Journal of Antimicrobial Chemotherapy (1999) 43, 491-496
© 1999 The British Society for Antimicrobial Chemotherapy
Collaboration of human phagocytes with LY 303366 for antifungal activity against Aspergillus fumigatus
a Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA b California Institute for Medical Research, San Jose, CA c Stanford University School of Medicine, Stanford, CA, USA
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Abstract |
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LY 303366, an inhibitor of 1, 3-ß-D-glucan synthase, was tested alone, or in co-culture with neutrophils or monocytes, for antifungal activity against Aspergillus fumigatus using the XTT metabolism assay. LY 303366 at 0.1 mg/L for 48 h significantly inhibited growth by conidia in a microtest plate XTT assay system. Inhibition was similar if the drug was removed after only 24 h. Microscopically this correlated with less growth and stunted malformed hyphae. LY 303366 (0.1 mg/L) also inhibited the further growth of germlings (43%) in a 24 h assay. Antifungal activity of neutrophils against 24 h control hyphal growth was limited at an effector: target ratio of 400:1. In co-cultures of neutrophils plus drug with hyphal growth from 24 h LY 303366 cultures the antifungal activity was additive. Neutrophils had a similar additive effect even if the drug were not present (i.e. when germinating conidia were pretreated with drug). Under conditions where monocytes did not have significant antifungal activity against hyphae, they collaborated with LY 303366 for significantly increased inhibition from 38% by LY 303366 alone to 67% by co-culture. Thus, LY 303366 has activity against germinating or germinated conidia of Aspergillus, human effector cells act co-operatively with LY 303366, and LY 303366 can sensitize germinating conidia for damage by host cells.
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Introduction |
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LY 303366 is a new echinocandin B antifungal agent with potent inhibition of 1,3-ß-D-glucan synthase in several pathogenic fungi.1, 2 With traditional methods of broth dilution susceptibility testing, altered Aspergillus growth was noted, but not a clear tube or killing endpoint. 2 However, with the NCCLS broth dilution method modified by the addition of Alamar Blue, LY 303366 had an MIC90 in the range of 0.03–5.0 mg/L for Aspergillus spp.1 Here we report results of measuring antifungal activity of LY 303366 against conidia and germlings of Aspergillus fumigatus using the XTT metabolic assay system. 3 Collaboration of neutrophils or monocytes with LY 303366 for additive activity against A. fumigatus was also measured using the XTT assay.
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Materials and methods |
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Aspergillus fumigatus
A patient isolate of A. fumigatus (96–92) was grown on Sabouraud's dextrose agar slants at 35°C for 24 h, then allowed to form conidia at room temperature for 24- 48 h. Conidia were harvested with distilled water, washed once, diluted in saline and counted. Conidia suspensions consisted primarily of single conidia (95%) or small groups of conidia with two or three conidia per group (5%). Over 95% of the conidia germinated when incubated in RPMI-1640 medium (Gibco, Grand Island, NY, USA) at 26°C or 37°C. Each study group was set up in quadruplicate cultures in every experiment.
Antifungal drug
LY 303366 powder was supplied by Eli Lilly and Co., Indianapolis, IN, USA (100 mg/vial). LY 303366 was suspended in methanol 2 g/L, diluted to 1 g/L with distilled water, sterilized by filtration and stored at 4°C. Diluent controls were prepared in identical manner but without LY 303366.
XTT
Inhibition of hyphal growth was measured by the colorimetric XTT- coenzyme Q method. 3 (2,3)-Bis-(2-methoxy-4-nitro-5-sulphenyl-(2H)-tetrazolium-5-carboxanilide) sodium salt (XTT) at 0.5 g/L plus 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q) at 0.04 g/L in phosphate-buffered saline (PBS) pH 7.4 (Sigma Chemical Co., St Louis, MO, USA) constituted the test solution. Viable cells reduce XTT to a reduced soluble form of XTT with a colour change from yellow to orange.
LY 303366 assay
Antifungal activity of LY 303366 alone was tested in several different configurations. LY 303366 was first tested by incubating conidia in RPMI-1640 with or without the drug in 24-well tissue culture plates, microcentrifuge tubes or wells of 96-well microtest plates at 37°C for 24 h. In a second type of test, conidia were first allowed to germinate overnight at 26°C, then germlings (conidia with germ tubes 10–20 times the diameter of a conidium in length) were incubated with or without LY 303366 for 24 h at 37°C.
Neutrophil assay
Polymorphonuclear neutrophils (PMN) and peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by 6% dextran-70 sedimentation followed by density gradient centrifugation of the buffy coat diluted 1:1 with RPMI-1640 on Histopaque 1077 (Sigma). The PBMC layers were harvested, washed and counted. The pelleted cells (PMN and some RBC) were collected in 0.85% NH4Cl to lyse RBC. PMN were washed, counted and suspended in RPMI-1640 plus 10% fresh autologous human serum (referred to as complete tissue culture medium). PMN were added to microtest plate wells containing washed A. fumigatus growth from conidia incubated with RPMI-1640 with or without LY 303366 at 37°C for 24 h. Some cultures received LY 303366 again and all cultures were incubated at 37°C in an incubator (5% CO2 + 95% air). Microtest plates were centrifuged, supernatants were aspirated and wells were washed twice with 0.2 mL of distilled water. PMN were lysed or killed as shown by debris and deformed cells, observed by microscopy. XTT test solution (0.2 mL) was added to each well and cultures were incubated at 37°C for 1 h in a CO2 incubator. An aliquot (0.1 mL) from each well was transferred to corresponding wells of another plate and the absorbance at 410 nm was recorded with a microtest plate reader (Dynatech MR250, Dynatech Lab. Inc., Chantilly, VA, USA).
Monocyte assays
PBMC at 3 x 106/mL in complete tissue culture medium were incubated for 1 or 2 h at 37°C in 60 mm plastic Petri dishes precoated with human serum (2.5 mL of cells per dish). Non-adherent cells were washed away with two washings of warm RPMI-1640 and then 2.5 mL of 0.1 M EDTA in PBS, diluted 1:1 with complete tissue culture medium was added. After 15 min at room temperature, adherent cells were collected, washed, counted and suspended in complete tissue culture medium, as previously described. 4 These cells (monocytes) were added (4 x 105 monocytes/well) to wells containing washed growth of A. fumigatus and the XTT assay was performed as above for PMN.
Quantification of growth inhibition
Absorbance of XTT alone at 410 nm (Shimadzu UV160 spectrophotometer, Shimadzu
Corp., Kyoto, Japan), was subtracted from absorbance of culture supernatants with metabolized
XTT to give the change in absorbance (
A) (24-well plate experiments). In
96-well plate experiments a microtest plate reader (MR250, Dynatech) was used to determine
the A at 410 nm. Percent inhibition of growth was calculated by the formula:
((Acontrol - Aexperimental)/Acontrol) x 100. Since there was a linear relationship
between inoculum and metabolism of XTT by respective cultures as measured by change in
absorbance, to be detailed, decreased A of XTT supernatants from LY
303366-treated cultures represented inhibition of growth.
Statistical analysis
Student's t-test was used for statistical analysis of the data and significance set at P < 0.05. The GB-STAT program (Microsoft, Redmond, WA, USA) for Bonferroni's adjustment to the t-test was used where appropriate.
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Results |
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Preliminary XTT assays
Various configurations were explored for measuring growth of A. fumigatus with the XTT assay. A wide range of conidia inocula were cultured, 1 mL per well of 24-well tissue culture plates for 24 h at 37°C; growth was transferred to microcentrifuge tubes, pelleted and washed at 5000g for 10 min, and then incubated with 1 mL of XTT for 1 h at 37°C. When metabolism of XTT was measured by absorbance at 410 nm there was a linear relationship between inoculum and metabolism of XTT (Figure). The main disadvantage of this method was the difficulty in completely removing sticky mycelial growth from tissue culture plate wells to the microcentrifuge tubes. Consequently the experiment was performed in 96-well microtest plates. A wide range of conidia inocula were cultured, 0.2 mL per well for 24 h at 37°C; the plates were centrifuged at 800g for 10 min, supernatants were aspirated and growth was washed with distilled water by centrifugation and observed microscopically. Washed growth was assayed in situ by the XTT method. A linear relationship similarly existed between inoculum size and metabolism of XTT over a range of 156–2500 conidia/well.
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Preliminary tests of LY 303366
With the 24-well tissue culture plate XTT assay and 2 x 105 conidia/well, LY 303366 at 5 mg/L inhibited growth by 70% in the 24 h assay. Inhibition of growth was associated with club-like malformation of hyphal growth when observed microscopically. Similar results (40% inhibition) were obtained with LY 303366 at 5 mg/L and 2 x 105 conidia/ tube in 1 mL cultures using microcentrifuge tubes and in-situ measurement of XTT metabolism.
In the microtest plate system, using an inoculum of 103 conidia/well, growth was inhibited in a concentration-dependent manner by LY 303366 where 2.5 mg/L gave 80% inhibition. If conidia were allowed to germinate at 26°C and then treated with LY 303366 at 37°C for 24 h, 0.1 mg/L inhibited growth by 43%. This indicated that LY 303366 inhibits the hyphal phase of growth as well as growth in the early germination stage. In view of the convenience and utility of the microtest plate system, it was used for co-culture experiments with PMN and monocytes with or without LY 303366.
Antifungal activity of PMN 1 LY 303366
After conidia and hyphae had been grown at 37°C (with or without LY 303366), cultures were washed and challenged with PMN in complete tissue culture medium with or without LY 303366 for 24 h at 37°C in a CO2 incubator. The results are given in Table I. LY 303366 at 0.1 mg/L for 48 h significantly inhibited (57%) growth of A. fumigatus. Damage by LY 303366 due to exposure for 24 h persisted for another 24 in absence of the drug as shown by growth inhibition of 54% (Table I). Higher concentrations of LY 303366 (0.5 and 1.0 mg/L) did not significantly increase these effects.
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When 4 x 105 PMN were added per well to hyphal growth (effector:target (E:T) ratio 400:1), PMN caused 29% inhibition of growth (Table I). In two experiments under these conditions, inhibition was 26 ± 4%. Lower numbers of PMN (E:T ratios of 100:1 to 10:1) caused 10–18% inhibition of growth in four experiments (data not shown); this inhibition by PMN alone could occasionally reach significance, depending on the S.D. When 4 x 105 PMN were added to hyphae that had developed in the presence of LY 303366 (0.1 mg/L), with or without more drug for 24 h, there was an additive inhibition of growth, 80% and 73% respectively (Table I). Higher concentrations of LY 303366 (0.5 or 1.0 mg/L) did not significantly alter these effects. Lower numbers of PMN (1 x 105 or 0.5 x 105) with LY 303366 at 0.1, 0.5 or 1.0 mg/L gave similar results to those shown in Table I.
Similar results were obtained when PMN from two different donors were used (results pooled). LY 303366 (0.1 mg/L) for 48 h resulted in 38 ± 9.1% inhibition (P < 0.01). PMN (4 x 105) alone for 24 h caused 17 ± 8% inhibition and the combination of LY 303366 and PMN produced an additive inhibition of 51.5 ± 0.7%.
A lower concentration of LY 303366 (0.01 mg/L) did not give an additive effect in this system. When the inoculum of conidia was reduced to 400/well and the number of PMN remained the same (4 x 105/well), the collaborative effect of PMN and LY 303366 (0.1 mg/L) was additive, 63% (Table II), i.e. approximately the sum of inhibition by PMN and by LY 303366 alone.
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Taken together, these results indicate that LY 303366 causes visual malformation and growth inhibition of A. fumigatus and this correlates with decreased metabolism of XTT, and that previous exposure to LY 303366 sensitizes A. fumigatus for significant damage by PMN.
Antifungal activity of monocytes plus LY 303366
Under the conditions of these assays, monocytes have less antifungal activity against hyphae than against PMN, therefore a ten-fold lower inoculum of conidia was used (100 conidia/well). LY 303366 at 0.1 mg/L for 48 h significantly inhibited growth (38%) (Table III). Monocytes (4 x 105/well) alone did not inhibit 24 h hyphal growth when co-cultured for 24 h. These results are specific to the method used; in other experiments (not shown) monocytes alone could be demonstrated to have anti-aspergillus activity under other study conditions (other metabolite assays, plates, times of incubation, and studies where fungal elements were added to adherent monocyte monolayers).
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Under the present, stringent conditions, monocytes could collaborate with LY 303366 (0.1 mg/L) and increase inhibition from 38% to 67% (Table III). Monocytes also collaborated with LY 303366 (0.01 mg/L) by increasing inhibition from 7% to 37% in co-cultures (Table III). Similar results were found in a second identical experiment with monocytes from a different donor, e.g. monocytes (0% inhibition) collaborated with LY 303366 (0.1 mg/L) (35% inhibition) to give 50% inhibition of growth.
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Discussion |
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Difficulties have been encountered with conventional in-vitro tests of echinocandins and pneumocandins against A. fumigatus.5 With the first of these to be extensively studied, cilofungin, our laboratory and others failed to detect anti-aspergillus activity by classical in-vitro methods;6 however, our laboratory noted,7 and others have confirmed (reviewed by Kurtz et al.5), anti-aspergillus activity in vivo. We report here that the XTT metabolic assay can be used to measure LY 303366 activity against A. fumigatus in a microtest plate system. With this system multiple parameters can be easily tested; it also allows for objectivity and statistical analysis.
The antifungal activity of neutrophils against hyphae of A. fumigatus in short-term 2 h challenge experiments has been measured using the MTT and XTT assays. 3 We have used this method for measuring antifungal activity of neutrophils in long-term 24 h experiments against hyphae. We defined antifungal activity in these experiments as percentage inhibition of growth instead of hyphal damage as for 2 h challenge experiments.3 Under these very challenging conditions, neutrophils inhibited mycelial growth. However, even compared at the same E:T ratios, PMN activity alone was variable between experiments; aspergillus strain, PMN donor and germling size (which we found could vary after 24 h incubation) are possible factors. At high or low levels of PMN activity, additive effects with drug were seen. On the other hand, monocytes, even against a ten-fold smaller inoculum, failed to inhibit mycelial growth.
To simulate in-vivo therapeutic conditions, hyphae exposed to antifungal activity of LY 303366 were co- cultured with neutrophils and the combined antifungal activity was measured. The results showed that the antifungal activity of this combination was additive. On the other hand, monocytes co-cultured with hyphae previously exposed to LY 303366 resulted in antifungal activity greater than the sum of the individual activities. These novel findings help explain the efficacy of this class of drugs in models of infection with A. fumigatus, 7,8 despite the absence of killing by drug alone in vitro.2,6 We speculate that the altered cell wall resulting from drug exposure makes the fungal cell more susceptible to effector cell activity, including oxidative metabolites.
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Notes |
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* Correspondence address. Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, San Jose CA 95128, USA. Tel: +1-408-998-4556; Fax: +1-408-998-2723.
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References |
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1 . Rennie, R., Sand, C. & Smith, S. (1996). In vitro activity of antifungal agents LY 303366 against Candida species, and other yeasts and Aspergillus species. In Program and Abstracts of the Thirty-Sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA, 1996. Abstract F45, p. 107. American Society for Microbiology, Washington, DC.
2 . Stevens, D. A., Martinez, M. & Devine, M. J. (1996). Antifungal activity of LY303366, an echinocandin beta glucan synthase inhibitor. In Program and Abstracts of the Thirty-Sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA, 1996. Abstract F46, p. 107. American Society for Microbiology, Washington, DC.
3 . Meshulam, T., Levitz, S. M., Christin, L. & Diamond, R. D. (1995). A simplified new assay for assessment of fungal cell damage with the tetrazolium dye, (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H )-tetrazolium-5-carboxamide (XTT). Journal of Infectious Diseases 172, 1153–6.[Web of Science][Medline]
4 . Douglas, S. D., Zuckerman, S. H. & Ackerman, S. K. (1981). Obtaining and culturing human monocytes. In Methods for Studying Mononuclear Phagocytes (Adams, D. O., Edelson, P. J. & Koren, H., Eds), pp. 33- 42. Academic Press, New York.
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Kurtz, M. B., Heath, I. B., Marrinan, J., Dreikorn, S.,
Onishi, J. & Douglas, C. (1994). Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-b -D-glucan
synthase. Antimicrobial Agents and Chemotherapy 38, 1480–9.
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Denning, D. W. & Stevens, D. A. (1991).
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8 . Verweij, P. E., Oakley, K. L., Morrisey, J., Morrisey, G. & Denning, D. W. (1998). Efficacy of LY303366 against amphotericin B-susceptible and -resistant Aspergillus fumigatus in a murine model of invasive aspergillosis. Antimicrobial Agents and Chemotherapy 42,873 –8.
Received 23 June 1998; returned 3 September 1998; revised 26 October 1998; accepted 20 November 1998
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