug amphotericin B on neutrophil chemotaxis and lack of suppression by fluocytosine, miconazole, griseofulvin, and Mycostatin (nystatin). Chemotaxis was assessed by the leading front technique (11). Suspensions of leukocytes were obtained by sedimentation of the erythrocytes of freshly drawn heparinized blood from healthy persons. The white cell-rich buffy coat was diluted with Hanks balanced salt solution (HBSS) containing 3 ml of 1 M HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.3) per 100 ml to a final concentration of 7 x 10' to 8 x 10"' polymorphonuclear leukocytes per ml. Samples of the cell suspensions were incubated for 30 min in a 37 C water bath with equal amounts of amphotericin B (Squibb & Sons, Princeton, N.J.), fluocytosine (Roche Laboratories, Nutley, N.J.), miconazole (Janssen R & D, New Brunswick, N.J.), griseofulvin (Schering Corp., Bloomfield, N.Y.), or Present address: Department of Pediatrics, University of UmeA, S. 901 85, Sweden.
Mycostatin (nystatin) (Lederle Laboratories, Pearl River, N.Y.) of different concentrations. All drugs were diluted in buffered HBSS. In the upper compartment of a modified Boyden chamber (Neuroprobe Corp., Bethesda, Md.) was deposited 0.4 ml of the leukocyte drug suspension, and this compartment was separated from the lower by a 5-,um membrane filter (Millipore Corp., Bedford, Mass.). Leukocytes from the same donors incubated with buffered HBSS served as controls. An Escherichia coli culture filtrate was used as the chemotactic agent (6). The chemotaxis of cells incubated with drugs was expressed as a percentage value of the chemotaxis of simultaneously run control leukocytes. In two experiments, the leading front had passed through the filter and the results of these assays were evaluated by counting the number of neutrophils on the bottom surface of the filters in 10 randomly selected fields, using a magnification of x360 and a reticule (5 by 5 ,um). Random migration was assessed by the agarose technique as described by Nelson et al. (9), using buffy-coat suspensions of leukocytes. Chemiluminescence during phagocytosis was measured by the method described by Allen et al. (1), using opsonized zymosan to stimulate neutrophil light production. The peak of chemiluminescence of neutrophils incubated with amphotericin B was expressed as a percentage of the peak chemiluminescence produced by control leukocytes from the same donor, incubated with opsonized zymosan without added amphotericin B. To study the effect of amphotericin B on neutrophil phagocytosis, the uptake of preopsonized, radiolabeled streptococci, group B, was measured at a bacteria/polymorphonuclear leukocyte ratio of 5 to 10:1, using a method re315
316
INFECT. IMMUN.
NOTES
cently developed in our laboratory (Peterson et al., submitted for publication). As shown in Table 1, incubation of leukocytes with amphotericin B inhibited chemotaxis. The concentrations used to demonstrate this effect of amphotericin B were within the range found in the plasma of patients treated with this drug. There was no effect of amphotericin B on random migration under agarose. No effect could be demonstrated by fluocytosine (1 to 100 ,ug/ml), miconazole (0.4 to 50 ,ug/ml), griseofulvin (3.1 to 50 ug/ml), or nystatin (0.8 to 100 ug/ ml), not even in concentrations much above those found in the plasma of patients treated with these drugs. Amphotericin B also inhibited neutrophil chemiluminescence, 5 ,ug/ml being moderately inhibitory (Fig. 1). Therefore, amphotericin B inhibits the oxidative metabolic response of granulocytes as well as chemotaxis. A 5-gg/ml concentration of amphotericin B did not affect phagocytosis, whereas 20 gg/ml was inhibitory. Because this concentration is higher than would be found in the plasma of patients treated with amphotericin B, the clinical relevance of this effect on phagocytosis is uncertain. Amphotericin B and nystatin, like the other polyene antibiotics, are fungicidal and have an affinity for sterols in the cell membrane, which becomes more permeable to water and solutes (3). The other three antifungal drugs are fungistatic; fluocytosine is incorporated into and modifies fungal ribonucleic acid (10) and miconazole has an effect primarily on the plasmalemma and the cell wall (10), whereas the mode of action for griseofulvin is not known in detail (4). The affinity for cell membranes may explain the effect of aniphotericin B on chemotaxis. Although nystatin has a similar mode of TABLE 1. Effect of exposure to amphotericin B on neutrophil responsiveness to chemotactic stimulus Concn of drug in which cells were incubated
Amphotericin B 5 ,ug/ml 2
gg/ml
Expt la
21,
3a
4b
5b
34 55 74 111 100
81 71 91 100 100
86
7 9 21 31 34
6
100
1.1
90
80 75
0 0 0'
70
60 50
40 F
30F 20F l0 1.
SX
0.5
2
5
20
Concentration of Amphotericin B (jig/ml) 1. FIG. Effect of exposure to amphotericitn B on neutrophil chemiluminescence (mean and range of five experiments). Results are expressed as a percentage of chemiluminescence by simultaneously run unincubated neutrophils from the same person.
action to that of amphotericin B, neutrophil cell membranes may differ in sensitivity to the two drugs. Future studies of the in vivo effect of amphotericin B on chemotaxis and chemiluminescence should be done to evaluate the clinical relevance of the in vitro findings. Such studies are complicated by the fact that patients receiving amphotericin B frequently have severe generalized mycotic infections secondary to an underlying disease or to immunosuppressive treatment. Finding abnormal chemotaxis in the granulocytes of these patients would be difficult to interpret. This work was supported in part by Public Health Service research grants AI06931, AI08821, and AI12402 from the National Institute of Allergy and Infectious Diseases, and by the J. C. Kempe Foundation, Sweden.
LITERATURE CITED
26 95 1,ug/ml 90 0.2 ,ug/ml 98 55 100 Hanks BSS The distance traveled by the leading front leukocytes expressed as a percentage of the simultaneously run control. Numbers indicate mean triplicate assays on three separate dates. b Number of leukocytes on bottom of filter. Results are expressed as number of cells in 10 highpower fields. 11
lb0
1. Allen, R. C., R. L. Stjernholm, and R. H. Steele. 1972.
Evidence for the generation of electronic excitation states in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem.
Biophys. Res. Commun. 47:679-684. 2. De Nollin, S., and M. Borgers. 1974. The ultrastructure of Candida albicans after in vitro treatment with miconazole. Sabouraudia 12:341-351. 3. Forsgren, A., D. Schmeling, and P. G. Quie. 1974. Effects of tetracycline on the phagocytosis function of human leukocytes. J. Infect. Dis. 130:412-415. 4. Franklin, T. J., and G. A. Snow. 1971. Biochemistry of antimicrobial action, p. 56-58. Academic Press Inc., New York.
VOL. 14, 1976 5. Goodman, L. S., and A. Gilman. 1975. The pharmacological basis of therapeutics, 5th ed., p. 1239-1241. Macmillan, New York. 6. Hill, H. R., R. D. Estenson, P. G. Quie, N. A. Hogan, and N. D. Goldberg. 1975. Modulation of human neutrophil chemotactic responses by cyclic 3'5'-guanosine monophosphate and cyclic 3'5'-adenosine monophosphate. Metabolism 24:447-456. 7. Lehrer, R. I. 1971. Inhibition by sulfonamides of the candidicidal activity of human neutrophils. J. Clin. Invest. 50:2498-2505. 8. Martin, R. R., G. A. Wau, R. B. Couch, H. Yeager, and
NOTES
317
V. Knight. 1974. Effects of tetracycline on leukotaxis. J. Infect. Dis. 129:110-116. 9. Nelson, R. D., P. G. Quie, and R. L. Simmons. 1975. Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J. Immunol. 115:1650-1656. 10. Polak, A. 1974. Effects of 5-fluorocytosine on protein synthesis and amino acid pool in Candida albicans. Sabouraudia 12:309-319. 11. Wilkinson, P. C. 1974. Chemotaxis and inflammation, p. 168-172. Churchill Livingston, London.
