Healthy, female C57BL/6 mice (6–8 weeks old) were purchased from the SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All of the animals were housed in a specific pathogen-free environment and maintained on standard mouse chow with free access to food and water. The study was approved by the Animal Care and Use Committee of the China Medical University (Shenyang, Liaoning, China; permit no. CMU62043010), and complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Zymosan A from Saccharomyces cerevisiae (Z4250), purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA), was dissolved in phosphate-buffered saline (PBS) to a final concentration of 3 mg/ml. Twenty mice were randomly allocated into two groups: the PBS and the 1,3-β-glucan group. All of the animals were anesthetized by an intraperitoneal injection of 2% pentobarbital sodium (45 mg/kg body weight). The trachea was exposed by opening the neck skin and blunt dissection. A 7-gauge needle was inserted transorally into the trachea. The mice in the 1,3-β-glucan or the PBS group were administered 0.1 ml zymosan solution (3 mg/ml) or 0.1 ml sterile PBS, respectively. The site of surgery was sutured and cleaned with penicillin. The mice were allowed to recover until they were sacrificed.

All of the mice were sacrificed 7 days following exposure to 1,3-β-glucan (Zymosan A) or PBS. The lungs were removed and washed twice in cold PBS. Bronchoalveolar lavage fluid (BALF) was obtained by cannulating the trachea, injecting and retrieving 1-ml aliquots of sterile physiological saline several times to obtain 6 ml liquid. The BALF was centrifuged at 200 × g for 8 min at 4°C; the pellet was washed and re-suspended with 0.5 ml RPMI-1640. Cell suspension (10 μl) was used to count the cells under a microscope. RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) was then added to a final concentration of 1×10⁵ cells/ml. BALF cells from mice in the PBS group were placed in 24-well tissue culture plates (Costar) in four wells. The plates were then placed in an incubator supplemented with 5% CO₂ at 37°C. Macrophages were allowed to adhere for 2 h, and the wells were then washed with RPMI-1640 thrice to rinse away most non-adherent cells.

The spleens were removed from the mice and placed on 35-mm plates with 4–5 ml mouse lymphocyte separation medium (EZ-Sep™ Mouse 1X). After being grinded and mechanically disrupted, the splenocytes were isolated and carefully transferred to a 15-ml centrifuge tube and covered with 200–500 μl RPMI-1640. Following centrifugation for 30 min at 360 × g, lymphocytes were obtained and washed once with RPMI-1640 supplemented with 10% FBS by centrifugation, followed by re-suspension in RPMI-1640. The cells were counted using a hemocytometer. Subsequently, cell concentration was adjusted to 5×10⁶. Lymphocytes (1 ml) from mice treated with PBS were transferred to the four wells containing macrophages.

Lymphocytes were cultured in 24-well flat-bottom plates pre-coated with macrophages. Macrophages from mice treated with PBS were the same in the four experimental groups. The four experimental groups were the following: the LG⁻G⁻, LG⁻G⁺, LG⁺G⁻ and LG⁺G⁺ group. LG⁻, lymphocytes isolated from PBS-treated mice; LG⁺, lymphocytes isolated from 1,3-β-glucan-treated mice; G⁻, lymphocytes co-cultured in vitro and treated with PBS; and G⁺, lymphocytes co-cultured in vitro treated with 1,3-β-glucan (100 μg/ml). Each group contained ConA to stimulate survival. The cells were cultured for 24 or 48 h at 37°C under a 5% CO₂ atmosphere.

Following 24 or 48 h of culture, mixtures of cells and culture media were transferred to 1.5-ml Eppendorf tubes, followed by centrifugation for 8 min at 4°C. Subsequently, culture media were stored for the investigation of cytokine protein levels. TRIzol reagent was added to the lymphocytes to avoid RNA degradation. The cells and culture media were maintained at −70°C.

The ELISA plate was coated with 100 μl capture antibody in coating buffer/well of the ELISA kit (eBioscience, San Diego, CA, USA) and incubated at 4°C overnight. The plate was washed with 250 μl washing buffer. The cells in each well were then blocked with 200 μl assay diluent and incubated for 1 h at room temperature. A volume of 100 μl culture supernatants or the different dilutions of standard (for standard curve) were added to each well and incubated for 2 h at room temperature. The cells in each well were incubated with 100 μl detection antibody for 1 h at room temperature, followed by incubation with 100 μl avidin-HRP for 30 min at room temperature. Substrate solution (100 μl)was added to each well for a 15-min incubation at room temperature. Stop solution (50 μl) was added to stop the reaction. The absorbance was read at 450 nm. ELISA was performed in triplicate.

Total RNA was extracted from lymphocytes using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNA concentration and the ratio of A260/280 were determined using an ultraviolet (UV) spectrophotometer. The primers were designed using Primer3 (http://frodo.wi.mit.edu/primer3) and the sequences were blasted (http://blast.ncbi.nlm.nih.gov/Blast.cgi). PrimeScript RT reagent kit (DRR037A; Takara, Shiga, Japan) and SYBR Premix Ex Taq II (DRR081A; Takara) were used for real-time RT-PCR. The primer sequences used were the following: Foxp3, sense 5′-CAGCTCTGCTGGCGAAAGTG-3′ and antisense, 5′-TCGTCTGAAGGCAGAGTCAGGA-3′; GAPDH, sense, 5′-CAATGTGTCCGTCGTGGATCT-3′ and antisense, 5′-GTCCTCAGTGTAGCCCAAGATG-3′. Total RNA (0.4 μg) of each group at 48 h was reverse transcribed in a volume of 20 μl and the following PCR conditions were used: 37°C for 15 min and 85°C for 5 sec. cDNA (2 μl) was used in a 25-μl PCR reaction volume. Each sample was assayed in triplicate. The difference of the amplification efficiency between the target gene and the housekeeping gene were identified by comparing the slopes of the standard curves. The PCR reactions were run on ABI 7500 (Applied Biosystems) using the following conditions: 95°C for 30 sec, and 40 cycles of 95°C for 5 sec and 60°C for 34 sec. Analysis was performed using the ABI 7500 system software.

The SPSS 16.0 software was used to perform statistical analyses. The differences between values were evaluated using one-way analysis of variance (ANOVA) followed by pair-wise comparison with the LSD and Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference. All of the experiments were repeated five times. The results were expressed as the means ± SEM.

To investigate the role of 1,3-β-glucan in the secretion of Th1 cytokines, macrophages isolated from mice treated with PBS were allocated into 4 groups. The levels of Th1 cytokines (IL-2 and IFN-γ) in the culture media of cells treated with 1,3-β-glucan in vitro (LG⁻G⁺ group) significantly decreased compared with the LG⁻G⁻ group 24 and 48 h following in vitro treatment (Figs. 1 and 2). The levels of IL-2 in the culture media of cells in the LG⁻G⁺ and LG⁺G⁺ groups significantly decreased compared with the LG⁻G⁻ and LG⁺G⁻ groups 24 and 48 h following treatment with 1,3-β-glucan or PBS in vitro (Fig. 1). The levels of IFN-γ in the LG⁺G⁺ group significantly decreased compared with the LG⁺G⁻ group at both time points (Fig. 2).

The levels of IFN-γ in the culture media of the cells treated with 1, 3-β-glucan in vivo (LG⁺G⁻ group) significantly increased compared with LG⁻G⁻ and LG⁻G⁺ groups 48 h following in vitro treatment (Fig. 2). The levels of IL-2 in the culture media of the cells treated with 1,3-β-glucan in vivo (LG⁺G⁻ group) significantly increased compared with LG⁻G⁻ and LG⁻G⁺ group 24 h following in vitro treatment (Fig. 1). This suggests that 1,3-β-glucan inhibits the secretion of Th1 cytokines in vitro.

The secretion of the Th2 cytokine IL-4 in response to 1,3-β-glucan treatment in vitro was examined. The levels of IL-4 increased in the culture media of the cells in the LG⁺G⁺ group compared with the LG⁺G⁻ group 24 h following in vitro treatment (Fig. 3). The levels of IL-4 in the culture media of the LG⁻G⁺ and LG⁺G⁺ groups increased compared with the LG⁻G⁻ and LG⁺G⁻ groups 48 h after treatment with 1,3-β-glucan or PBS in vitro (Fig. 3). This result suggests that 1,3-β-glucan increases the expression levels of IL-4 in vitro.

The expression levels of anti-inflammatory cytokines were then assessed. The expression levels of IL-10 in the culture media of the LG⁻G⁺ and LG⁺G⁺ groups significantly increased compared with the LG⁻G⁻ and LG⁺G⁻ groups 24 and 48 h following in vitro treatment (Fig. 4). Thus, 1,3-β-glucan increases the secretion of IL-10 in vitro.

The expression levels of TGF-β in the culture media of the LG⁺G⁺ group increased compared with the LG⁺G⁻ group at 24 h. At 48 h, the levels of TGF-β in the LG⁻G⁺ and LG⁺G⁺ groups increased compared with the LG⁻G⁻ and LG⁺G⁻ groups (Fig. 5). These results suggest that 1,3-β-glucan alters the Th1/Th2 balance through an IL-10 dependent rather than an TGF-β-dependent pathway.

As shown in Fig. 6, although the mRNA expression of Foxp3 increased in response to 1,3-β-glucan treatment in vitro, no statistically significant difference among the 4 cell groups was observed. This result suggests that 1,3-β-glucan exposure may increase the expression of Foxp3 in lymphocytes.