A sub-inhibitory concentration of amphotericin B enhances candidastatic activity of interferon-{gamma}- and interleukin-13-treated murine peritoneal macrophages

Agnès Coste1,2, Marie D. Linas1,2,*, Sophie Cassaing1,2, José Bernad1, Sandrine Chalmeton1,2, Jean P. Séguéla1,2 and Bernard Pipy1

1Laboratoire des Macrophages, Médiateurs de l’Inflammation et Interactions Cellulaires, UPRES-EA 2405, INSERM IFR 31, C.H.U., Rangueil, 1 avenue Jean Poulhès; 2Département de Parasitologie et Mycologie, Centre Hospitalier Universitaire, Hopital Rangueil, 1 avenue Jean Poulhès, 31403 Toulouse Cedex 4, France

Received 3 September 2001; returned 13 November 2001; revised 18 December 2001; accepted 4 January 2002.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We studied the effects of interferon-{gamma} (IFN-{gamma}), a Th1 cytokine, and interleukin-13 (IL-13) or interleukin-4 (IL-4), Th2 cytokines, on the antifungal activity of resident murine peritoneal macrophages against Candida albicansin vitro’. IFN-{gamma}, IL-13 and IL-4 treatment enhanced the candidastatic functions of the macrophages. Reactive oxygen intermediates (ROIs) seem to be directly involved in the increase of anti-Candida activity in macrophages treated with Th1 or Th2 cytokines. Study of unopsonized C. albicans phagocytosis showed that IFN-{gamma} reduces the uptake process whereas the Th2 cytokines increase it. This difference is correlated to mannose receptor expression, which is decreased by IFN-{gamma} but increased by the Th2 cytokines. So, the effects on phagocytosis and candidastatic activity of IFN-{gamma}-treated macrophages are dissociated. In contrast, the phagocytic ability of macrophages pretreated ‘in vitro’ with IL-4 or IL-13 played a complementary role to the ROIs, in reduction of yeast proliferation by macrophages. In consequence, the macrophages treated with IL-13 and IL-4 develop a higher fungistatic activity than macrophages activated by IFN-{gamma}. Amphotericin B associated with IL-13 or IFN-{gamma}, but not with IL-4, enhanced the yeast growth inhibition activity of macrophages. The ROIs were involved in the additive effect of IFN-{gamma} with amphotericin B, whereas another mechanism was implicated in the increase of candidastatic activity of macrophages treated with IL-13 in association with amphotericin B.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Candida albicans is an opportunistic fungal pathogen that represents a serious problem in immunocompromised patients and patients undergoing immunosuppressive therapies,1,2 particularly those with impaired phagocytic cell function (mainly neutrophils and monocytes or macrophages). Several classes of antifungal compounds, including triazoles and amphotericin B, are available for management of invasive candidiasis. Nevertheless, the use of these drugs raises several problems. In severely immunocompromised patients the treatment of systemic mycoses by classical antifungal drugs has relatively low efficacy.3,4 The severe toxicity and low tolerance of patients to agents such as amphotericin B limit the doses administered, restricting their use and their efficacy in these patients. In immunocompromised patients, the lack of effective functions of phagocytic cells, which cooperate with antifungal drugs to clear the microorganism, seems to be a crucial factor that hampers the efficacy of the available antifungal agents.5,6

Cell-mediated immunity is believed to be an important mechanism in acting against opportunistic infections, especially those due to C. albicans. The mechanisms of defence against C. albicans are essentially dependent on the capacity of macrophages to phagocytose the yeasts and to exert their fungicidal activity by releasing large amounts of highly toxic molecules such as reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs). These macrophage functions are largely regulated by T helper (Th) lymphocytes through the production of cytokines. Thus, T cells play a central role in the regulation of immune responses against C. albicans.7,8 Th cells can be subdivided into two main subsets (Th1 and Th2) that are distinguished by different patterns of secreted cytokines.9,10 Th1 cytokines [such as interferon-{gamma} (IFN-{gamma})] initiate and participate in cell-mediated immune reactions, including the activation of inflammatory macrophages; they enhance the phagocytic function and killing of C. albicans by phagocytic leucocytes.11,12 Thus, IFN-{gamma} enhances superoxide and nitric oxide (NO) production by phagocytic cells. ROIs and RNIs produced by phagocytic cells are cytotoxic factors that act directly as antifungals.13 In contrast, cytokines produced by Th2 cells [such as interleukin-4 (IL-4) and -13 (IL-13)] are involved in humoral immune reactions, activating mast cells and eosinophils. These cytokines inhibit Th1 development and deactivate phagocytic effector cells and thus exhibit anti-inflammatory properties. It has been shown by others authors that IL-4 diminishes the fungicidal activity of macrophages against Candida14 as well as phagocytosis of the blastospores.15

However, the regulatory role of the Th1 and Th2 cytokines on macrophage functions in defence against C. albicans is complex. Indeed, recent studies indicate that endogenous IL-4 is required for the development of protective CD4+ Th type 1 cell responses to C. albicans.16 Furthermore, the macrophage mannose receptor (MMR) is both a major phagocytic and an endocytic receptor sufficient to mediate phagocytosis of unopsonized C. albicans.17,18 IL-4 and IL-13 increase both cell-surface MMR expression and mannose receptor-mediated endocytosis, whereas the prototypical Th1 cytokine, IFN-{gamma}, decreases both surface expression and endocytosis. However, these usually antagonistic cytokines cooperate in increasing heat-killed Saccharomyces phagocytosis via the mannose receptor.19

Although no clearly established recommendations have been published, the use of cytokines as therapeutic adjuvants in the prevention and/or treatment of invasive fungal infections has been advocated. Some studies established intracellular interaction between cytokines and sub-inhibitory concentrations of an antifungal agent, against C. albicans, and determined which mechanisms are likely to be responsible for the observed candidastatic activity of monocytes or macrophages. So, it was shown that the treatment of the macrophages with GM-CSF potentiates the action of fluconazole20,21 and that in vivo administration of M-CSF in mice, increases the efficacy of amphotericin B by enhancing the antifungal activities of macrophages and neutrophils.22

The purpose of this study was to define the intracellular interaction between Th1 (IFN-{gamma}) or Th2 (IL-4 and IL-13) cytokines and a sub-inhibitory concentration of amphotericin B against C. albicans, and to determine which mechanisms are likely to be responsible for the enhanced candidastatic activity of resident murine peritoneal macrophages.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents

Amphotericin B powder was purchased from Bristol Myers-Squibb (Woerden, The Netherlands). L-(NG)-Monomethyl arginine (L-NMMA), superoxide dismutase (SOD), 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) and mannosylated bovine serum albumin (mBSA) were obtained from Sigma (Saint Quentin, France). [3H]Uracil (specific activity, 44 Ci/mmol) was purchased from Amersham Pharmacia Bio-tech (Saclay, France). Recombinant murine IFN-{gamma} was obtained from Boehringer Mannheim (Roche, Basel, Switzerland), IL-4 was purchased from R&D Systems and IL-13 was provided by Sanofi Synthelabo (Labège, France). The monoclonal antibodies (anti-IFN-{gamma}, anti-IL-4 and anti-IL-13) were purchased from Biosource International (Montrouge, France). Medium 199 with Hanks’ salts and special serum-free medium (SFM) were prepared with Gibco products (Cergy Pontoise, France).

C. albicans

A single strain of C. albicans was used throughout these experiments. It was isolated from a blood culture of a patient at Toulouse-Rangueil Hospital and identified by common laboratory criteria. The MIC of amphotericin B for the isolate when tested by the NCCLS micromethod23 was 0.1 mg/L. The yeasts were maintained at –80°C in Sabouraud broth with glycerol. For the experiments, the yeasts were cultured on Sabouraud dextrose agar plates containing gentamicin 0.1 g/L and chloramphenicol 0.05 g/L for 24 h at 37°C.

Antifungal agents

Amphotericin B powder was dissolved in dimethyl sulphoxide (DMSO) and distilled water to give a standard solution of 1000 mg/L. Further dilutions were made in SFM medium supplemented with 1.8% glucose. The final concentration of DMSO in the cell culture was always <0.001%.

To determine the sub-inhibitory concentration of amphotericin B to use during our work, the strain studied was grown in the presence of various concentrations of the antifungal. The minimal concentration of amphotericin B necessary to inhibit the growth of the strain was 0.1 mg/L. A concentration of 0.05 mg/L inhibited proliferation too significantly (80%) to allow the effect of the various treatments on the inhibition of yeast proliferation to be determined. A concentration of 0.01 mg/L gave an inhibition of 20%. This concentration seemed to be suitable to reveal, during later work, any modification of the growth index by the cytokines.

Isolation of murine peritoneal macrophages

Murine resident peritoneal cells were harvested from female Swiss mice (6–10 weeks of age) maintained in a sterile environment. The peritoneal cavity was washed with 5 mL of sterile medium 199 with Hanks’ salts. The cells collected were centrifuged for 10 min at 400g and the cell pellet was suspended in SFM supplemented with 2 mM glutamax (Gibco-BRL, Cergy Pontoise, France), penicillin 100 U/mL and streptomycin 100 mg/L (Bio-Whittaker, Gagny, France). Then, the macrophages were allowed to adhere in 96- or 24-well Falcon culture plates for 2 h at 37°C under 5% CO2 in air. The non-adherent macrophages were removed by washing with phosphate-buffered saline (PBS) (Gibco-BRL) and the remaining adherent cells were incubated in SFM. After 2 h of adhesion, >98% of the adherent cells were non-specific esterase positive and had the morphological appearance of macrophages when examined by May–Grunwald and Giemsa staining.

Cytokine treatment

Macrophages were incubated for 18 h at 37°C in a humidified atmosphere with 5% CO2, with 20 IU of recombinant murine IFN-{gamma}, with 10 ng of IL-4 or with 10 ng of IL-13 per millilitre.

Determination of yeast proliferation

C. albicans growth in monolayer cultures was measured by monitoring [3H]uracil incorporation (1 µCi/well) into RNA of viable yeast cells.24 [3H]Uracil incorporation into C. albicans can be used as a sensitive index of proliferation.

For each assay, 2 x 105 macrophages per well were cultivated for 2 h in 96-well Falcon plates in SFM at 37°C and 5% CO2, stimulated by the cytokines (IFN-{gamma}, IL-13 or IL-4) for 18–24 h and used for infection as follows. Macrophage monolayers were infected with C. albicans to give a Candida: macrophage ratio of 1:200 for 6 h at 37°C in a humidified atmosphere containing 5% CO2. Various durations (4, 6, 8, 18 and 24 h) of proliferation were tested. A 6 h proliferation was found to be the most sensitive in our model. In some experiments, amphotericin B (0.01 mg/L) and labelled uracil (1 µCi) were added to each well. To evaluate the involvement and the mechanisms of action of these different modulators (Th1 and Th2 cytokines and amphotericin B), macrophages were pre-treated 30 min before the experiment with SOD (a specific inhibitor of superoxide anion production)25 or L-NMMA (a specific competitor of L-arginine for NO production).26 Macrophages were challenged with blastospores (6 h), then the supernatant from each well containing non-adherent blastospores was collected and centrifuged. The pellet was washed twice with PBS. The radioactivity incorporated into yeast RNA was counted with BCS scintillation fluid (Amersham). The monolayers containing adherent or phagocytosed yeasts were washed twice with PBS, disrupted with 1 M NaOH and neutralized later by 1 M HCl. Subsequently, scintillation liquid (Amersham) was added to each vial and the radioactivity counted in a liquid scintillation counter (Pharmacia LKB 1217). Therefore, the number of disintegrations per minute (dpm) counted provided a direct assessment of Candida proliferation in each assay. Therefore, the sum of radioactivity measured in the supernatant and in the monolayers was proportional to the yeast proliferation.

Evaluation of the phagocytosis percentage from [3H]uracil incorporation

To investigate the mechanism of C. albicans ingestion by murine peritoneal macrophages, we used [3H]uracil-prelabelled yeasts. Three C. albicans colonies were dispersed in 1 mL of Sabouraud broth containing 150 µCi of [3H]uracil. After 24 h of growth at 37°C with stirring, the labelled blastospores were centrifuged and resuspended in SFM supplemented with 1.8% glucose. For each assay, macrophage monolayers in 24-well plates were treated with the cytokines (IFN-{gamma}, IL-13 or IL-4) for 18–24 h, then infected with viable labelled C. albicans for 1 and 2 h (ratio of macrophage:yeast, 1:1) at 37 and 5°C. At 37°C both internalization of C. albicans by the macrophages and Candida–cell adhesion occurred. At 5°C the phagocytosis process is blocked and we measured adherence alone. After each incubation period (1 and 2 h), the culture medium was collected, the monolayers washed twice with PBS and the washings retained. The monolayers were disrupted with 1 M NaOH then neutralized by 1 M HCl. The radioactivity of the supernatant and washings and the radioactivity contained in the cellular lysate were counted independently by the use of BCS scintillation fluid (Amersham) in a liquid scintillation counter. The difference between the radioactivity measured in the monolayers at 37 and 5°C was proportional to phagocytosis. The radioactivity measured in the supernatant was proportional to the quantity of extracellular yeasts. The percentage of cells phagocytosed in comparison with the quantity of total yeasts was determined according to the following formula:

% Phagocytosis = {Delta}(37°C–5°C) dpm monolayers/[{Delta}(37°C–5°C) dpm monolayers + dpm supernatant]

Assay for ROI production

The oxygen-dependent respiratory burst of murine macrophages stimulated with yeast was measured by chemilumin-escence.27 Macrophages (2 x 105) were plated into 5 mm diameter plastic culture dishes. After cell activation, ROI production by macrophages in the presence of C. albicans was measured by chemiluminescence in the presence of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) with a thermostatically (37°C) controlled luminometer (Wallac 1420 Victor,2 Finland). The generation of chemiluminescence was mon-itored continuously for 1 h after incubation of the cells with luminol (66 µM) in the absence (basal conditions) and in the presence of 2 x 106/mL cells and/or amphotericin B (0.01 mg/L). In some experiments, macrophages were incubated for 10 min before yeast and amphotericin B were added in the presence of different inhibitors. To assess superoxide anion (O2) production, chemiluminescence was measured in the presence of SOD (scavenger for O2) and to evaluate NO production, chemiluminescence was measured in the presence of L-NMMA (inhibitor of NO production). None of the inhibitors affected cell viability at the concentrations used. Statistical analysis was based on the area under the curve expressed in counts x seconds.

Quantification of mannose receptors

For each assay, 2 x 105 macrophages were cultured for 2 h in 96-well plates in SFM at 37°C and 5% CO2, and were treated with cytokines (IFN-{gamma}, IL-13 or IL-4) for 24 h. The murine peritoneal macrophages were exposed to the MMR ligand, mBSA (840 nM) labelled with fluorescein. After 1 h of incubation at 4°C the cells were washed three times with PBS to remove free mBSA molecules. The fluorescence was analysed with a fluorimeter.

Statistical analysis

The data are expressed as means ± S.E.M. of three separate experiments with three replications per experiment. For each experiment, the data were subjected to one-way analysis of variance followed by the means multiple comparison method of Tukey. P < 0.05 was considered as the level of statistical significance.


    Results
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Modulating effect of amphotericin B on the candidastatic activity of IFN-{gamma}-, IL-13- or IL-4-pretreated macrophages

The C. albicans growth-inhibitory activity of macrophages pretreated with IFN-{gamma} (20 IU/mL), IL-4 (10 ng/mL) or IL-13 (10 ng/mL) for 18 h at 37°C against C. albicans was tested. The inhibition of C. albicans proliferation was evaluated after 6 h challenge with the yeast in the presence or absence of amphotericin B (0.01 µg/mL).

Pretreatment of resident macrophages with all three cytokines significantly raised their ability to inhibit proliferation of C. albicans relative to that of unpretreated macrophages (Table 1). Addition of amphotericin B led to a significantly enhanced anti-proliferative effect for macrophages without exposure to cytokines and those pretreated with IFN-{gamma} and IL-13.


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Table 1.  Effect of amphotericin B alone or in association with IFN-{gamma}, IL-4 or IL-13 on C. albicans proliferation
 
Amphotericin B had no effect on the anti-proliferative activity of macrophages pretreated with IL-4. To determine that the increase in the candidacidal functions of the macrophages was due specifically to the cytokines IL-13, IL-4 and IFN-{gamma}, proliferation was also studied in the presence of monoclonal antibodies directed against these cytokines (Table 2). In this case, the effects due to the various treatments were inhibited. Thus, the enhancement of the candidastatic functions of the macrophages really is the consequence of the various treatments.


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Table 2.  Effect of monoclonal antibodies directed against IFN-{gamma}, IL-13 and IL-4 on C. albicans growth activity of macrophages treated with these cytokines
 
Effect of IFN-{gamma}, IL-13 and IL-4 alone or associated with amphotericin B on unopsonized C. albicans phagocytosis

Peritoneal macrophages were incubated overnight with IFN-{gamma} (20 IU/mL), IL-4 (10 ng/mL) or IL-13 (10 ng/mL) at 37°C. The results presented correspond to the phagocytic capacity of the macrophages measured after 1 h of challenge with [3H]uracil-prelabelled C. albicans. After 2 h of macrophage–yeast contact we observed similar results (data not shown).

Figure 1 shows that when the macrophages were treated with IFN-{gamma}, the number of cells ingested decreased significantly (P < 0.05) compared with resident macrophages, which had a phagocytosis index of 54%. Conversely, IL-4 and IL-13 significantly increased (P < 0.05) the capacity of macrophages to take up C. albicans compared with resident macrophages.



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Figure 1. Effect of Th1 (IFN-{gamma}) and Th2 cytokines (IL-4, IL-13) on phagocytosis of C. albicans. The phagocytosis capacity of resident macrophages (Macro) and of Th1 or Th2 cytokine-treated macrophages was studied in the absence or presence of amphotericin B (AMB) 0.01 mg/L, after a C. albicans challenge of 1 h. The ratio of C. albicans: macrophages was 1:1. Values are means ± S.E.m. of three separate experiments. The asterisks indicate a significant difference, *P < 0.05, compared with the control (resident macrophages).

 
The C. albicans phagocytosis process was neither modified by the presence of amphotericin B, nor did the agent modify the effect of cytokines on phagocytosis.

Quantification of mannose receptors in IFN-{gamma}-, IL-13- or IL-4-pretreated macrophages

As the mannose receptor is involved in the phagocytosis of pathogenic microorganisms such as unopsonized C. albicans, we quantified the numbers of MMRs by studying the binding of fluorescein-labelled mBSA to the phagocytes for 1 h at 4°C. Macrophages were pretreated with IFN-{gamma} (20 IU/mL), IL-4 (10 ng/mL) or IL-13 (10 ng/mL) for 18–24 h at 37°C. Results are shown in Figure 2.



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Figure 2. Effect of Th1 (IFN-{gamma}, 20 IU/mL) and Th2 (IL-4, 10 ng/mL; IL-13, 10 ng/mL) cytokines on mannose receptor expression. The expression of MMRs was determined by binding to cells at 4°C of fluorescently labelled mBSA (840 nM). Values are means ± S.E.m. of three separate experiments. The asterisks indicate a significant difference, *P < 0.05 and **P < 0.01, compared with the control (resident macrophages; Macro).

 
In comparison with the control (resident macrophages) fluorescence on the macrophages was significantly decreased after treatment with IFN-{gamma} and significantly increased in the presence of IL-4 and IL-13, indicating a down-regulation of MMR expression by the Th1 cytokine and an up-regulation by the Th2 cytokines.

These data were confirmed by fluorescence microscopy, which demonstrated greatly increased fluorescence on macrophages in the presence of Th2 cytokines compared with the control (resident macrophages). Inversely, fluorescence on the IFN-{gamma}-treated macrophages was less intense compared with the control (data not shown).

Regression analysis of the data in the experiments shown in Figures 1 and 2 showed a linear association between mannose receptor fluorescence and phagocytosis by macrophages, demonstrating a true correlation between expression of man-nose receptors as influenced by different cytokines and the capacity of these cells to take up C. albicans (r2 = 0.942).

Role of oxidizing agents (ROIs and NO) in the candidastatic function of the IFN-{gamma}-, IL-13- or IL-4-pretreated macrophages

To evaluate the role of the ROIs and NO in the candidastatic function of the IFN-{gamma}-, IL-13- or IL-4-pretreated macrophages, we added the specific inhibitors L-NMMA (a specific competitor of L-arginine), and SOD (a specific inhibitor of superoxide anion production) to the macrophage–C. albicans growth proliferation system. Inhibitors were introduced 30 min before C. albicans infection.

Table 3 shows that the anti-proliferative properties of macrophages pretreated with cytokines was antagonized by SOD but not by L-NMMA. This indicates that ROIs are involved in the microbicidal function of the IFN-{gamma}-, IL-13- or IL-4-treated macrophages and that NO is not implicated in the inhibition of C. albicans growth by the cytokine-pretreated macrophages.


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Table 3.  Role of ROIs and NO on the candidacidal activity of macrophages pretreated with IFN-{gamma}, IL-4 or IL-13 in the presence or absence of amphotericin B (AMB) 0.01 mg/L
 
In the presence of amphotericin B no extra enhancement of the candidacidal activity of phagocytes pretreated with cytokines was evident in the presence of SOD except in IL-13-pretreated macrophages.

Production of oxidizing agents by the IFN-{gamma}-, IL-13- or IL-4-pretreated macrophages in the presence or absence of amphotericin B and during C. albicans infection

We used chemiluminescence to investigate production of oxidizing agents by macrophages, with and without overnight cytokine pretreatment. After infection of the cytokine-pre-treated macrophages with C. albicans, chemiluminescence was evaluated in the presence or absence of amphotericin B over a period of 1 h. The inhibitors SOD and L-NMMA were used to determine whether NO or O2 was produced.

In the absence of yeast, basal chemiluminescence of macrophages treated with IFN-{gamma} increased two-fold (5300.0 ± 242.2) in comparison with the resident macrophages (2523.3 ± 141.8), but no difference was seen for macrophages pre-treated with IL-4 (2474.2 ± 100.4) or IL-13 (2400.1 ± 70.0).

The addition of yeasts to the macrophage monolayer did not increase the production of oxidant agents by resident macrophages (2612.5 ± 97.3) but significantly increased pro-duction by the macrophages treated by the Th1 or Th2 cytokines (Table 4).


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Table 4.  Production of oxidizing agents by macrophages pretreated with Th1 (IFN-{gamma}, 20 IU/mL) or Th2 (IL-4, 10 ng/mL; IL-13, 10 ng/mL) cytokines, in the presence or absence of amphotericin B (AMB) and mixed with C. albicans
 
Amphotericin B had no effect on chemiluminescence of resident macrophages or of cytokine-pretreated macrophages.

Chemiluminescence in IFN-{gamma}-treated macrophages was inhibited by 80% in the presence of SOD and by 70% in the presence of L-NMMA, indicating that IFN-{gamma} induced both O2 and NO production. In contrast, chemiluminescence in IL-4- and IL-13-pretreated macrophages was inhibited significantly by SOD but not by L-NMMA, indicating that these cytokines principally induced O2 production.

Involvement of phagocytosis and oxidizing agent production in the inhibition of the C. albicans proliferation by treated macrophages

Multiple linear regression analysis of the data in the experiments shown in Tables 1 and 4 and Figure 1 showed a linear association between inhibition of proliferation exerted by the macrophages treated with Th2 cytokines, the increase of oxidizing agent production and the increase of phagocytosis (r2 = 0.9781). Conversely, we showed that the inhibition of proliferation exerted by the macrophages treated with IFN-{gamma} was only dependent on the increase of ROI production by these cells. Therefore, phagocytosis and the candidastatic activity developed by the macrophages activated by the IFN-{gamma} could be dissociated.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we demonstrated that macrophages treated with IFN-{gamma} (Th1 cytokine), IL-13 and IL-4 (Th2 cytokines) were rendered fungicidal against C. albicans. IFN-{gamma}, IL-13 and IL-4 directly enhanced the macrophage effector functions to fight C. albicans proliferation. Thus, our results confirm studies which show that IFN-{gamma} enhances the antifungal activity of macrophages against opportunistic fungal pathogens such as Candida species and Aspergillus fumigatus.2831 The in-creased effect of IL-13 on the antimicrobial activity of macrophages against C. albicans was demonstrated for the first time. In our experimental conditions, we showed that IL-4, another Th2 cytokine that shares a common sub-unit of its receptor with IL-13, stimulates the macrophage anti-Candida properties as well as IL-13. Indeed, we demonstrated that IL-13- or IL-4-pretreated macrophages significantly inhibited C. albicans growth.

Phagocytosis plays an important role in the microbicidal function of the macrophages against C. albicans. We show that the treatment of macrophages with IL-13 or IL-4 increases the capacity of these cells to ingest unopsonized C. albicans. Thus, in the presence of these two cytokines, phagocytosis plays a role in the modulation of yeast pro- liferation. In contrast, treatment of the macrophages with IFN-{gamma} decreases their capacity to ingest unopsonized C. albicans. Thus, unopsonized phagocytosis is not thought to participate in the inhibition of yeast proliferation by the macrophages treated with IFN-{gamma}.

It is known that the mannose receptor is involved in the phagocytosis of unopsonized pathogenic microorganisms such as C. albicans. This receptor recognizes glycosylated mol-ecules with terminal mannose, fucose, or N-acetylglucosamine moieties and efficiently internalizes soluble and particulate ligands through the endocytic and phagocytic pathways, respectively.3234 We noted that IL-4 and IL-13 induced an increase of mannose receptor expression. Our results support the works of Raveh et al.19 who showed that IL-14 or IL-13 up-regulate mannose receptor expression. We showed also that C. albicans phagocytosis was inhibited by mannose receptor agonists (mannosylated albumin) proving the in-volvement of these receptors in the Candida phagocytosis process. Thus, the increase of the phagocytosis capacity of cells treated with IL-13 and IL-4 was related to an increase in the expression of mannose receptors, which are the pre-ponderant receptors implied in the unopsonized C. albicans phagocytosis process. In contrast, the treatment of macrophages with IFN-{gamma} significantly reduced the expression of MMR expression. Our results support the works of Harris et al.35 and Alan et al.36 who showed that IFN-{gamma} represses mannose receptor expression. The inhibition of mannose receptor expression by IFN-{gamma} is correlated with a reduction in the capacity of these cells to take up C. albicans.

In spite of its down-regulator activity on mannose receptor expression, IFN-{gamma} plays an important role in the activation of macrophage functions against C. albicans. So, we studied macrophage ROI and NO production, known to be involved in the microbicidal function of macrophages.25,37,38 We found that pretreatment of macrophages with IFN-{gamma}, with or without the yeast, increases the macrophages oxidative hyperactivity leading to an enhancement of ROIs and NO release. We also showed that pretreatment of mouse resident peritoneal macrophages with IL-13 or IL-4 enhanced O2 release in response to infection by C. albicans indicating that these cytokines pre-activate the capacity of macrophages to produce O2 and not NO. This result is supported by previous studies which show that IL-4 and IL-10 inhibit NO release of mouse peritoneal and splenic macrophages.39

We determined the role of O2 and NO produced by IFN-{gamma}-, IL-4- and IL-13-treated macrophages in the inhibition of C. albicans growth. We found no direct correlation between NO production by the IFN-{gamma}-treated macrophages and their anti-C. albicans activity. In contrast, the use of SOD suggests that O2 was involved in the fungicidal activity of the macrophages treated by the Th1 (IFN-{gamma}) or Th2 (IL-4 and IL-13) cytokines. Thus, the inhibition of C. albicans growth by the macrophages treated by the two cytokine families appears to be dependent on superoxide anion production.

In conclusion, the phagocytosis capacity of macrophages treated by IL-4 or IL-13 plays a complementary role to ROIs in the control of the yeast proliferation by macrophages. This double effect of IL-4 and IL-13 could explain our results, which showed that the murine peritoneal macrophages treated by IL-13 or IL-4 develop a fungistatic activity against C. albicans greater than the macrophages activated by the IFN-{gamma}. Conversely, in the IFN-{gamma}-pretreated macrophages, phagocytosis and microbicidal activity via ROIs were dis-sociated.

We also studied the potential role of IFN-{gamma}, IL-4 or IL-13 to oppose C. albicans growth when used in combination with the antifungal drug amphotericin B at a sub-inhibitory concentration. The data presented in this report indicated that associating this antifungal agent with macrophages limited the yeast proliferation efficiently. This inhibitory activity was not dependent on the increase of ROI production by the macrophages under the effect of amphotericin B as suggested by Wilson et al.40 These authors reported that incubation of macrophages with amphotericin B resulted in enhanced res-piratory burst activity leading to O2 release. This difference in the results could be explained by the fact that these authors worked with a higher concentration of amphotericin B whereas the sub-inhibitory concentration used in our work was not sufficient to increase the ROI production of macrophages. Indeed, a concentration >0.1 mg/L is necessary to increase the ROI production of macrophages. In our study the concentration used was 10 times smaller.

Our study demonstrated that amphotericin B plus IFN-{gamma} or IL-13 enhanced the growth-inhibitory activity of macrophages against C. albicans. In contrast, we did not show any additive benefits of amphotericin B combined with IL-4 in fighting fungal proliferation. The additive effect of amphotericin B combined with IFN-{gamma} in the inhibition of the C. albicans growth is dependent on superoxide anion production. We also showed that superoxide anion was not increased by amphotericin B. Thus, amphotericin B could increase the permeability of the fungal membrane to superoxide anions and this allows the latter compounds to enter and destroy the organism more easily. On the contrary, we demonstrated that SOD does not completely eliminate the additive benefits of amphotericin B combined with IL-13. This means that the additive mechanism is superoxide anion independent. This indicates the existence of another factor in the mechanism. This factor would be produced only by the IL-13-pretreated macrophages. In association with the superoxide anion this factor would increase the amphotericin B effect on C. albicans growth. IL-4 would not induce the production of this factor and the superoxide anion alone would not be sufficient to potentialize the microbicidal activity of the antifungal drug. These results indicate that according to the cytokine used in the presence of amphotericin B, different signalling pathways would be involved in the inhibition of the C. albicans growth.

In conclusion, the present ‘in vitro’ study demonstrated that IFN-{gamma}, IL-13 and IL-4 treatment enhanced the candidacidal functions of macrophages. Both IFN-{gamma} and IL-13 combined with a sub-inhibitory concentration of amphotericin B further enhanced this candidacidal activity. According to the treatment used, the increase of the fungicidal functions of the macrophages depends on the superoxide anion production and/or phagocytosis via the regulation of mannose receptor expression.


    Footnotes
 
* Corresponding author. Tel: +33-5-6132-2893; Fax: +33-5-6132-2293; E-mail: md.linas{at}toulouse.inserm.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Fridkin, S. K. & Jarvis, W. R. (1996). Epidemiology of nosocomial fungal infections. Clinical Microbiology Review 9, 499–511.[ISI]

2 . Viscoli, C., Girmenia, C., Marinus, A., Colette, L., Martino, P., Vandercam, B. et al. (1999). Candidemia in cancer patients: a prospective, multicenter surveillance study by the Invasive Fungal Infection Group (IFIG) of the European Organization for Research and Treatment of Cancer (EORTC). Clinical Infectious Diseases 28, 1071–9.[ISI][Medline]

3 . Lortholary, O. & Dupont, B. (1997). Antifungal prophylaxis during neutropenia and immunodeficiency. Clinical Microbiology Reviews 10, 477–504.[Abstract]

4 . Walsh, T. J., Lee, J. W. & Roilides, E. (1992). Experimental antifungal chemotherapy in granulocytopenic animal models of disseminated candidiasis: approaches to understanding investigational antifungal compounds for patients with neoplastic diseases. Clinical Infectious Diseases 14, Suppl. 1, S139–47.[ISI][Medline]

5 . Roilides, E., Dignani, Anaissie, E. J. & Rex, J. H. (1998). The role of immunoreconstitution in the management of refractory opportunistic fungal infections. Medical Mycology 36, Suppl. 1, 12–25.[ISI][Medline]

6 . Stevens, D. A. (1998). Combination immunotherapy and antifungal chemotherapy. Clinical Infectious Diseases 26, 1266–9.[ISI][Medline]

7 . Romani, L. & Howard, D. H. (1995). Mechanisms of resistance to fungal infections. Current Opinion in Immunology 7, 517–23.[ISI][Medline]

8 . Romani, L., Puccetti, P. & Bistoni, F. (1996). Biological role of helper T-cell subsets in candidiasis. Chemical Immunology 63, 115–37.[ISI][Medline]

9 . Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. & Coffman, R. L. (1986). Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. Journal of Immunology 136, 2348.[Abstract/Free Full Text]

10 . Abbas, A. K., Murphy, K. M. & Sher, A. (1996). Functional diversity of helper T lymphocytes. Nature 383, 787.[ISI][Medline]

11 . Brummer, E., Morrison, C. J. & Stevens, D. A. (1985). Recombinant and natural gamma-interferon activation of macrophages in vitro: different dose requirements for induction of killing activity against phagocytizable and non phagocytizable fungi. Infection and Immunity 49, 724–30.[ISI][Medline]

12 . Marodi, L., Schreiber, S., Anderson, D. C., MacDermott, R. P., Korchak, H. M. & Johnston, R. B. (1993). Enhancement of macrophage candidacidal activity by interferon-gamma. Increased phagocytosis, killing, and calcium signal mediated by a decreased number of mannose receptors. Journal of Clinical Investigation 91, 2596–601.[ISI][Medline]

13 . Mencacci, A. & Romani, L. (1995). Rationale for cytokine and anti-cytokine therapy of Candida albicans infections. Journal of Mycology Medicine 5, 25–30.

14 . Cenci, E., Romani, L., Mencacci, A., Spaccapelo, R., Schiaffella, E., Puccetti, P. & Bistoni, F. (1993). Interleukin-4 and interleukin-10 inhibit nitric oxide-dependent macrophage killing of Candida albicans. European Journal of Immunology 23, 1034–8.[ISI][Medline]

15 . Roilides, E., Kadiltsoglou, I. & Walsh, T. J. (1997). Interleukin-4 suppresses antifungal activity of human mononuclear phagocytes against Candida albicans in association with decreased uptake of blastoconidia. FEMS Immunology and Medical Microbiology 19, 169–80.[ISI][Medline]

16 . Mencacci, A. & Romani, L. (1998). Endogenous interleukin 4 is required for the development of protective CD4+ T helper type 1 cell responses to Candida albicans. Journal of Experimental Medicine 187, 307–17.[Abstract/Free Full Text]

17 . Felipe, I., Bim, S. & Loyola, W. (1989). Participation of mannose receptors on the surface of stimulated macrophages in the phagocytosis of glutaraldehyde-fixed Candida albicans, in vitro. Brazilian Journal of Medical Biology Research 22, 1251–4.

18 . Marodi, L., Kaposzta, R., Campbell, D. E., Polin, R. A., Csongor, J. & Johnston, R. B., Jr (1994). Candidacidal mechanisms in the human neonate. Impaired IFN-gamma activation of macrophages in newborn infants. Journal of Immunology 153, 5643–9.[Abstract/Free Full Text]

19 . Raveh, D., Kruskal, B. A., Farland, J. & Ezekowitz, R. A. B. (1998). Th1 and Th2 cytokines cooperate to stimulate mannose-receptor-mediated phagocytosis. Journal of Leukocyte Biology 64, 108–13.[Abstract]

20 . Baltch, A. L., Smith, R. P., Franke, M. A., Ritz, W. J., Michelsen, P. B. & Bopp, L. H. (2001). Effects of cytokines and fluconazole on the activity of human monocytes against Candida albicans. Antimicrobial Agents and Chemotherapy 45, 96–104.[Abstract/Free Full Text]

21 . Natarajan, U., Randhawa, N., Brummer, E. & Stevens, D. A. (1998). Effect of granulocyte-macrophage colony-stimulating factor on candidacidal activity of neutrophils, monocytes or monocyte-derived macrophages and synergy with fluconazole. Journal of Medical Microbiology 47, 359–63.[Abstract]

22 . Tetsuya, K., Uchida, K. & Yamagichi, H. (2000). Therapeutic efficacy of human macrophage colony-stimulating factor, used alone and in combination with antifungal agents, in mice with systemic Candida albicans infection. Antimicrobial Agents and Chemotherapy 44, 19–23.[Abstract/Free Full Text]

23 . National Committee for Clinical Laboratory Standards. (1995) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Tentative Standard M27-T. NCCLS, Villanova, PA.

24 . Blackburn, J. (1992). The development of a radiometric assay for monocyte phagocytosis and killing. Microbiology Immunology 105, 331–6.

25 . Bogdan, C., Rollinghoff, M. & Diefenbach, A. (2000). Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Current Opinion in Immunology 12, 64–76.[ISI][Medline]

26 . Liew, F. Y., Millott, S., Parkinson, C., Palmer, R. M. J. & Moncada, S. (1990). Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from l-arginine. Journal of Immunology 140, 4793.2.

27 . Allen, R. C. & Loose, L. D. (1976). Phagocytic activation of luminol dependent cheluminescence in rabbit alveolar and peritoneal macrophages. Biochemical and Biophysical Research Communications 69, 245–52.[ISI][Medline]

28 . Roilides, E., Holmes, A., Blake, C., Pizzo, P. A. & Walsh, T. J. (1995). Effects of granulocyte colony-stimulating factor and interferon-{gamma} on antifungal activity of human polymorphonuclear neutrophils against pseudohyphae of different medically important Candida species. Journal of Leukocyte Biology 57, 651–6.[Abstract]

29 . Roilides, E., Holmes, A., Blake, C., Venzon, D., Pizzo, P. A. & Walsh, T. J. (1994). Antifungal activity of elutriated human monocytes against Aspergillus fumigatus hyphae: enhancement by granulocyte-macrophage colony-stimulating factor and interferon-gamma. Journal of Infectious Diseases 170, 894–9.[ISI][Medline]

30 . Roilides, E. & Pizzo, P. A. (1992). Modulation of host defenses by cytokines evolving adjuncts in prevention and treatment of serious infections in immunocompromised hosts. Clinical Infectious Diseases 15, 508–24.[ISI][Medline]

31 . Roilides, E., Uhlig, K., Venzon, D., Pizzo, P. A. & Walsh, T. J. (1993). Enhancement of oxidative response and damage caused by neutrophils to Aspergillus fumigatus hyphae by granulocyte colony-stimulating factor and interferon-gamma. Infection and Immunity 61, 1185–93.[Abstract]

32 . Ezekowitz, R. A. B., Sastry, K., Bailly, P. & Warner, A. (1990). Molecular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and phagocytosis of yeasts in COS cells. Journal of Experimental Medicine 172, 1785–94.[Abstract]

33 . Kitz, D. J., Stahl, P. D. & Little, J. R. (1992). The effect of a mannose binding protein on macrophage interactions with C. albicans. Cellular and Molecular Biology 38, 407–12.[ISI][Medline]

34 . Pontow, S. E., Kery, V. & Stahl, P. D. (1992). Mannose receptor. International Review of Cytology 137B, 221–44.

35 . Harris, N., Super, M., Rits, M., Chang, G. & Ezekowitz, R. A. (1992). Characterization of the murine macrophage mannose receptor: demonstration that the downregulation of receptor expression mediated by interferon-gamma occurs at the level of transcription. Blood 80, 2363–73.[Abstract]

36 . Alan, R., Ezekowitz, B., Hill, M. & Gordon, S. (1986). Interferon alpha/beta selectively antagonises down-regulation of mannosyl-fucosyl receptors on activated macrophages by interferon gamma. Biochemical and Biophysical Research Communications 136, 737–44.[ISI][Medline]

37 . Bogdan, C. (2000). The function of NO in the immune system. In Handbook of Experimental Pharmacology: Nitric Oxide (Mayer, B., Ed.), pp. 443–93. Springer, Heidelberg.

38 . Fang, F. C. (1997). Mechanisms of nitric oxide-related antimicrobial activity. Journal of Clinical Investigation 99, 2818–25.[Free Full Text]

39 . Bistoni, F. & Romani, L. (1993). Interleukin-4 and 10 inhibit nitric-oxide-dependent macrophage killing of Candida albicans. European Journal of Immunology 23, 1034–8.[ISI][Medline]

40 . Wilson, E., Thorson, L. & Speert, D. P. (1991). Enhancement of macrophage superoxide anion production by amphotericin B. Antimicrobial Agents and Chemotherapy 35, 796–800.[ISI][Medline]