Effects of glycyrrhizin, an active component of licorice roots, on Candida albicans infection in thermally injured mice

Abstract

Due to the generation of burn-associated CD8⁺ CD11b⁺ TCR γ/δ⁺ type 2 T cells (burn-associated type 2 T cells), the susceptibility of thermally injured mice to infection with C. albicans has been shown to be increased by up to 50-fold when compared with normal mice. Glycyrrhizin (GR), an active component of licorice roots, reduced the susceptibility of thermally injured mice to C. albicans infection to levels observed in normal mice. Thermally injured mice inoculated with CD4⁺ T cells from GR-treated mice were also resistant to C. albicans infection. The following demonstrated that susceptibility to fungal infection was similar in thermally injured mice and normal mice inoculated with T6S cells (a clone of burn-associated type 2 T cells). This susceptibility of T6S mice (normal mice inoculated with T6S cells) was reversible by (i) administration of GR, (ii) inoculation of CD4⁺ T cells from GR-treated mice, and (iii) injection of a mixture of MoAbs targeted against type 2 cytokines (IL-4 and IL-10). After stimulation with anti-CD3 MoAb, splenic T cells from thermally injured and T6S mice, treated with GR or inoculated with CD4⁺ T cells from GR-treated mice, did not have type 2 cytokines in culture supernatants. They were present in splenic T cell cultures from thermally injured and T6S mice that were treated with saline or inoculated with naive T cells. These results suggest that GR, by inducing CD4⁺ T cells which suppress type 2 cytokines produced by burn-associated type 2 T cells, improves the resistance of thermally injured mice to C. albicans. An anti-type 2 T cell action of the CD4⁺ T cells derived from GR-treated mice was previously described.

INTRODUCTION

Normal individuals can experience severe infections, but the majority of these infections occur in patients with compromised host defences, such as trauma and burn victims [1], patients with malignancies [2], transplant recipients [3] and patients with AIDS [4]. Infection is one of the most important factors in mortality following thermal injury [1]. Candida albicans has been reported to be a severe pathogen in thermally injured patients [5], while common but rarely disseminating in immunocompetent individuals. With the current practice of early wound excision, early adequate fluid resuscitation and better control of infection, the incidence of fatal Candida infection has declined in burned patients [6,7]. However, fungaemia remains as a major cause of mortality and morbidity in burned patients [5].

The major reason for increased susceptibility of thermally injured patients to these infections is the immunological abnormalities associated with thermal injury [8–10]. In previous studies from our laboratory [11], the susceptibility of burned mice to infection with herpes simplex virus type 1 (HSV-1) was shown to be 100 times higher than normal mice. The increased susceptibility of burned mice to HSV-1 infection was shown to be associated with the production of burn-associated CD8⁺CD11b⁺ TCR γ/δ⁺ type 2 T cells (burn-associated type 2 T cells) [11], since burned and normal mice inoculated with these type 2 T cells were equally susceptible [11]. In addition, the impaired resistance of burned mice to HSV-1 infection was shown to be reversed when they were treated with a mixture of MoAbs directed against IL-4 and IL-10 (type 2 cytokines) [11]. This suggests that type 2 T cell-associated cellular responses (type 2 T cell responses) are important in the impaired resistance of thermally injured mice to infection with HSV-1. It has also been shown that thermally injured mice treated with glycyrrhizin (GR) resisted HSV-1 infection [12]. This compound has been shown to be an inducer of anti-type 2 T cells [12–14], which are able to counteract the activity of the burn-associated type 2 T cells [11–14]. Anti-type 2 T cells induced by GR were characterized as CD4⁺CD28⁺ TCR α/β⁺Vicia villosa lectin-adherent T cells [12–14]. They were different from the other CD4⁺ T cell subsets, such as T helper type 1 (Th1 cells) and T helper type 2 cells (Th2 cells) by their cytokine-producing profile and adherence to V. villosa lectin [11–14]. When thermally injured mice were treated intraperitoneally with 10 mg/kg of GR 2 and 4 days after burn injury, these anti-type 2 T cells were detected in their spleens 5 days after burn injury and were present for 11 days post-thermal injury [14]. Also, anti-type 2 T cells were detected in spleens of normal mice 1 day after the last treatment with GR [12]. In this experiment, GR was administered twice to normal mice (2 and 4 days before removal of spleen cells). These cells peaked 2 days after the last GR treatment [12]. Anti-type 2 T cells were also produced in vitro when splenic mononuclear cells (SMNC) from normal mice were cultured with 0.1–10 μg/ml of GR [13].

GR has been used clinically in Japan for patients with active chronic hepatitis [15–17]. GR has been shown to be anti-inflammatory [16], augment natural killer cells [16] and induce interferon [18]. GR also influences infection with human cytomegalovirus, herpes simplex virus, HIV and influenza virus in the appropriate experimental system [19–22]. In this study, the effect of GR on the impaired resistance of burned mice against C. albicans infection was examined. The results showed that the impaired resistance of burned mice to C. albicans was reversed by GR. It was also suggested that GR might protect thermally injured mice from C. albicans by inhibiting type 2 cytokine production from burn-associated type 2 T cells.

MATERIALS AND METHODS

Mice, cells, media and C. albicans

Eight-week-old BALB/c mice (Jackson Labs, Bar Harbor, ME) were used in the experiments. All procedures involving animal experiments were approved by the Animal Care and Use Committee of the University of Texas Medical Branch at Galveston (ACUC approval number 93-04-030). T6S cells, a clone of burn-associated CD8⁺CD11b⁺ TCR γ/δ⁺ type 2 T cells established in our laboratory [23], were serially maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mml-glutamine and antibiotics (complete medium) in the presence of 10 U/ml rIL-2. T6S cells were shown to suppress the proliferation of lymphocytes stimulated with allogeneic lymphocytes in a mixed lymphocyte reaction [23]. T6S cells also produced type 2 cytokines (IL-4 and IL-10) in their culture fluids when they were stimulated with anti-CD3 MoAb [23]. However, IL-2 and interferon-gamma (IFN-γ) were not produced by T6S cells stimulated with the same MoAb [23]. A strain of C. albicans was isolated from a patient at the University of Texas Medical Branch and serially maintained on agar plates of Sabouraud dextrose medium.

Reagents

GR was supplied by Minophagen Pharmaceutical Co., Ltd (Tokyo, Japan). This compound consists of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid [15]. GR was dissolved in saline at appropriate concentrations and 0.2 ml of the solution was administered intraperitioneally to mice 2 and 4 days after burn injury (therapeutic treatment) or 1 day before, then 2 and 4 days after burn injury (prophylactic and therapeutic treatment). Also, GR was administered to normal mice 2 and 4 days before spleens were harvested, according to the previous studies [12]. MoAbs for IL-2, IFN-γ, IL-4, IL-10, and CD3ε were purchased from Pharmingen (San Diego, CA). Murine recombinant IL-2 (rIL-2), rIFN-γ, rIL-4, and rIL-10 were purchased from PeproTech (Rocky Hill, NJ) Amphotericin B were obtained commercially from Sigma Chemical Co. (St Louis, MO).

Thermal injury

Thermally injured mice were prepared, as described previously [23,24]. Briefly, before being subjected to burn injury, all mice (24–26 g) were anaesthetized intraperitoneally with pentobarbital (40 mg/kg). The back hair was removed with an electric clipper. A custom insulated mould with a 2.5 × 3.5-cm window was pressed firmly against the shaved area and the bare region was exposed for 9 s to the gas flame from a Bunsen burner equipped with a flame dispersing cap. This procedure resulted in the production of a third-degree burn over approx. 15% of the total body surface area on these mice. Immediately after the burn injury, 4 ml of saline were administered to each mouse for fluid resuscitation. Thermally injured mice were housed until they were needed in the experiments. Control mice were anaesthetized and their backs were shaved, but the shaved regions were not exposed to a gas flame.

Preparation of splenic lymphocytes

SMNC were prepared from normal mice that had been treated or not treated with GR (i.p., 10 mg/kg) by Ficoll–Hypaque sedimentation, as described previously [12,14]. The SMNC from mice treated with saline were used as controls. To prepare whole T cells, CD4⁺ T cells and CD8⁺ T cells, SMNC (5 × 10⁷ cells/ml) were passed through a T cell enrichment column (R&D Systems, Minneapolis, MN), the CD4 T cell subset column (R&D Systems) or the CD8 T cell subset column (R&D Systems), respectively. After washing with media, harvested cells were used in the experiments. The purities of all three cell preparations (whole T cells, CD4⁺ T cells and CD8⁺ T cells) were > 96%, as previously described [12]. These cell preparations were injected intravenously into burned or T6S mice infected with C. albicans. In other experiments, splenic T cells were prepared from normal, burned mice or T6S mice that had been treated or not treated with GR, respectively, and burned or T6S mice inoculated with 5 × 10⁶ cells/mouse of CD4⁺ T cells. These cells (2 × 10⁶ cells/ml) were resuspended in complete medium and stimulated with anti-CD3 MoAb (2.5 μg/ml) for 48 h and supernatants measured for cytokines.

Infection experiments

One day after thermal injury, mice were infected intravenously with appropriate amounts of C. albicans. An inoculum of 1 × 10⁷ cells/mouse of C. albicans in normal mice and 2 × 10⁵ cells/mouse in burned mice was shown to be equivalent to 1 LD₅₀ [25]. The susceptibility of thermally injured mice to C. albicans infection was 50 times greater than that of normal mice. In the first experiments, mice at 1 day after thermal injury (n = 20 each) or normal mice (n = 10) were infected with 1 × 10⁶ cells/mouse of C. albicans (5 LD₅₀ in burned mice and 0.1 LD₅₀ in normal mice), and they were treated intraperitioneally with various doses of GR (0.1–50 mg/kg) 2 days before, 1 and 3 days post-fungal infection. As controls, normal mice and burned mice were treated intraperitoneally with saline (0.2 ml/mouse). As a positive control, infected mice were treated twice daily with amphotericin B (20 mice, 1 mg/kg, i.p.) for a total of 7 days beginning 1 day after the infection [26]. In the second series of experiments, normal unburned mice infected with 5 × 10⁷ cells/mouse of C. albicans (5 LD₅₀ in normal mice and 250 LD₅₀ in burned mice) were treated intraperitoneally with 10 mg/kg of GR 2 days before and 1 and 3 days after infection. As a control, normal mice exposed to C. albicans were treated intraperitoneally with saline (0.2 ml/mouse). As a positive control, amphotericin B was administered intraperitoneally to mice twice daily for a total of 7 days beginning 1 day after the C. albicans infection. In another experiment, mice at 1 day after thermal injury (n = 20 each) were inoculated with whole T cells (1 × 10⁷ cells/mouse), CD4⁺ T cells (5 × 10⁶ cells/mouse) or CD8⁺ T cells (5 × 10⁶ cells/mouse) from spleens of normal mice treated intraperitoneally with GR. Two hours after the inoculation, these mice were exposed to 5 LD₅₀ of C. albicans (1 × 10⁶ cells/mouse, 0.1 LD₅₀ in normal mice). As controls, burned mice (n = 10 each) inoculated with whole T cells, CD4⁺ T cells or CD8⁺ T cells from spleens of normal mice treated with saline, were infected with the same amount of C. albicans. Instead of being subjected to thermal injury, normal unburned mice were inoculated intravenously with 2 × 10⁶ cells/mouse of T6S cells and designated as T6S mice. Four groups of normal mice (n = 10 each) were inoculated intravenously with 1 × 10⁷ cells/mouse of splenic T cells from normal mice or 2 × 10⁶ cells/mouse of T6S cells. Twenty-four hours after inoculation, these mice were infected with appropriate amounts of C. albicans. As controls, four groups of normal mice (n = 10 each) treated with saline or mice 1 day after thermal injury were infected with appropriate amounts of C. albicans. In addition, T6S mice (n = 20 each) were treated with a mixture of MoAbs against IL-4 and IL-10 (s.c., 200 μg/mouse, 2 h before, then 2 and 4 days after infection), or inoculated intravenously with CD4⁺ T cells from GR-treated mice (5 × 10⁶ cells/mouse) 2 h before infection. The protective effect of various treatments on C. albicans infection in normal or thermally injured mice was identified when differences in mean survival time in days (MSD) and survival rate were measured. The survival rate of each group of mice was determined at 28 days after infection.

To measure the amount of C. albicans in kidneys

To determine the growth of C. albicans in organs, groups of 10 mice treated with GR or without GR were exposed to 5 LD₅₀ dose of C. albicans. Kidneys were removed from these mice 7 and 14 days after the infection, disrupted with a glass homogenizer [27], and suspended in saline at a 10% concentration (w/v). Serial 10-fold dilutions of the suspensions were performed and 1 ml of the dilution was plated onto a 10 × 10-cm Petri dish with Sabouraud dextrose agar. Forty-eight hours after cultivation at 27°C, the number of colonies on the agar plates was counted [27].

Anti-fungal activity of GR in vitro

To determine the effect of GR on the growth of C. albicans in vitro, 1 ml of saline suspended with 1 × 10² cells of C. albicans and 1 ml of saline that contained 1–1000 μg/ml of GR were plated onto a 10 × 10-cm Petri dish with Sabouraud dextrose agar. Following 48 h of cultivation at 27°C, the numbers of colonies were counted [27]. In order to determine the effect of GR on the viability of C. albicans, 1 ml of saline that contained 1–1000 μg/ml of GR was mixed with 1 ml of saline containing 1 × 10⁵ cells/ml of C. albicans. After 24 h at 4°C, these solutions were diluted and assayed using a colony-forming assay on the agar plates. As a control, the same C. albicans concentration was mixed with saline or 1 ml of saline that contained 0.1–1 μg/ml of amphotericin B and incubated for 24 h at 4°C.

Production and assay of cytokines

For the in vitro induction of IL-2, IFN-γ, IL-4 and IL-10, 2 × 10⁶ cells/ml of splenic T cells from (i) normal and burned mice treated with or without GR, (ii) burned mice inoculated with 5 × 10⁶ cells/mouse of CD4⁺ T cells from mice treated with GR, (iii) burned mice inoculated with 1 × 10⁷ cells/mouse of splenic T cells from normal mice, (iv) T6S mice treated with or without GR, (v) T6S mice inoculated with CD4⁺ T cells from mice treated with GR, and (vi) T6S mice inoculated with splenic T cells from normal mice, were stimulated with anti-CD3 MoAb (2.5 μg/ml) at 37°C in complete medium supplemented with 30 mm HEPES buffer and 5 × 10⁻⁵m 2-mercaptoethanol (2-ME) [11]. Culture fluids harvested 48 h after the stimulation were assayed for cytokines using a standard ELISA technique and an enhanced protein-binding ELISA plate (Costar, Cambridge, MA). The detection limit for the respective cytokines by ELISA varied between 5 and 50 pg/ml. The assay was performed in triplicate, and the results expressed as the mean level.

Statistical analysis

The survival of mice exposed to C. albicans was statistically analysed by log rank test (% survival of mice) or by the analysis of variance (anova, MSD of mice), followed by Fisher's protected least significant difference test. Other data are expressed as mean ± s.d. and the results were analysed by anova. P < 0.05 was considered significant.

RESULTS

GR improves the resistance of thermally injured mice to C. albicans

An inoculation of 1 × 10⁶ cells/mouse of C. albicans produced 100% mortality in burned mice and 0% mortality in normal mice (Fig. 1). This amount of C. albicans was previously determined to be a 5 LD₅₀ in burned mice and a 0.1 LD₅₀ in normal mice [25]. However, 80% of burned mice exposed to the same amount of C. albicans survived when treated intraperitoneally with a 10-mg/kg dose of GR 1 day before, then 2 and 4 days post-burn injury (Fig. 1) (P < 0.001). In addition, when GR was given to mice 2 and 4 days post-burn injury, 70% of them survived (Fig. 1) (P < 0.001). A 30% survival rate was observed in burned mice treated with amphotericin B, as a positive control (Fig. 1). The survival effect of GR in thermally injured mice was studied for dose-dependency at doses ranging from 0.1 to 10 mg/kg. The maximum efficacy of GR was observed when burned mice infected with C. albicans were treated with at least a 10-mg/kg dose. On the other hand, GR did not protect normal mice infected with 5 LD₅₀ (5 × 10⁷ cells/mouse) of C. albicans. Amphotericin B resulted in a 50% survival rate in these mice (P < 0.005). These results suggest that the activity of GR on C. albicans infection in mice was different from the anti-fungal effect of amphotericin B, and that recovery from the burn-associated impairment of anti-fungal immunity might be involved in the effect of GR. In subsequent experiments, the effect of GR on the growth of C. albicans in kidneys of normal and burned mice was determined. When 1 × 10⁶ cells/mouse of C. albicans were challenged, 7.6 × 10⁴ cells/kidney in normal mice and 1.3 × 10⁶ cells/kidney in burned mice were present at 7 days. However, only 1.2 × 10⁴ cells/organ were detected in kidneys of burned mice treated with GR (P < 0.01). When normal mice infected with same concentration were treated with GR, the growth of C. albicans in kidneys did not differ from normal mice treated with saline. Similar results were obtained when the numbers of the organisms were assayed in kidneys of these mice at 14 days after infection (P < 0.001). This suggests that the severity of C. albicans infection in burned mice was reduced by GR treatment.

Fig. 1.

Glycyrrhizin (GR) improved the resistance of thermally injured mice against C. albicans infection. Mice 1 day after thermal injury or normal mice were infected with 1 × 10⁶ cells/mouse of C. albicans (5 LD₅₀ in burned mice and 0.1 LD₅₀ in normal mice). A 10-mg/kg dose of GR was administered to mice 1 day before, then 2 and 4 days after burn injury (○). GR was also given to mice 2 and 4 days post-burn injury (Δ). As controls, normal (□) and burned mice (•) were treated with saline (0.2 ml/mouse, i.p.). As a positive control, a 1-mg/kg dose of amphotericin B was administered intraperitoneally to burned mice twice daily for a total of 7 days beginning 1 day after the infection of C. albicans (▴).

Effect of GR on C. albicans in vitro

When 1 × 10² cells/plate of C. albicans were cultured for 48 h at 27°C in Sabouraud dextrose agar and 1–1000 μg/ml of GR were added, the replication of the pathogen was not altered (data not shown). Next, 1 × 10⁵ cells/ml of C. albicans were incubated with 1 ml of saline containing 1–1000 μg of GR per ml for 24 h at 4°C. This did not alter the growth of C. albicans. Thus, GR had no activity on the growth or viability of C. albicans in vitro. This suggests that immune responses were important in the protection of thermally injured mice exposed to C. albicans by GR. Therefore, in subsequent studies, cell populations from GR-treated mice were adoptively transferred to burned mice exposed C. albicans.

The effect of splenic T cells from GR-treated mice on C. albicans infection in burned mice

Whole T cells, CD4⁺ T cells and CD8⁺ T cells were prepared from spleens of normal mice treated with GR. These cells were transferred to burned mice infected with 5 LD₅₀ of C. albicans (recipients). As shown in Table 1, when mice were inoculated with T and CD4⁺ T cells from GR-treated normal mice, 90–100% of them survived (P < 0.001). This is in comparison with no survival in mice inoculated with naive T cells, CD4⁺ T cells and CD8⁺ T cells from normal mice or CD8⁺ T cells from GR-treated mice. These results suggest that CD4⁺ T cells have a role in the protection of burned mice infected with C. albicans by GR.

Table 1.

Effect of various splenic T cells on C. albicans infection in burned mice

The role of type 2 T cells in burned mice on C. albicans

T6S cells are a clone of cells that are burn-associated CD8⁺CD11b⁺ TCR γ/δ⁺ type 2 T cells [23]. Both T6S cells and burn-associated type 2 T cells produce type 2 cytokines after stimulation with anti-CD3 MoAb [11,23]. The susceptibility of T6S mice, mice inoculated with T6S cells, to HSV-1 infection has been shown to be similar to that of burned mice [23]. Therefore, the susceptibility of T6S mice to C. albicans infection was compared with that of thermally injured mice. It was hypothesized that if both groups of mice had the same susceptibility to C. albicans infection, the burn-associated type 2 T cells may play a role in the increased susceptibility. Therefore, T6S mice, burned mice, mice inoculated with naive T cells and normal mice treated with saline were studied. Each group of mice (n = 40) were divided into four subgroups (n = 10 each) and infected with various doses of C. albicans. As shown in Fig. 2, 100% of T6S mice died when infected with 1 × 10⁶ cells/mouse, while 5 × 10⁷ cells/mouse were required to kill 100% of the normal mice or the mice inoculated with naive splenic T lymphocytes. The dose required to kill 50% of T6S mice was 3 × 10⁵ cells/mouse and the concentration in thermally injured mice was 2 × 10⁵ cells/mouse. These results suggest that the increased susceptibility of burned mice (or T6S mice) to C. albicans infection was influenced by burn-associated type 2 T cells (or T6S cells).

Fig. 2.

Increased susceptibility of normal mice inoculated with T6S cells (T6S mice). Four groups of normal mice (n = 10 each) were inoculated intravenously with 1 × 10⁷ cells/mouse of splenic T cells from normal mice (Δ) or 2 × 10⁶ cells/mouse of T6S cells (○). Twenty-four hours later, these mice were infected with appropriate amounts of C. albicans. As controls, four groups of normal mice (n = 10 each) treated with saline (▴) or mice 1 day after thermal injury (•) were infected with appropriate amounts of C. albicans. The survival rates of these mice were calculated 4 weeks after the infection.

Effect of GR, CD4⁺ T cells from GR-treated mice and a mixture of MoAbs targeted to type 2 cytokines on the susceptibility of T6S mice to C. albicans

To examine the effect of GR on C. albicans infection in burned mice, T6S mice were studied instead of thermally injured mice. As shown in Fig. 3, 80–90% of T6S mice, infected with 1 × 10⁶ cells/mouse of C. albicans (0.1 LD₅₀ in normal mice and 5 LD₅₀ in burned mice), died when they were treated with saline or inoculated with naive T cells, while 100% of T6S mice treated with GR survived (P < 0.001). In addition, 85–95% of T6S mice infected with the same dose survived when they were inoculated with CD4⁺ T cells from GR-treated normal mice or treated with a mixture of MoAbs against IL-4 and IL-10 (Fig. 3) (P < 0.001). Therefore, it appeared that the resistance of burned mice to C. albicans infection was impaired by the type 2 cytokines from burn-associated type 2 T cells. GR or CD4⁺ T cells induced by GR protect mice against C. albicans infection by inhibiting of type 2 cytokine production by T6S cells.

Fig. 3.

Effect of glycyrrhizin (GR), CD4⁺ T cells from spleens of GR-treated mice and a mixture of MoAbs against type 2 cytokines on the susceptibility of T6S mice exposed to C. albicans. T6S mice were normal mice inoculated with 2 × 10⁶ cells/mouse of a clone of burn-associated type 2 T cells. T6S mice were infected with 1 × 10⁶ cells/mouse of C. albicans (corresponded to 0.1 LD₅₀ in normal mice and 5 LD₅₀ in burned mice) and inoculated with naive T cells (▪; 1 × 10⁷ cells/mouse), CD4⁺ T cells from GR-treated mice (○; 5 × 10⁶ cells/mouse). T6S mice infected with the pathogen were also treated with GR (□; 10 mg/kg) or a mixture of MoAbs against IL-4 and IL-10 (▴; 200 μg/mouse each) or saline (•). Survival of these mice was observed daily for 4 weeks after the infection.

Effect of GR or CD4⁺ T cells from GR-treated mice on the production of cytokines by splenic T cells from burned mice or T6S mice

The production of cytokines by splenic T cells from burned and T6S mice, previously treated with GR or inoculated with CD4⁺ T cells from GR-treated mice, was examined. Forty-eight hours after stimulation with anti-CD3 MoAb, splenic T cells from burned mice and T6S mice, treated with GR or inoculated with CD4⁺ T cells from GR-treated mice, failed to have type 2 cytokines in their culture fluids. However, cytokines were produced by splenic T cells from burned mice and T6S mice treated with saline or inoculated with naive T cells (Table 2). These results suggest that the production of type 2 cytokines from burned mice or T6S mice was regulated by GR through induction of CD4⁺ T cells inhibiting the production of type 2 cytokines by burn-associated type 2 T cells.

Table 2.

Effect of glycyrrhizin (GR) or CD4⁺ T cells from GR-treated mice on the production of cytokines by splenic T cells from burned mice or T6S mice

DISCUSSION

Non-specific immune responses, expressed by activation of polymorphonuclear neutrophils [28–30] and macrophages [31,32], have been shown to be involved in the resistance to the early phase infection with C. albicans. A major influence in host resistance against systemic C. albicans infection is a type 1 T cell-associated cellular response (type 1 T cell response) [33–38]. Type 1 T lymphocytes produce type 1 cytokines (IL-2 and IFN-γ) following appropriate stimulation [34]. These type 1 cytokines are able to activate and enhance killing activities of effector cells (cytotoxic T lymphocytes, natural killer cells, macrophages and neutrophils) targeted to cells infected with C. albicans [34–39]. However, type 1 T cell responses are generally suppressed by type 2 cytokines (IL-4 and IL-10) [40–42] released from T helper type 2 cells (Th2 cells) or CD8⁺ type 2 T cells [43–45]. Type 1 T cell responses are diminished in individuals dominated by type 2 T cell-associated cellular responses (type 2 T cell responses) [44,45]. Therefore, antagonists for type 2 T cell responses (anti-IL-4 MoAb and soluble IL-4 receptor) have been shown to have a protective effect in mice infected with C. albicans [46,47].

In previous studies, patients with severe thermal injuries and burned mice were noted to have a predominance of type 2 T cell responses [11,48]. CD8⁺CD30⁺ T cells were characterized as human type 2 T cells [48]. CD8⁺CD11b⁺ TCR γ/δ⁺ IL-4- and IL-10-producing T cells were identified as the type 2 T cells generated in burned mice [11]. The impaired resistance of thermally injured mice to HSV-1 could be transferred to normal unburned mice by the adoptive transfer of burn-associated type 2 T cells [11]. In addition, the resistance of thermally injured mice to HSV-1 infection was enhanced to levels observed in normal mice, following treatment with a mixture of MoAbs directed against type 2 cytokines [11]. In addition, severe combined immunodeficient (SCID) mice inoculated with peripheral blood lymphocytes (PBL) from burned patients (patient PBL–SCID chimeras) were susceptible to C. albicans infection, while SCID mice inoculated with PBL from healthy donors resisted the same infection [48]. When SCID chimeras were reconstituted with patient PBL depleted of burn-associated type 2 T cells, the resistance of these chimeras to infection improved [48]. This indicates that burn-associated type 2 T cells and/or the type 2 cytokines produced increased the susceptibility of thermally injured individuals.

Previously, the generation of anti-type 2 T cells was observed when normal mice [12] or spleen cells [13] were stimulated with GR. These anti-type 2 T cells inhibited the production of type 2 cytokines by burn-associated type 2 T cells [13]. They were characterized as CD4⁺CD28⁺ TCR α/β⁺V. villosa lectin-adherent T cells [12–14]. Anti-type 2 T cells induced by GR had similar properties, phenotypically, to contrasuppressor cells reported by Gershon et al. in 1981 [49]. Anti-type 2 T cells induced by GR did not belong in other CD4⁺ T cell subsets (e.g. Th1 cells or Th2 cells), based upon their abilities to produce cytokines [12–14]. Following stimulation with anti-CD3 MoAb, anti-type 2 T cells induced by GR secreted only IFN-γ into their culture fluids. No IL-2, IL-4 and IL-10 were present in the culture fluids. Therefore, the anti-type 2 T cells induced by GR differed from other CD4⁺ T cell populations by their cytokine-producing profile.

In the present study, the activity of GR in thermally injured mice infected with lethal doses of C. albicans was detected. The protective effect of GR against C. albicans infection resulted in (i) increased survival rates, (ii) prolongation of MSD, and (iii) decreased growth of C. albicans in kidneys of burned mice compared with that of appropriate controls. The effect of GR was dose-dependent, ranging from 0.1 to 50 mg/kg, with the maximum observed when burned mice were treated intraperitoneally with a 10-mg/kg dose of the compound. The anti-fungal effect of GR was not detected in normal mice infected with 5 LD₅₀ of C. albicans. Also, GR did not exhibit any direct inhibition on the viability and growth of C. albicans. These results suggest that GR modulated host defences influenced by burn injury, and protected burned mice against C. albicans. In addition, the susceptibility of normal mice to C. albicans infection was shown to be increased, to that observed in burned mice, when they were inoculated with T6S cells. This increased susceptibility of T6S mice to C. albicans infection was reversed when they were treated with the mixture of MoAbs directed against type 2 cytokines. These results indicate that resistance of burned mice was impaired by type 2 cytokines released from burn-associated type 2 T cells. After stimulation with anti-CD3 MoAb, splenic T cells from burned mice had type 2 cytokines in their culture fluids. However, IL-4 and IL-10 were not present in supernatants of splenic T cells from burned mice treated with GR. Also, when burned mice were inoculated with CD4⁺ T cells from GR-treated mice, the production of type 2 cytokines from splenic T cells of these mice was abrogated. Further, the resistance of burned mice to C. albicans infection reversed when they were inoculated with CD4⁺ T cells from normal mice treated with GR. However, the inoculation of naive T cells and CD8⁺ T cells from GR-treated mice did not improve the resistance of burned mice. Similar results were obtained in T6S mice treated with GR or inoculated with CD4⁺ T cells from GR-treated mice.

These results indicate that the activity of GR in burned mice exposed to C. albicans was due to the following: (i) the resistance of burned mice to infection was impaired by production of type 2 cytokines from burn-associated type 2 T cells; (ii) burned mice treated with GR resisted C. albicans infection; and (iii) CD4⁺ T cells from GR-treated mice protected T6S mice infected with C. albicans. In addition, it was previously demonstrated that GR induced a CD4⁺ T cell subset that suppressed the production of type 2 cytokines by burn-associated type 2 T cells [12,13]. Therefore, GR, as a regulator of type 2 T cell responses (or an inducer of CD4⁺ T cells antagonistic to the production of type 2 cytokines by burn-associated type 2 T cells), could possibly be active in thermally injured patients, who have a predominant type 2 T cell response against C. albicans infections.