Abstract
We evaluated the effect of the combination of micafungin and polymorphonuclear leukocytes (PMN) against hyphae of Candida albicans and Candida dubliniensis. Micafungin enhanced the PMN oxidative burst dose dependently. The combination was synergistic (C. albicans) or additive (C. dubliniensis); when PMN were pretreated with granulocyte-macrophage colony-stimulating factor, the combination was more effective.
When infecting parenchymal tissues, both Candida albicans and Candida dubliniensis switch between blastoconidia and hyphae (10). This constitutes an important virulence factor, probably because hyphae are too large for phagocytosis. Within the tissue, then, the hyphae will be subject both to extracellular attack by phagocytes, mainly neutrophils or polymorphonuclear leukocytes (PMN), and to the drug that is being administered. Therefore, the effects of the combination of new antifungal drugs with the fungicidal functions of these immune cells are clinically relevant. Micafungin (MCFG) is a new candin already licensed in Japan for systemic use. In this study, we have evaluated the activity of MCFG on the PMN oxidative burst and the fungicidal activity of a combination of MCFG with PMN, pretreated or not with granulocyte-macrophage colony-stimulating factor (GM-CSF), against hyphae of C. albicans and C. dubliniensis.
The strains used were C. albicans ATCC 90028 and C. dubliniensis CBS 7987. Fungi were seeded from frozen stocks on Sabouraud agar (Merck, Darmstadt, Germany) plates and grown for 3 days. Five colonies were transferred to 10 ml of liquid Sabouraud medium and incubated overnight at 35°C with shaking. After being washed, blastoconidia were counted on a hemocytometer and their concentration was adjusted to 10 × 106 blastoconidia/ml of Hanks' balanced salt solution (HBSS) containing 50% fetal bovine serum. Following incubation at 37°C with shaking for 30 to 60 min, 95% of the Candida cells showed pseudohyphae.
Drug.
MCFG was kindly donated by Fujisawa GmbH, Münich, Germany. MCFG was dissolved at 0.5 mg/ml in saline solution and stored at −24°C. For the fungicidal-activity experiments, the concentration of 0.05 μg/ml was selected in order to achieve a low, but constant, fungicidal effect.
Superoxide anion release assay.
The oxidative burst evidenced by the production of O2− was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome c. Freshly purified PMN were obtained from the German Cancer Research Center, Heidelberg, Germany. Buffy coats from healthy donors were centrifuged over a Ficoll layer, the pellet was resuspended in phosphate-buffered saline, and the erythrocytes were sedimented by use of dextran. Remaining erythrocytes were lysed by incubating the cells in lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA; pH 7.4) at 4°C for 15 min. After being washed, the cells were resuspended in HBSS without Ca2+ or Mg2+ and counted on a hemocytometer. PMN were then added at a final concentration of 106 cells/ml to 300 μl of a suspension containing 50 μM ferricytochrome c (from horse heart; Sigma) in the absence or presence of stimuli. As stimuli, C. albicans or C. dubliniensis hyphae were added to the corresponding wells at a final concentration of 106 hyphae/ml. In addition, 0.5 μM N-formyl-methionyl-leucyl-phenylalanine (Sigma) were used as a control. After incubation at 37°C with 5% CO2 for 30 min, 200 μl was transferred to a fresh plate and read in a spectrophotometer at a wavelength of 550 nm with reference filter at 690 nm. The extinction coefficient of ferricytochrome c at 550 nm was taken as 29.5 × 104 mol−1 cm−1.
Fungicidal activity.
PMN were purified from blood of healthy volunteers by dextran sedimentation (Amersham Pharmacia Biotech AB, Uppsala, Sweden) followed by centrifugation over Ficoll (Biochrom AG, Berlin, Germany) as previously described (3). After hypotonic lysis of the remaining erythrocytes, PMN were counted on a hemocytometer and adjusted to 5 × 106 PMN/ml of RPMI 1640 supplemented with 10% autologous serum. GM-CSF (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany) was added at 100 ng/ml, and both untreated and GM-CSF-treated PMN were incubated for 3 h at 37°C with 5% CO2. Hyphae of C. albicans or C. dubliniensis were diluted to 106 hyphae/ml of HBSS supplemented with 10% fetal bovine serum and incubated in the absence or presence of 0.05 μg of MCFG/ml for 3 h at 37°C with shaking. Thirty minutes before the incubation time was over, PMN were added to a final concentration of 106 PMN/ml, and the concentration of MCFG was adjusted accordingly for a final volume of 500 μl. To evaluate the viability of the fungal cells, the Life/Dead yeast viability kit (Molecular Probes, Leiden, The Netherlands) was used. First, a 1:10 dilution of FUN-1 was prepared in dimethyl sulfoxide (Serva Electrophoresis GmbH, Heidelberg, Germany). From this dilution, a mixture containing 50 μM (each) FUN-1 and Calcofluor white in HBSS supplemented with 4% glucose was prepared according to the manufacturer's instructions. After the samples were centrifuged at 1,000 × g for 3 min, 450 μl of supernatant was discarded, and the pellet was resuspended in the remaining 50 μl. Fifty microliters of the Life/Dead yeast viability kit solution was added, and the mixture was mixed thoroughly. After incubation for 30 min at 30°C in the dark, the samples were transferred to a microscopic slide for evaluation. The fungal cells were counted under the fluorescence microscope using an excitation wavelength of 480 nm. The total number of fungal cells was determined with a filter with ∼350-nm excitation and ∼445-nm emission. The viability of the cells was then evaluated with a long-path filter with ∼485-nm excitation and >520-nm emission. Candida cells were counted as alive when orange-red intravacuolar structures could be observed (8). The percentage of killing was calculated according to the formula percent killing = 100 − 100 · Ca/Ct, where Ca is the number of living Candida cells counted and Ct is the total number of cells counted.
Statistical analysis.
Each experiment was performed with the PMNs of one donor, and, for the superoxide anion (O2−) release assay, duplicate wells for each condition were set. The average value for these replicate wells was taken as the value for that particular donor and experiment. The values of each experiment were then used in the data analysis to calculate the mean ± standard error of mean for all experiments conducted under the same conditions. Seven donors were used in the O2− release experiments, whereas six or seven donors were used for the fungicidal-activity assays. Differences between mean values were statistically evaluated by repeated-measures analysis of variance (ANOVA) followed by Dunnett's correction for multiple comparisons (O2− release assays) or Bonferroni's correction (fungicidal-activity assays). A two-sided P value of <0.05 indicated statistical significance. In the fungicidal-activity assays, synergism was calculated as previously described (4). In brief, for each experiment, the sum of the hyphal damage produced by the PMN alone and the drug alone was calculated and compared with the effect of treatment with a combination of PMN and drug. Synergism was defined as an antifungal effect of the combination which was greater than the effect of PMN alone plus the effect of the drug alone. Significance of this effect was evaluated by paired Student t test. An additive effect was defined as an antifungal effect of the combination which was greater than the effect produced by either PMN or drug alone but which did not reach synergism.
Effects of MCFG on PMN oxidative burst.
The effects of MCFG on the oxidative burst produced by PMN in response to various stimuli was evaluated by a superoxide anion (O2−) assay (Table 1). MCFG enhanced the oxidative burst of PMN in a dose-dependent fashion. An enhanced burst was seen both in the presence and absence of other stimuli. Under the conditions studied, the tested fungi did not independently enhance the oxidative burst.
TABLE 1.
O2− produced by PMN in the presence of increasing concentrations of MCFG
| Stimulus | Mean O2− concna (nM) ± SE produced by PMN in the presence of MCFG at a concn (μg/ml) of:
|
||||
|---|---|---|---|---|---|
| 0 | 0.05 | 1 | 5 | 50 | |
| None | 0.95 ± 0.17 | 1.02 ± 0.18 | 1.01 ± 0.17 | 1.02 ± 0.16 | 1.32 ± 0.23* |
| FMLPb | 2.11 ± 0.33 | 2.21 ± 0.34 | 2.13 ± 0.31 | 3.00 ± 0.54* | 3.38 ± 0.66* |
| C. albicans | 0.82 ± 0.11 | 0.82 ± 0.09 | 0.82 ± 0.10 | 1.06 ± 0.12* | 1.15 ± 0.15* |
| C. dubliniensis | 0.84 ± 0.10 | 0.86 ± 0.09 | 0.88 ± 0.09 | 1.12 ± 0.12* | 1.23 ± 0.14* |
*, significant difference (P < 0.01) from the O2− released in the absence of MCFG, as evaluated by repeated-measures ANOVA with Dunnet's correction for multiple comparisons (n = 7).
FMLP, N-formyl-methionyl-leucyl-phenylalanine.
Effects of the combination of MCFG with PMN.
The percentages of fungal cells killed by subinhibitory concentrations of MCFG (0.05 μg/ml), PMN, or combinations of both are shown in Table 2. Against C. albicans hyphae, the combination of MCFG with PMN, pretreated or not with GM-CSF, was synergistic. Moreover, the combination of MCFG with GM-CSF-pretreated PMN was more effective than that with untreated PMN. Against hyphae of C. dubliniensis, the combination of PMN with MCFG was synergistic only when PMN were preincubated with GM-CSF, while the combination with untreated PMN was additive.
TABLE 2.
Percentages of dead fungal cells in the presence or absence of MCFG and/or PMN, pretreated or not with GM-CSF
| Species and condition | MCFG concn (μg/ml) | Mean % killing ± SEa |
P vs reference conditionb for:
|
|
|---|---|---|---|---|
| MCFG + untreated-PMN | MCFG + GM-CSF-treated PMN | |||
| C. albicans (n = 6) | ||||
| Medium | 0.05 | 2.7 ± 1.0 | <0.001 | <0.001 |
| Untreated PMN | 0 | 51.5 ± 4.6 | <0.05 | <0.001 |
| 0.05 | 63.8 ± 6.2 (S) | NA | <0.05 | |
| GM-CSF-pretreated PMN | 0 | 59.0 ± 5.0 | NS | <0.01 |
| 0.05 | 74.5 ± 5.8 (S) | <.05 | NA | |
| C. dubliniensis (n = 7) | ||||
| Medium | 0.05 | 6.4 ± 1.1 | <0.001 | <0.001 |
| Untreated PMN | 0 | 52.7 ± 4.0 | <0.01 | <0.001 |
| 0.05 | 63.4 ± 5.0 (A) | NA | NS | |
| GM-CSF-pretreated PMN | 0 | 56.6 ± 3.4 | NS | <0.001 |
| 0.05 | 70.2 ± 4.0 (S) | NS | NA | |
Mean percentages of dead fungal cells killed by the PMN and/or MCFG. A, additive; S, synergistic.
P values were obtained by performing statistical comparisons by repeated-measures ANOVA with Bonferroni's correction. NA, not applicable; NS, not significant.
The present study has evaluated the effects of the combination of a new antifungal drug, MCFG, with PMN against C. albicans and C. dubliniensis hyphae. Since the oxidative microbicidal mechanisms of PMN are of main importance against fungi (5), we have assessed the effect of MCFG on the production of O2−, the first metabolite produced during the oxidative burst. MCFG primed the PMN oxidative burst in response to all the stimuli evaluated in a dose-response manner, starting with 5 μg/ml. It has been previously reported that any PMN purification technique induces a basal O2− production (7). While doses up to 5 μg of MCFG/ml did not affect significantly this basal O2− production, the highest dose used, 50 μg/ml, increased it.
In addition to the oxidative burst, we evaluated the fungicidal activity of the combination of MCFG with PMN. We used MCFG at very low concentration, since preliminary experiments showed that higher concentrations of the drug alone were too effective, preventing us from observing the effects of its combination with PMN. The combination of MCFG and PMN was more effective than either agent alone against both species, reaching significant synergism against C. albicans. A number of factors may be responsible for this synergy: One possibility is that MCFG may affect the presence or expression of some cell wall-associated proteins that the fungus uses to resist phagocytic attack, such as glycosylphosphatidylinositol-anchored proteins (9) or cell wall-associated superoxide dismutases (6). Another possibility is that a small decrease in the contents of β-1,3-glucan in the cell wall might render the Candida hyphae more susceptible to PMN antifungal mechanisms.
Several authors have proposed coadjuvant treatment of fungal infections with GM-CSF, or with granulocyte transfusions elicited from donors by treatment with this cytokine. The rationale for this is that GM-CSF treatment enhances the fungicidal functions of phagocytes against a number of fungi (see, for instance, references 1 and 2). We have therefore evaluated the fungicidal effect of the combination of MCFG with GM-CSF-pretreated PMN to assess whether the drug would have beneficial or deleterious effects on PMN treatment with the cytokine.
In our conditions, the combination of MCFG with GM-CSF-pretreated PMN was even more effective than the combination with untreated PMN against hyphae of both species. In fact, against C. dubliniensis, the combination of MCFG with PMN reached significant synergism when the cells were pretreated with GM-CSF. Therefore, the results from this study support the hypothesis that the use of GM-CSF as a coadjuvant might have beneficial effects in the treatment of Candida infections. Nevertheless, additional data from animal models of Candida infection are needed prior to clinical trials.
In conclusion, MCFG primes the PMN for an increased oxidative burst and shows additive or synergistic effects with PMN in killing Candida hyphae, which are even greater when PMN are pretreated with GM-CSF. From these results, it appears that MCFG has a beneficial effect on the host's PMN response. Besides, GM-CSF treatment or transfusion of PMN elicited with this cytokine might be of additional use.
Acknowledgments
We are grateful to Markus Munder (DKFZ, Heidelberg, Germany) for providing us with the purified PMN at a specific concentration that we used in our oxidative burst analysis.
This project was supported by a research grant provided by Fujisawa GmbH, Munich, Germany, which kindly provided micafungin.
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