Abstract

Proinflammatory cytokines have been proposed as adjunctive therapeutic agents to enhance the host immune response during infections caused by opportunistic fungi. The study compared the differential in vitro priming effects of interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on hyphal damage of opportunistic fungi mediated by isolated neutrophils (polymorphonuclear leukocytes, PMNL) and buffy coat cells (polymorphonuclear leukocytes/peripheral blood mononuclear cells, PMNL/PBMC) from healthy donors. IFN-γ (1000 U/mL) effectively primed both PMNL and PMNL/PBMC for enhanced hyphal damage of Aspergillus fumigatus, Fusarium solani, and Candida albicans. G-CSF (100 ng/mL) increased hyphal damage mediated by both PMNL and PMNL/PBMC against F. solani, and GM-CSF (100 ng/mL) augmented the antifungal activity of PMNL/PBMC against hyphal forms of both F. solani and C. albicans. IFN-γ may be superior to G-CSF or GM-CSF for enhancing the microbicidal activity of PMNL and PMNL/PBMC against opportunistic fungi.

Recent clinical surveys have documented the increased frequency of potentially life-threatening fungal infections in immunocompromised patients. Candida and Aspergillus species have been consistently noted as the most important fungal pathogens [1]. Neutropenia is recognized as the most important risk factor for invasive candidiasis, invasive aspergillosis, and other mold infections, such as fusariosis [2]. Despite aggressive antimicrobial therapy, marked morbidity and mortality are common clinical sequelae of these infections.

Whereas inhaled fungal conidia are destroyed by pulmonary alveolar macrophages primarily by nonoxidative mechanisms (and thereby preventing germination of the conidia) [3], the hyphal and pseudohyphal forms of fungi are damaged by both neutrophils (polymorphonuclear leukocytes, PMNL) and monocytes via an oxidative mechanism [4, 5]. Thus, PMNL play a key role in host defense once conidia have germinated into hyphae. Recent studies have demonstrated that specific proinflammatory cytokines, such as interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), are able to up-regulate certain functional properties of PMNL, including respiratory burst activity, antibody-dependent cellular cytotoxicity (ADCC), degranulation, and FcγRI (CD64) expression [6–11]. GM-CSF and IFN-γ also up-regulate distinct properties of monocytes, such as respiratory burst activity, phagocytosis, and FcγR expression [11–14]. Priming for these different cellular functions, particularly enhancement of respiratory burst activity, has been shown to increase killing of hyphal forms of Aspergillus fumigatus [10, 12], as well as hyphal and pseudohyphal forms of Candida albicans [9]. Furthermore, cytokines have been found to have differential priming effects on the distinct functions of PMNL and monocytes [6, 8, 9, 11]. Different microorganisms or different biologic growth forms of the same microorganism may elicit dissimilar responses from PMNL or monocytes primed by a particular cytokine [9].

Despite the fact that IFN-γ, G-CSF, and GM-CSF appear to share a number of biologic activities, there are few reports in which these cytokines have been compared with each other under the same experimental conditions [9–12, 14]. Because hyphal forms of pathogenic fungi are invariably present within lesions [9], we compared the relative ability of IFN-γ, G-CSF, and GM-CSF to prime PMNL and peripheral blood mono-nuclear cells (PBMC) in vitro for hyphal damage of three medically important opportunistic fungi: A. fumigatus, Fusarium solani, and C. albicans.

Materials and Methods

Organisms

The isolates of A. fumigatus, F. solani, and C. albicans used in this study are well-characterized clinical isolates from patients with invasive fungal infections at Fred Hutchinson Cancer Research Center and have been used extensively in our laboratory. Stock fungal isolates were maintained by standard microbiologic methods. Conidia (A. fumigatus and F. solani) and blastoconidia (C. albicans) were suspended in RPMI 1640 without phenol red (Life Technologies GIBCO BRL, Grand Island, NY) and containing 10 mM HEPES, pH 7.4 (RPMI/HEPES). Conidia and blastoconidia were counted in a hemocytometer and adjusted to a final concentration of 1 × 105 conidia/mL and 1 × 104 blastoconidia/mL. Aliquots of 100 μL were dispensed into 96-well flat-bottom plates (Costar, Cambridge, MA) and incubated for 16 h to form hyphae and pseudohyphae (A. fumigatus at 37°C, and F. solani and C. albicans at 25°C). More than 95% of conidia and blastoconidia germinated (hyphal length, 160–200 μm; pseudohyphal length, 80–100 μm).

Reagents

Recombinant human G-CSF (1 × 108 U/mg) was provided by Amgen (Thousand Oaks, CA), recombinant human GM-CSF (5 × 107 U/mg) was purchased from Immunex (Seattle), and recombinant human IFN-γ (1 × 107 U/mg) was purchased from R&D Systems (Minneapolis). 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT; Sigma, St. Louis) was prepared fresh daily in Dulbecco's PBS (PBS; Life Technologies Gibco BRL) at 0.5 mg/mL with heating at 55°C for 30 min. Then 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q; Sigma) was added at 50 μg/mL, and the solution was passed through a 0.22-μm filter.

PMNL purification

Venous blood was collected from healthy human volunteers using 0.2% K2 EDTA as anticoagulant. PMNL were isolated by sequential centrifugation on histopaque-1077 (Sigma), sedimentation in Dextran (Sigma) in 0.9% sodium chloride, and hypotonic lysis of remaining erythrocytes. After separation, PMNL were suspended in RPMI/HEPES. PMNL purity was >98% and cell viability was >98% as determined by trypan blue exclusion.

Buffy coat preparation (PMNL/PBMC)

Venous blood collected from healthy human volunteers using 0.2% K2 EDTA as anticoagulant was centrifuged at 2500 g for 20 min; then the layer of white blood cells overlying the erythrocytes was extracted and the remaining erythrocytes removed by hypotonic lysis. After separation, buffy coat cells were suspended in RPMI/HEPES. Differential counts were performed after Wright's stain was applied. Cell viability assessed by trypan blue exclusion was >98%.

Priming PMNL and PMNL/PBMC with cytokines

PMNL or PMNL/PBMC suspended in RPMI/HEPES were incubated with IFN-γ (1000 U/mL), G-CSF (100 ng/mL), or GM-CSF (100 ng/mL) for 60 min at 37°C. After treatment, the antifungal activities of PMNL and PMNL/PBMC were evaluated without further washing. Untreated PMNL and PMNL/PBMC were subjected to the same experimental conditions. These priming doses were based on preliminary studies with PMNL that demonstrated optimal priming at 100 ng/mL for G-CSF and GM-CSF and 1000 U/mL for IFN-γ. Furthermore, a 60-min preincubation yielded maximal in vitro priming effects for each of these cytokines (data not shown).

Hyphal damage assay

Hyphal damage was assessed by a modification of a tetrazolium dye assay described previously by Meshulam et al. [15]. PMNL or PMNL/PBMC at effector-to-target (E:T) ratios of 10:1 and 1:1 suspended in RPMI/HEPES plus 20% homologous serum were aliquoted in 100-μL volumes in a 96-well flat-bottom plate, for a final volume of 200 μL per well. All conditions were performed in quadruplicate. The leukocytes were gently sedimented to the bottom of the well by centrifugation at 400 g for 3 min and then gently rocked at 37°C for 2 h. Blank wells containing only RPMI/HEPES plus serum and control wells with only hyphae or cells were also prepared and subjected to the same experimental conditions. After incubation, 100 μL of fluid was aspirated from the wells using a multichannel pipettor, and leukocytes were lysed by addition of 100 μL of ice-cold distilled water, followed by centrifugation at 2000 g for 7 min at 1°C. This leukocyte lysis procedure was repeated once. Microscopic examination confirmed that leukocytes were completely lysed by this procedure.

Fungal viability was assessed by adding 100 μL of XTT solution (0.5 mg/mL XTT plus 50 μg/mL coenzyme Q) to each well and rocking the plates for 1 h at 37°C. Absorbance at 450 nm was determined by a microplate spectrophotometer (Automated Microplate Reader, model EL309; Bio-Tek Instruments, Winooski, VT). The percentage of fungal damage was defined by the equation {1 − [(A450 of fungi incubated with cells − A450 of cells alone)/A450 of fungi alone]} × 100.

Statistical analysis

Statistical analysis was performed using paired Student's two-tailed t test. Statistical significance was defined as P < .05.

Results

Differential priming effects of IFN-γ, G-CSF, and GM-CSF on PMNL-mediated hyphal damage

Baseline damage of A. fumigatus hyphae mediated by control (nonprimed) PMNL was 40% ± 8% (mean ± SE) at a 10:1 E:T ratio and 28% ± 8% at a 1:1 E:T ratio. IFN-γ significantly enhanced PMNL-mediated fungicidal activity against A. fumigatus hyphae at both E:T ratios, with 51% ± 7% (P = .04) hyphal damage observed at a 10:1 E:T ratio and 44% ± 7% (P = .001) at a 1:1 E:T ratio. In contrast, PMNL-mediated hyphal damage was not significantly affected by incubation with either G-CSF or GM-CSF (figure 1).

Figure 1

Damage of hyphae (Aspergillus fumigatus and Fusarium solani) and pseudohyphae (Candida albicans) by purified polymorphonuclear leukocytes (PMNL) (A–C; n = 6 donors) and buffy coat cells (PMNL/peripheral blood mononuclear cells [PBMC]) (D–F; n = 5 donors) in vitro. Damage of hyphae and pseudohyphae was assessed by XTT assay (see Materials and Methods) at effector-to-target ratios of 10:1 (striped bars) and 1:1 (open bars). Leukocyte-mediated fungicidal activity against A. fumigatus (A and D), F. solani (B and E), and C. albicans (C and F) was determined after no priming or priming with G-CSF (100 ng/mL), GM-CSF (100 ng/mL), or IFN-γ (1000 U/mL), respectively. Data are reported as hyphal/pseudohyphal damage expressed as % (mean ± SE) (see Materials and Methods). * Significant difference in leukocyte-mediated hyphal damage vs. nonprimed control (no cytokine) (P < .05).

Figure 1

Damage of hyphae (Aspergillus fumigatus and Fusarium solani) and pseudohyphae (Candida albicans) by purified polymorphonuclear leukocytes (PMNL) (A–C; n = 6 donors) and buffy coat cells (PMNL/peripheral blood mononuclear cells [PBMC]) (D–F; n = 5 donors) in vitro. Damage of hyphae and pseudohyphae was assessed by XTT assay (see Materials and Methods) at effector-to-target ratios of 10:1 (striped bars) and 1:1 (open bars). Leukocyte-mediated fungicidal activity against A. fumigatus (A and D), F. solani (B and E), and C. albicans (C and F) was determined after no priming or priming with G-CSF (100 ng/mL), GM-CSF (100 ng/mL), or IFN-γ (1000 U/mL), respectively. Data are reported as hyphal/pseudohyphal damage expressed as % (mean ± SE) (see Materials and Methods). * Significant difference in leukocyte-mediated hyphal damage vs. nonprimed control (no cytokine) (P < .05).

Control (nonprimed) PMNL induced 26% ± 3% and 16% ± 5% damage to hyphal forms of F solani at 10:1 and 1:1 E:T ratios, respectively. Both IFN-γ and G-CSF significantly enhanced PMNL-mediated damage of F. solani hyphae at a 10:1 E:T ratio. IFN-γ increased F. solani hyphal damage to 45% ± 4% (P = .001), and G-CSF augmented hyphal damage to 38% ± 3% (P = .02). In contrast, PMNL-mediated damage to F. solani hyphae was not significantly affected by GM-CSF (figure 1).

Baseline damage of pseudohyphae of C. albicans mediated by control (nonprimed) PMNL was 43% ± 5% at a 10:1 E:T ratio and 30% ± 5% at a 1:1 E:T ratio. IFN-γ significantly enhanced PMNL-mediated fungicidal activity against C. albicans pseudohyphae to 57% ± 4% (P = .04). Neither G-CSF or GM-CSF significantly affected the ability of PMNL to damage C. albicans pseudohyphae (figure 1).

Differential priming effects of IFN-γ, G-CSF, and GM-CSF on buffy coat-mediated hyphal damage

To assess cytokine priming of a mixed leukocyte population to induce hyphal damage, buffy coat cells were isolated from 5 independent donors. Differential counting revealed mean percentage compositions of 58% (range, 48%–66%) for neutrophils, 31% (range, 18%– 39%) for lymphocytes, 12% (range, 8%–19%) for monocytes, and <1% for eosinophils.

Baseline damage of A. fumigatus hyphae mediated by control (nonprimed) PMNL/PBMC was 41% ± 8% at a 10:1 E:T ratio and 17% ± 6% at a 1:1 E:T ratio. IFN-γ significantly enhanced PMNL/PBMC-mediated fungicidal activity against A. fumigatus hyphae with 54% ± 8% (P = .02) hyphal damage observed at a 10:1 E:T ratio. In contrast, PMNL/PBMC-mediated hyphal damage was not significantly affected by incubation with either G-CSF or GM-CSF (figure 1).

Baseline damage by control (nonprimed) PMNL/PBMC to F. solani hyphae was 50% ± 7% at a 10:1 E:T ratio and 31% ± 7% at a 1:1 E:T ratio. All three cytokines significantly increased PMNL/PBMC-mediated damage of F. solani hyphae at a 10:1 E:T ratio. Hyphal damage increased to 74% ± 8% (P = .009), 65% ± 6% (P = .003), and 67% ± 8% (P = .01) after PMNL/PBMC priming with IFN-γ, G-CSF, and GM-CSF, respectively. None of the cytokines significantly enhanced PMNL/PBMC-mediated hyphal damage when examined at a 1:1 E:T ratio (figure 1).

Control (nonprimed) PMNL/PBMC induced 62% ± 8% and 52% ± 14% damage to C. albicans pseudohyphae at 10:1 and 1:1 E:T ratios, respectively. Both IFN-γ and GM-CSF significantly enhanced PMNL/PBMC-mediated damage of C. albicans pseudohyphae at a 10:1 E:T ratio, with 86% ± 7% (P = .04) damage observed after priming with IFN-γ and 81% ± 5% (P = .03) after priming with GM-CSF. In contrast, PMNL/PBMC-mediated pseudohyphal damage was not significantly affected by G-CSF (figure 1).

Discussion

This study demonstrated that IFN-γ, G-CSF, and GM-CSF can modulate the fungicidal activity of leukocytes against hyphal and pseudohyphal forms of opportunistic fungal pathogens. However, this modulatory effect on leukocyte-mediated fungicidal activity is variable, being both cytokine-dependent and organism-specific.

IFN-γ significantly enhanced PMNL-mediated damage to the hyphal and pseudohyphal forms of the three opportunistic fungi studied. The colony-stimulating factors, G-CSF and GM-CSF, were less effective in priming PMNL for hyphal damage. G-CSF significantly increased PMNL-mediated damage only of F. solani hyphae, whereas GM-CSF failed to modulate the ability of isolated PMNL to induce damage to the hyphal and pseudohyphal forms of A. fumigatus, F. solani, or C. albicans.

Recently, it has been reported that primed PMNL and monocytes display their greatest oxidative activity under conditions in which cell-to-cell contact is promoted [11]. Furthermore, lymphocytes, especially NK cells, have been shown to respond to C. albicans exposure by releasing cytokines that activate PMNL [14]. To study the cooperative interaction among PMNL, monocytes, and lymphocytes, we examined the ability of non-primed and primed buffy coat (PMNL/PBMC) preparations to induce hyphal damage of opportunistic fungal pathogens. As was observed with isolated PMNL, IFN-γ significantly enhanced PMNL/PBMC-mediated antifungal activity against all three of the opportunistic fungi studied. GM-CSF significantly increased PMNL/PBMC-mediated fungicidal activity against both F. solani and C. albicans but not A. fumigatus. Significant G-CSF priming of PMNL/PBMC for antifungal activity was limited to F. solani.

Previously, we reported that G-CSF administered in vivo to healthy human subjects augmented PMNL-mediated activity against conidia of medically important fungal pathogens [7]. This study extends our understanding of cytokine modulation of PMNL and PMNL/PBMC activity against opportunistic fungi. Among the 3 clinically available cytokines compared in parallel in this study, IFN-γ was the most effective in priming PMNL and PMNL/PBMC for hyphal and pseudohyphal damage. These findings provide further experimental support for the use of cytokines as an adjunct to conventional antifungal therapy in the treatment of infections due to opportunistic fungal pathogens and argue for controlled clinical trials to define the utility of this potential therapeutic modality.

Acknowledgments

We thank Stuart M. Levitz and Elin Rodger for helpful advice and discussions.

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Presented in part: Infectious Diseases Society of America 36th annual meeting (abstract 341: Clin Infect Dis 1998; 27:985).
Informed consent was obtained from healthy human volunteers prior to the start of the study. Studies were performed in accordance to human experimentation guidelines established by the US Department of Health and Human Services and by the Human Subjects Division of the Investigational Review Board of the University of Washington.
Grant support: Amgen, Inc., and NIH (HL-53515 [to D.C.D.] and AI-01411 [to J.-A.H.v.B.]).