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Previous Article | Next Article 
Infection and Immunity, November 2003, p. 6472-6478, Vol. 71, No. 11
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.11.6472-6478.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Third Pediatric Department, Aristotle University, Thessaloniki, Greece 54642,1 Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland 208922
Received 26 August 2002/ Returned for modification 25 October 2002/ Accepted 5 August 2003
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ABSTRACT |
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INTRODUCTION |
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Little is known about phagocytic host defenses against S. apiospermum. Studies of host defenses against filamentous fungi have focused principally upon Aspergillus fumigatus. The main immune component that has been found responsible for host defenses against A. fumigatus consists of phagocytes, including circulating polymorphonuclear leukocytes (PMNs) and monocytes as well as monocyte-derived macrophages (MDMs) (29, 31, 38). These cells are capable of damaging hyphal elements through oxygen-dependent and -independent mechanisms (38). The oxygen-dependent mechanisms consist of a series of reactions starting with the production of superoxide anion (O2-), which is dismutated into hydrogen peroxide (H2O2). Myeloperoxidase (MPO) then catalyzes the conversion of H2O2 and halides to generate hypohalides, such as hypochlorite (HOCl) and chloramines, which exert potent antifungal activities (1, 15). Cationic peptides (represented by two families, the defensins and the cathelicidins) constitute part of the oxygen-independent microbicidal arsenal in phagocytic cells. These peptides also have potent antifungal activity (6, 26, 45).
The extent to which these mechanisms of phagocytic host defenses apply to Scedosporium spp. is unknown. We therefore investigated the human phagocytic cell responses to conidia and hyphae of amphotericin B-resistant and -susceptible isolates of this emerging fungal pathogen.
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MATERIALS AND METHODS |
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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 
-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:
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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 x 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 x 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 x 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] x 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.
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RESULTS |
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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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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.
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DISCUSSION |
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S. apiospermum is a filamentous fungus characterized by oval, 6- to 12-µm-long conidia. The in vitro susceptibility of this fungus to conventional and novel antifungal drugs has been studied extensively (4, 5, 10, 23, 40). Despite interlaboratory discrepancies, in general these studies suggest that S. apiospermum is frequently resistant to amphotericin B. Determination of antifungal drug MICs for the two clinical isolates used in this study showed that strain SA54A was 32-fold more resistant to amphotericin B than strain SA1216 (amphotericin B MICs = 4 and 0.125 µg/ml, respectively).
As macrophages are the first phagocytic cells to encounter conidia of A. fumigatus, their primary role in antifungal defense appears to be prevention of conidial germination (38). When macrophages fail to prevent germination, hyphae transfix these cells and grow through them. In that case, PMNs and circulating MNCs attempt to damage hyphae extracellularly by potent oxidative and nonoxidative fungicidal mechanisms (31, 38).
The fact that host defenses against S. apiospermum are poorly understood prompted us to evaluate the antifungal activities of phagocytes against conidia and hyphae of this organism. To gain insight into the immunopathogenesis of infection due to S. apiospermum, two clinical strains (isolated from human infections with very different outcomes and characterized by distinct colonial morphologies and antifungal drug susceptibility features) were used to compare the innate immune responses to each of them.
Phagocytosis of S. apiospermum conidia by MDMs does not differ from phagocytosis of A. fumigatus conidia by MDMs, as assessed in similar experiments using the same methodology in our laboratory (27). While macrophages phagocytosed conidia of the two S. apiospermum isolates in equivalent manners, hyphae of the two isolates studied showed distinct differences in their interactions with and susceptibilities to PMNs and MNCs. For example, when the oxidative burst in response to opsonized hyphae of both isolates was evaluated the SA54A isolate elicited a significantly higher amount of O2- from PMNs than SA1216. While MNCs displayed a significant oxidative burst in response to opsonized hyphae of both isolates, the level of O2- generated in response to SA54A tended to be higher than that seen with SA1216. Although SA54A elicited production of more O2- than did SA1216, this did not translate into more hyphal damage, suggesting a relatively high level of resistance of this strain to oxidative injury.
When hyphal damage induced by both PMNs and MNCs was assessed, the level of damage inflicted on isolate SA54A was found to be significantly lower than that seen with SA1216. Under some experimental conditions this difference in susceptibility to hyphal damage approached 40%. As the patient infected with SA54A had a fatal outcome whereas the patient infected with SA1216 was cured, we hypothesized that the difference in the hyphal damage was related to distinct differences in the susceptibilities of these isolates to certain components of the antifungal host defense mechanism. To further investigate this, we used oxidative pathway antifungal products and inhibitors or scavengers of various steps of PMN antifungal functions to determine whether this difference resides in one or more of these pathways.
Hyphae of the SA1216 strain appeared to be slightly more susceptible to HOCl at 
0.1 mM than those of SA54A. This finding in a cell-free system correlates with the finding of a higher level of susceptibility of SA1216 to MPO products (as shown by AZI inhibition) in our PMN-hyphal system. By (presumably) acting on the hyphal surface, both antifungal oxidative compounds of phagocytes damaged hyphae of the susceptible strain more efficiently, a finding similar to that of the higher degree of susceptibility to amphotericin B, an agent also acting on the surface of the fungi (ergosterol of the fungal membrane).
The differences in susceptibility to amphotericin B between these isolates of S. apiospermum parallel the differences in vulnerability to oxidative metabolites and to hyphal damage. As susceptibility to amphotericin B-induced membrane injury is dependent upon lipoperoxidation (3), it is possible that the greater susceptibility to oxidative injury by some isolates of S. apiospermum is related. Resistance to lipoperoxidative injury by amphotericin B may be enhanced by increased expression of CAT (32). However, this appears not to be the mechanism of resistance of SA54A to amphotericin B or to H2O2 since there was a concentration-dependent response to H2O2 and even a trend toward heightened H2O2 activity at 1 mM. Assessment of potential differences in membrane sterol composition may further elucidate these mechanisms.
MAN receptors have been shown to mediate attachment and subsequent phagocytosis of various fungi (13, 33, 34, 39) and can be inhibited by the use of MAN (18). It appears that MAN receptors are not involved in the attachment of PMNs to S. apiospermum hyphae.
Although expected to create a cellular environment similar to that of chronic granulomatous disease PMNs by inhibiting the NADPH oxidase activity (21), DPI exerted a direct adverse effect on survival of Scedosporium hyphae, thus precluding an assessment of its effects on PMN hyphal damage. This may have been due to an effect of DPI on NADPH-like enzymes of Scedosporium hyphae (21).
Further, in previous studies CAT was successfully used to disrupt H2O2 at concentrations similar to those used in the present study (9, 41). H2O2 has been found to play a role in inducing destruction of fungi, including damage to A. fumigatus conidia and hyphae (8, 9, 22, 44). Using H2O2 as the substrate, MPO generates hypohalides, such as HOCl and chloramines, which are highly toxic compounds (1, 15). It is unclear whether the antifungal role of H2O2 is due to its own fungicidal activity or to the activity of its downstream metabolic products. Wagner et al. reported similar results when assessing the damage to C. albicans pseudohyphae caused by PMNs (37). They proposed that CAT might be excluded from the area of attack at the fungal surface, since it has been previously demonstrated that proteins with a molecular mass over 50 kDa are excluded from sites of attachment of macrophages (42) and CAT is a high-molecular-mass protein.
SOD (alone or in combination with CAT) as well as DMSO did not induce significant differences in hyphal damage between the two strains. It is possible that SOD with or without CAT is unable to eliminate all superoxide and H2O2 in the presence of PMNs. As noted in Table 1, a concentration of H2O2 of <0.1 mM was able to inflict hyphal injury on Scedosporium spp. It is possible that at low concentrations, the oxidative metabolites O2-, H2O2, HOCl, and MPO function additively or synergistically to mediate hyphal damage in the presence of these inhibitors.
Furthermore, HEP has been shown to bind to -defensins (members of the cationic proteins) and inhibit their function (2, 16). We did not find an inhibitory effect of HEP on PMN-induced hyphal damage to either the SA54A or SA1216 isolate. Supporting our results, Diamond et al. suggested that although cationic proteins may be involved in damaging Rhizopus oryzae hyphae, they do not appear to have an effect on PMN-induced damage to A. fumigatus hyphae (9). Since S. apiospermum is more closely related to A. fumigatus than to R. oryzae, it is probable that -defensins are not a key component in cell damage of this organism.
Among all seven inhibitors and scavengers used, only AZI exerted an inhibitory effect on PMN-induced hyphal damage and this inhibition was significant only with SA1216 isolate. Since the aim of these experiments was to maximize differences in hyphal damage between the two isolates, we used a relatively high E:T ratio. Smaller differences would be expected at lower E:T ratios. The concentration of AZI used in this study has been shown previously to inhibit MPO enzyme without altering other PMN functions (8, 9, 36). This finding suggests that the isolate SA54A is resistant to MPO products. This resistance may occupy an important role in the differences in susceptibility to innate host defense and, thus, may be partially responsible for the differences in clinical outcome for the patients infected with these isolates.
MPO deficiency is a common phagocytic disorder in the general population and is asymptomatic in the vast majority of persons. These individuals are not at significant risk for invasive infection due to filamentous fungi. However, when exposed to an uncommon pathogen such as S. apiospermum, an MPO-deficient host may be at greater risk for refractory infection.
In conclusion, this study has demonstrated a selective activity of phagocytic host defense against S. apiospermum, enhancing our understanding of the immunopathogenesis of infection due to this emerging fungal pathogen. The study findings indicate the need for further studies on the effects of immunomodulatory agents to augment host response and thus improve therapy to combat the devastating infections caused by S. apiospermum.
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ACKNOWLEDGMENTS |
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We thank Joanne Peter for performing antifungal drug susceptibility studies.
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FOOTNOTES |
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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).