Departments of 1 Clinical Pharmacy and 2 Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 26 South Dunlap Street, Memphis, TN 38163; 3 National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA
Received 27 June 2002; returned 6 September 2002; revised 11 October 2002; accepted 1 November 2002
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Abstract |
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Keywords: amphotericin B, monocyte, cytokine, chemokine
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Introduction |
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Materials and methods |
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The deoxycholate salt of amphotericin B (AmB-DOC) was obtained from Bristol-Myers Squibb (New York, NY, USA). RPMI 1640 medium was obtained from Sigma (St Louis, MO, USA). Low-endotoxin fetal bovine serum was purchased from Summit Biotech (Fort Collins, CO, USA) and Gibco-BRL/Invitrogen (Carlsbad, CA, USA). The human mononuclear cell line THP-1 (ATCC TIB 202) was cultured as described previously.6
AmB-DOC was reconstituted in sterile water according to the manufacturers directions. Heat treatment was carried out as described previously.5 Amphotericin B concentrations >10 µg/mL have been associated with low cell viability.6 Therefore, concentrations from 0.625 to 10 µg/mL were used in this study. Cell viability was assessed by erythrosin B exclusion, as described previously.6
In vitro antifungal activity
Candida albicans ATCC 90028, Candida krusei ATCC 44507 and Candida glabrata ATCC 32312 were obtained from the American Type Culture Collection (Rockville, MD, USA), Candida tropicalis LM-64 was contained in the National Center for Natural Products Research collection (Oxford, MS, USA) and C. albicans 12-99 was a gift from Dr Spencer Redding (University of Texas Health Science Center, USA). Susceptibility testing was carried out using a modified version of the NCCLS methods.7
Cytokine and chemokine measurements
Tumour necrosis factor (TNF)-, interleukin (IL)-1ß, IL-1
, IL-1 receptor antagonist (Ra), macrophage inflammatory protein (MIP)-1
, MIP-1ß and monocyte chemoattractant protein (MCP)-1 concentrations were determined with commercial ELISA kits (R&D Systems, Minneapolis, MN, USA). After exposure to experimental conditions for 2 h supernatants were collected and stored at 70°C until assay. Duplicate measurements were made for each of two wells from three individual experiments.
Statistics and presentation of data
Data are expressed as means ± S.E.M. Data sets were compared by use of Students two-tailed, paired t-test. The Bonferroni correction was used for multiple comparisons. Significance was considered to be achieved when the P value was <0.05.
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Results |
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Viability of THP-1 cells was unchanged (virtually 100% viable) after a 2 h exposure to AmB-DOC concentrations up to 2.5 µg/mL. Higher concentrations produced dose-dependent cell death. In contrast, viability was unchanged (100%) at HT-AmB concentrations up to 10 µg/mL.
Heat treatment of amphotericin B does not alter its activity against C. albicans
No difference in antifungal activity against Candida species was observed between the two preparations. As shown in Table 1, the IC50 and MIC for both preparations tested in RPMI were virtually identical.
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AmB-DOC at concentrations up to 5 µg/mL produced dose-dependent increases in IL-1ß, TNF-, MIP-1
and MIP-1ß. A dose-dependent response was observed for IL-1
at AmB-DOC concentrations up to 2.5 µg/mL. Loss of dose-dependent responses at higher concentrations correlated with decreased cell viability. Dose-dependent increases in IL-1ß, IL-1
, TNF-
and MIP-1ß were observed in response to HT-AmB at concentrations up to 10 µg/mL, whereas no clear increase in MIP-1
in response to HT-AmB was observed (Table 2). These responses were substantially less than those observed with corresponding concentrations of AmB-DOC. No clear response to either AmB-DOC or HT-AmB was observed for IL-1Ra or MCP-1 (data not shown).
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Discussion |
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To ensure that antifungal activity of amphotericin B was not compromised by heat treatment we measured the MIC and IC50 for both AmB-DOC and HT-AmB against several Candida species. Consistent with previous reports for both C. albicans2 and Cryptococcus neoformans,4 we found no appreciable difference in these parameters between the two preparations.
Amphotericin B has been shown to induce the production of IL-1ß and TNF- in human and murine monocytes.6,810 Infusion-related fever, and rigors associated with amphotericin B administration are believed to be a result of the production of these cytokines.9,10 Other cytokines and chemokines that have been shown to be induced by amphotericin B include IL-1Ra, IL-8, MIP-1
, MIP-1ß and MCP-1.8 Whereas these molecules may contribute to amphotericin B-associated toxicity, they may also contribute favourably to the antifungal activity of amphotericin B through their immunomodulatory actions. Consistent with the work of Hartsel et al.,3 we found amphotericin B-induced TNF-
production in THP-1 cells to be abrogated by heat treatment of amphotericin B. We also observed similar results for IL-1ß, MIP-1
and MIP-1ß. Concentrations of these secreted cytokines increased in a dose-dependent fashion in response to doses of AmB-DOC up to 5 µg/mL. These responses diminished in conjunction with reduced cell viability. Dose-dependent responses were also observed for HT-AmB, but at higher concentrations than those observed with AmB-DOC.
We also observed a dose-dependent response to AmB-DOC for IL-1, but only up to a dose of 2.5 µg/mL. The diminished response observed with higher AmB-DOC doses is likely to reflect the cell-associated nature of this cytokine, where expression would be expected to more closely reflect cell viability compared with secreted cytokines. This is the first demonstration of the production of IL-1
in response to amphotericin B.
We did not observe a dose-dependent increase in IL-1Ra or MCP-1 in response to AmB-DOC after exposure to either amphotericin B preparation for 2 h. In previous studies, production of these cytokines was measured at 6 h (for MCP-1) and at 24 and 48 h (for IL-1Ra) after AmB-DOC exposure.6,8 In the present study, we chose to measure cytokine and chemokine concentrations at 2 h after exposure, as this reflects the period of time in which amphotericin B-associated infusion-related toxicity is observed.1,9 In light of the anti-inflammatory properties of MCP-1 and IL-1Ra, it is probable that these molecules are produced to counter the initial pro-inflammatory response elicited by amphotericin B in these cells.
In conclusion, heat treatment of AmB-DOC abrogates amphotericin B-induced toxicity as well as cytokine and chemokine production in THP-1 cells, while not compromising the antifungal activity of this agent. In light of these findings, as well as previous studies demonstrating reduced toxicity with this amphotericin B preparation, heat treatment of amphotericin B may prove to be a cost-effective approach to improving the therapeutic index of this antifungal agent.
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Acknowledgements |
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Footnotes |
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References |
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3 . Hartsel, S. C., Baas, B., Bauer, E., Foree, L. T. Jr, Kindt, K., Preis, H. et al. (2001). Heat-induced superaggregation of amphotericin B modifies its interaction with serum proteins and lipoproteins and stimulation of TNF-alpha. Journal of Pharmaceutical Sciences 90, 12433.[CrossRef][ISI][Medline]
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7 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A, Vol. 17, No. 9. NCCLS, Villanova, PA, USA.
8 . Rogers, P. D., Stiles, J. K., Chapman, S. W. & Cleary, J. D. (2000). Amphotericin B induces expression of genes encoding chemokines and cell adhesion molecules in the human monocytic cell line THP-1. Journal of Infectious Diseases 182, 12803.[CrossRef][ISI][Medline]
9 . Goodwin, S. D., Cleary, J. D., Walawander, C. A., Taylor, J. W. & Grasela, T. H. (1995). Pretreatment regimens for adverse events related to infusion of amphotericin B. Clinical Infectious Diseases 20, 75561.[ISI][Medline]
10 . Chia, J. K. & Pollack, M. (1989). Amphotericin B induces tumor necrosis factor production by murine macrophages. Journal of Infectious Diseases 159, 1136.[ISI][Medline]