Human Phagocytic Cell Responses to Scedosporium apiospermum (Pseudallescheria boydii): Variable Susceptibility to Oxidative Injury

Isolation of phagocytes.PMNs were purified from whole blood of healthy young adult volunteers. The blood was heparinized and immediately allowed to settle with 3% dextran T500 (Pharmacia Biotech AB, Uppsala, Sweden) in a 2:1 (vol/vol) proportion. PMNs were separated by centrifugation over a Ficoll (lymphocyte separation medium; Gibco BRL Life Technologies Ltd., Paisley, Scotland) cushion. Contaminating erythrocytes were hypotonically lysed with distilled water, and the PMN suspension was washed in Hanks' balanced salt solution (Gibco BRL Life Technologies Ltd.) without Ca²⁺ and Mg²⁺ (HBSS⁻). The cells were resuspended in HBSS⁻ and counted on a hemocytometer by trypan blue staining.

For O₂⁻ release and hyphal damage assays, mononuclear leukocytes (MNCs) were separated from the blood of healthy adult volunteers by centrifugation over a Ficoll cushion, washed, and resuspended in HBSS⁻. Cells were counted on a hemocytometer by trypan blue staining, and the percentage of monocytes in the total number of MNCs was defined by May-Grunwald-Giemsa staining.

For phagocytosis assays, monocytes were separated from the blood of healthy adult volunteers by elutriation (28) and resuspended in complete medium (CM) that consisted of RPMI 1640 supplemented with 25% human AB serum (Gibco), 100 U of penicillin/ml, and 100 μg of streptomycin/ml. Cells were counted on a hemocytometer and then used in the phagocytosis assays. The percentage of monocytes was consistently >95%.

Fungal isolates.Two well-characterized clinical isolates of S. apiospermum were used for these studies. SA54A was isolated from a heart biopsy of a patient with a fatal disseminated infection, and SA1216 was a leg skin biopsy isolate from a patient with a localized subcutaneous infection that was successfully treated. These isolates were cultured and identified to the species level in the Fungus Testing Laboratory of the University of Texas Health Sciences Center at San Antonio and confirmed in two other laboratories (Medical Mycology Research Laboratory, Medical College of Virginia Campus, Virginia Commonwealth University, and the Immunocompromised Host Laboratory, National Cancer Institute). These isolates have also been extensively investigated in previous studies of antifungal drug resistance, pathogenesis, and in vivo host response (11, 12, 40; T. J. Walsh, E. Roilides, C. Gonzalez, D. Shetty, J. Peter, H. Katsifa, and C. A. Lyman, Abstr. 12th Int. Symp. Infect. Immunocompromised Host, Int. J. Infect. Dis. 6:2S49, 2002).

There were distinct differences observed in the colonial morphologies of the two S. apiospermum isolates used in this study when the isolates were grown on potato dextrose agar (PDA; E. Merck AG, Darmstadt, Germany) plates at 37°C for 7 days. Colonies of the SA54A isolate on a PDA plate appeared grey-white in color and lanose, with a slithery mycelium. In contrast, the SA1216 colonies appeared brown-grey in color, with low aerial mycelium. On the reverse side, the SA54A colonies had a dark center with annular rings and dark radial lines surrounded by a white periphery. The SA1216 colonies had a dark brown-grey color, with an annular ring and no radial lines. There were no morphological differences apparent by light microscopy.

The susceptibilities of the clinical isolates to amphotericin B, itraconazole, voriconazole, and posaconazole were determined as the MICs of these drugs according to the National Committee for Clinical Laboratory Standards reference method for broth dilution antifungal susceptibility testing of filamentous fungi (25). Amphotericin B had distinct MICs for the two isolates of S. apiospermum. The MIC of amphotericin B for isolate SA54A was 32-fold higher (MIC = 4.0 μg/ml) than that for SA1216 (MIC = 0.125 μg/ml). The MICs of itraconazole, voriconazole, and posaconazole for SA54A (0.125, 0.125, and 0.125 μg/ml, respectively) and for SA1216 (0.25, 0.06, and 0.25 μg/ml, respectively) differed by ≤2-fold.

To prepare conidia from frozen stocks, fungi were inoculated on PDA plates and grown for 7 days. Conidia were harvested by gently scraping the surfaces of the plates. Conidia were suspended in HBSS, filtered through sterile gauze, and counted on a hemocytometer. They were kept at 4°C for no longer than 3 weeks.

Oxidative pathway products and inhibitors or scavengers of PMN function.The two fungicidal products of the superoxide pathway (H₂O₂ and HOCl) were studied at various concentrations. In addition, a number of selective inhibitors of PMN function were examined. These included (i) mannose (MAN) (blocking MAN receptors that mediate attachment) (18), (ii) azide (AZI) (a potent and specific inhibitor of MPO) (8, 9, 36), (iii) catalase (CAT) (an agent that disrupts H₂O₂) (9, 41), and (iv) heparin (HEP) (a compound that binds to α-defensins and inhibits their function) (2, 16). MAN (100 mM) was dissolved in H₂O and stored at −20°C. AZI (0.1 mM) was dissolved in H₂O and stored at 4°C. CAT (6,000 U/ml) was stored at 4°C and diluted in HBSS immediately before use. All of the above-named compounds were purchased from the Sigma Chemical Company (St. Louis, Mo.). HEP (Leo Pharmaceutical Products, Ballerup, Denmark) (0.5 U/ml) was diluted in HBSS and stored at 4°C. PMNs were incubated in the presence of 0.1 mM AZI or HBSS at 37°C for 15 min before being added to germinated conidia at an effector-to-target cell (E:T) ratio of 20:1. MAN, CAT, and HEP were added to wells containing hyphae immediately before the addition of PMNs at the same E:T ratio.

In addition, the effects of diphenyleneiodonium (DPI) (21), superoxide dismutase (SOD) (1), and dimethyl sulfoxide (DMSO) (7) on PMN function were examined. DPI and SOD were purchased from Sigma, whereas DMSO was purchased from Riedel-de-Haen, Seelze, Germany. DPI (75 μM) was dissolved in H₂O and stored at −20°C. SOD (2,000 U/ml) was dissolved in H₂O and stored at 4°C. DMSO (1 μM) was stored at the ambient temperature and diluted in HBSS immediately before use. DPI, SOD, SOD plus CAT, and DMSO each were added to wells containing hyphae immediately before the addition of PMNs at an E:T ratio of 20:1. At the concentrations employed in these studies, none of the above-named compounds (excluding H₂O₂ and HOCl) had an adverse effect on the viability of PMNs (assessed as the exclusion of trypan blue staining from the PMNs). Control wells contained inhibitors and hyphae without PMNs.

Phagocytosis assay.For early differentiation to MDMs, 1 million monocytes purified as mentioned above and resuspended in CM were placed on 18-mm-diameter sterile round glass coverslips in 12-well plates and incubated at 37°C with 5% CO₂ for 2 to 3 days. After this incubation, the wells with the coverslips in them were washed with warm HBSS and 1 ml of a suspension containing 10⁶ conidia/ml of CM was added. After incubation at 37°C with 5% CO₂ for 1 h, the wells were again washed with warm HBSS three times. For measurement of the percentage of phagocytosis and the phagocytic index, the coverslips were removed, dried, and subjected to modified May-Grunwald-Giemsa staining. Cells having or not having phagocytosed conidia were counted microscopically as previously described (14). Conidia that remained attached after three washes were counted only if they had been ingested at a rate of more than 50%. The results were calculated according to the following formulas: $$mathtex$$\[\mathrm{Percentage\ of\ phagocytosis}\ {=}\ 100\left(\frac{B{+}C{+}D{+}E{+}F}{A{+}B{+}C{+}D{+}E{+}F}\right)\]$$mathtex$$(1)$$mathtex$$\[\mathrm{Phagocytic\ index}\ {=}\left(\frac{B{+}2C{+}3D{+}4E{+}5F}{B{+}C{+}D{+}E{+}F}\right)\]$$mathtex$$(2) where the phagocytic index represents the average number of phagocytosed conidia per phagocytosing cell, A represents the number of cells that ingested no conidia, B represents the number of cells that ingested one conidium, C represents the number that ingested two conidia, D represents the number that ingested three conidia, E represents the number that ingested four conidia, and F represents the number that ingested more than four conidia.

Superoxide anion release assay.The oxidative burst evidenced by the production of O₂⁻ was measured as the SOD-inhibitable reduction of cytochrome c. A total of 200 μl of a suspension containing 1.5 × 10⁶ conidia/ml in yeast nitrogen base medium (YNB; Difco Laboratories, Detroit, Mich.) was plated in each of 96 flat-bottom wells in cell culture clusters (Corning Inc., New York, N.Y.) and incubated at 32°C for 18 h. In the case of O₂⁻ assays with serum-opsonized hyphae, YNB was replaced by 50% pooled human serum in HBSS and incubated for 30 min at 37°C for opsonization. Pooled human serum was obtained from healthy young adult volunteers and kept at −35°C for no longer than 1 month. The plates were washed three times, and phagocytes were added at an E:T ratio of 1:1 to a final volume of 300 μl of HBSS containing 50 μM cytochrome c (Sigma) (from horse heart) with serum-opsonized or unopsonized hyphae. Basal O₂⁻ production was assessed in the absence of stimuli. After incubation at 37°C with 5% CO₂ for 1 h, 200 μl was read in a spectrophotometer at a wavelength of 550 nm. The extinction coefficient of ferricytochrome c at 550 nm was taken as 29.5 × 10⁴ M⁻¹ cm⁻¹.

Hyphal damage assay.Phagocyte-induced hyphal damage was assessed with XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide sodium salt; Sigma) plus coenzyme Q₀ (2,3-dimethoxy-5-methyl-1,4-benzoquinone; Sigma). A total of 200 μl of a suspension containing 7.5 × 10⁴ conidia/ml in YNB was plated in each of 96 flat-bottom wells in cell culture clusters (Corning) and incubated at 32°C for 18 h. YNB was then replaced by HBSS, and phagocytes (either PMNs or MNCs) were added at different E:T ratios. Following incubation at 37°C with 5% CO₂ for 2 h, cells were lysed by washing with H₂O and shaking for 5 min three times. A total of 150 μl of phosphate-buffered saline (Biochrom KG, Berlin, Germany) containing 0.25 mg of XTT/ml with 40 μg of coenzyme Q₀/ml was added, and plates were again incubated at 37°C with 5% CO₂ for 1 h. A total of 100 μl of aliquots from each well was transferred to a new plate and read at 450 nm with a reference wavelength of 690 nm. Antifungal activity was calculated according to the following formula: percentage of hyphal damage = [(1 − X)/C] × 100, where X represents the optical density of test wells and C represents the optical density of control wells with hyphae only. Each set of conditions was tested in duplicate, control wells were set up in quadruplicate, and the results were averaged.

In studies examining the antifungal activity of individual oxidative compounds (H₂O₂ and HOCl), conidia were germinated as described above. YNB was then replaced by HBSS, and H₂O₂ or HOCl was added at different concentrations. Following incubation at 37°C with 5% CO₂ for 2 h, 150 μl of phosphate-buffered saline containing 0.25 mg of XTT/ml with 40 μg of coenzyme Q₀/ml was added and plates were again incubated at 37°C with 5% CO₂ for 1 h. Supernatants were read, and antifungal activity was calculated as described above.

Statistical analysis.Each experiment was performed with the cells of one donor and studied in duplicate or quadruplicate wells for each condition. The mean value of these replicate wells was taken as the value for the particular donor and experiment. The means of the replicate wells of each experiment were then used in the data analysis to calculate the means ± standard errors of the means (SE) of all the experiments at the same conditions. The statistical program GraphPad Instat (GraphPad Inc., San Diego, Calif.) was used for analysis. Unless otherwise stated, paired Student t tests with Bonferroni's correction for repeated measures were used for comparisons between the two strains, as these data followed a normal distribution. Repeated measurement of analysis of variance (ANOVA) with Dunnett's correction for multiple comparisons was used for comparisons of each strain with the control. A two-sided P value of <0.05 indicated statistical significance.

Phagocytosis of conidia by MDMs.To evaluate the ability of MDMs to phagocytose conidia of S. apiospermum, MDMs were incubated with conidia at an E:T ratio of 1:1 for 1 h (Fig. 1). No differences were detected in the percentages of MDMs that phagocytosed conidia of the two strains (21.2% ± 3.4% for strain SA1216 and 22.3% ± 2.7% for strain SA54A). Similarly, the phagocytic indices were similar for the two strains (1.3 ± 0.08 and 1.3 ± 0.05 for SA1216 and SA54A, respectively).

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FIG. 1.

Percentages of phagocytosis (A) and phagocytic indices (B) of S. apiospermum conidia by MDMs. Strains SA1216 (open columns) and SA54A (dashed columns) were used as targets of phagocytosis. Vertical bars represent means ± SE of the results of six experiments.

Oxidative burst in response to hyphae.The O₂⁻ released by PMNs and MNCs in response to the presence of both serum-opsonized and nonopsonized hyphae of both isolates was measured (Fig. 2). Opsonization of hyphae of both isolates with human serum induced a significant increase in PMN O₂⁻ production over that seen with nonopsonized hyphae (P < 0.001). Moreover, serum-opsonized hyphae of the SA54A isolate elicited significantly more O₂⁻ from PMNs than those of SA1216 (P < 0.001).

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FIG. 2.

Superoxide anion expressed as nanomolar concentrations of O₂⁻ produced by 10⁶ PMNs (upper panel) or MNCs (lower panel) in 1 h in response to isolates SA1216 and SA54A of S. apiospermum. Open columns, O₂⁻ produced by phagocytes in response to nonopsonized hyphae; dashed columns, O₂⁻ produced by phagocytes stimulated with opsonized hyphae. The results are presented as means ± SE of the results of six experiments with PMNs and four experiments with MNCs. The symbol * indicates a level of O₂⁻ produced by phagocytes in response to nonopsonized hyphae significantly different from that produced in response to serum-opsonized hyphae (P < 0.001). The symbol § indicates a level of O₂⁻ produced by monocytes in response to nonopsonized SA1216 hyphae significantly different from that produced in response to opsonized SA1216 hyphae (P = 0.011). The symbol † indicates a level of O₂⁻ produced by PMNs in response to opsonized hyphae of SA54A significantly different from that produced in response to opsonized hyphae of SA1216 (P < 0.001).

When the oxidative burst of MNCs was evaluated (Fig. 2, lower panel), opsonization of hyphae with pooled human serum significantly increased superoxide release over that seen with nonopsonized hyphae of isolates SA1216 and SA54A (P < 0.011). There also was a tendency for the opsonized SA54A isolate to elicit more O₂⁻ from MNCs than the SA1216 isolate (P = 0.07).

Damage of hyphae.Hyphal damage induced by PMNs (Fig. 3, upper panel) or by MNCs (Fig. 3, lower panel) was assessed at E:T ratios of 1:1, 5:1, 10:1, and 20:1. When challenged with either type of phagocytic cell, the isolates under investigation were damaged in a concentration-dependent manner, with a statistically significant linear trend (P < 0.001 for both strains and both types of phagocytes, as determined by repeated measurement of ANOVA and posttest for linear trend).

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FIG. 3.

Percentages of hyphal damage to strain SA1216 (open bars) or SA54A (dashed bars) of S. apiospermum induced by PMNs (upper panel) or MNCs (lower panel). Vertical columns represent means ± SE of the results of eight experiments with PMNs and six experiments with MNCs. The symbol * indicates a significant difference between SA54A and SA1216 in the levels of hyphal damage to these two strains induced by PMNs or MNCs at corresponding E:T ratios (P < 0.001).

However, the SA54A isolate was more resistant to PMN- and MNC-mediated hyphal damage than the SA1216 isolate. For instance, while PMNs caused 53.7% ± 6.4% hyphal damage to SA1216 at an E:T ratio of 5:1 they caused only 26.7% ± 4.5% hyphal damage to SA54A hyphae (P < 0.001, as determined by repeated measurement using ANOVA with Dunnett's correction for multiple comparisons). With MNCs at an E:T ratio of 5:1, the hyphal damage was 80.2% ± 7.9% and 44.2% ± 5.9% for SA1216 and SA54A, respectively (P < 0.001).

Effects of superoxide pathway products.There was a direct concentration-response relationship between the oxidative metabolites (H₂O₂ and HOCl) and percent hyphal damage to both isolates of S. apiospermum. Although the SA54A isolate exhibited slightly more susceptibility to H₂O₂-mediated hyphal damage at 1.0 mM, SA1216 was more susceptible to HOCl-mediated hyphal damage at 0.1 mM (Table 1). At high concentrations, however, both strains were highly susceptible (hyphal damage > 94%) to both H₂O₂ and HOCl.

Effects of inhibitors and scavengers of PMN antifungal functions on hyphal damage.To further investigate the differences of isolates in host response, we assessed the damage to both isolates induced by PMNs at an E:T ratio of 20:1 in the presence of various inhibitors and scavengers of PMN functions, i.e., DPI, MAN, CAT, SOD, DMSO, AZI, and HEP. In these experiments, MAN, CAT, SOD (or CAT plus SOD), and HEP had no effect on PMN-induced hyphal damage to the two isolates (Table 2). In addition, DMSO (2 and 20 mM) had no significant effect on PMN-induced hyphal damage to the two isolates. None of the above-named compounds had any adverse effect on damage to hyphae of either strain. By comparison, at 5 μM DPI alone had a direct adverse effect on hyphae of both strains, causing 62.2% damage to the SA54A isolate and 41.8% to SA1216. When DPI was added to PMNs and hyphae, the level of hyphal damage was similar to that seen with DPI alone.

In contrast, AZI exerted a significant effect on PMN-induced damage to SA1216 hyphae. At 0.1 mM, a concentration previously reported to inhibit MPO activity (8, 9, 36), the presence of AZI resulted in a significant inhibition (from 79.8% ± 7.1% to 26.6% ± 3.5%) of the damage to isolate SA1216 hyphae (P < 0.01). The presence of AZI resulted in only an insignificant reduction (from 34.9% ± 8.0% to 26.6% ± 3.5%) of the damage to SA54A hyphae caused by PMNs (P > 0.05). In the presence of AZI, the levels of damage to both isolates induced by PMNs became equivalent and SA1216 then displayed resistance to hyphal injury similar to that demonstrated by SA54A.