IL-4 and IL-13 regulate the induction of indoleamine 2,3-dioxygenase activity and the control of Toxoplasma gondii replication in human fibroblasts activated with IFN-γ

2.1 Cytokine effect on intracellular replication of T. gondii

The levels of T. gondii replication in human 2C4 fibroblasts activated with rIFN-γ, rIFN-β and / or rTNF-α are presented in Table 1. The stimulation of 2C4 cells with 100 and 500 IU / ml rIFN-γ resulted in significant inhibition of intracellular tachyzoite replication of 36.4 and 43.8 %, respectively. A smaller but statistically significant inhibitory effect of rTNF-α (60 IU / ml) and rIFN-β (1000 IU / ml) on T. gondii growth was observed in different experiments. The combination of 100 IU / ml rIFN-γ plus 60 IU /  ml TNF-α resulted in an additive effect showing a total of 48.7 % inhibition on tachyzoite replication. In contrast, no additive effect was observed when 500 IU / ml rIFN-γ was combined with rTNF-α. No augmentation of rIFN-γ activity was induced by association with rIFN-β. As previously reported 4, 11, in the presence of excess tryptophan (1 mM) the anti-Toxoplasma activity induced by IFN-γ was mostly reversed. In contrast, the inhibitory effect induced by rTNF-α and rIFN-β was only marginally affected by the addition of tryptophan (data not shown).

rIL-4 antagonized the rIFN-γ activity in 2C4 cells, reducing by 67.0 % the inhibitory effect of rIFN-γ on parasite replication. In contrast, pre-incubation with rIL-10 did not interfere with the control of T. gondii replication in human fibroblasts activated with rIFN-γ. No antagonistic effect of rIL-4 or rIL-10 was observed in the rIFN-β, rTNF-α- or rIFN-γ plus rTNF-α-induced inhibition of tachyzoite growth (Table 2).

2.2 Cytokine control of INDO activity

IFN-γ-induced tryptophan degradation by INDO has been shown to be a major mechanism involved in control of T. gondii replication in human fibroblasts 4, 11, 13. Therefore, we decided to evaluate the effect of different cytokines on tryptophan degradation by INDO. The INDO activity was estimated as the level of tryptophan consumed and kynurenine formed in the culture supernatants of fibroblasts activated with different cytokines. Our results show that the increase of INDO activity induced by different cytokines was inversely correlated with the parasite replication in 2C4 cells. rIFN-γ strongly stimulated INDO activity, with a corresponding fall in tryptophan and elevation of kynurenine levels in the fibroblast supernatants. In contrast to rIFN-γ, rIFN-β and rTNF-α had a weak stimulatory effect on INDO activity. Incubation with rIFN-β (1,000 IU / ml), rTNF-α (60 IU / ml) and rIFN-γ (100 and 500 IU / ml) resulted in 12.8, 29.6, 59.1 and 85.7 % of tryptophan reduction, respectively. Activation of the cells with rIFN-γ (100 IU / ml) plus rTNF-α (but not with rIFN-β) resulted in a statistically significant augmentation of tryptophan degradation (Table 3).

Pre-incubation with rIL-4 resulted in 37.6 % inhibition of kynurenine formation and 34.5 % of tryptophan degradation in 2C4 cells activated with rIFN-γ. Similar results were obtained with human lung fibroblasts (data not shown). No reduction of INDO activity by rIL-4 was observed when 2C4 cells were stimulated with either rIFN-β, rTNF-α or with the combination of rIFN-γ plus rTNF-α. Similarly, at higher rIFN-γ concentration (500 IU / ml) the INDO activity was not inhibited by rIL-4 (data not show). In contrast to rIL-4, pre-incubation with rIL-10 did not interfere with the rIFN-β, rTNF-α or rIFN-γ-induced INDO activity in the 2C4 cells (Table 4).

Further, radioactive tryptophan was used to confirm the HPLC results showing the rIL-4 antagonistic effect on INDO activity (Table 4). The activation of 2C4 cells with rIFN-γ resulted in formation of radioactive kynurenine. Thus, the peak of kynurenine, obtained in the HPLC analysis, was indeed generated from tryptophan degradation. In agreement with results obtained with non-radioactive tryptophan, pre-incubation with rIL-4 resulted in 35.0 % inhibition of radioactive formation induced by rIFN-γ (data not shown).

2.3 Cytokine control of INDO mRNA expression

As shown in Fig. 1, INDO mRNA transcripts were not detectable in untreated fibroblasts. The activation of 2C4 cells with rIFN-β, rTNF-α and rIFN-γ resulted in induction of INDO mRNA. A more intense expression of INDO mRNA was observed in 2C4 fibroblasts activated with rIFN-γ. Incubation of cells with 100 IU / ml rIFN-γ plus 60 IU / ml rTNF-α resulted in an additive effect on INDO mRNA expression (Fig. 1 A and C). The cytokine-induced INDO mRNA expression was measured by densitometric analysis and is shown in Table 1 B and D. As noticed in the INDO activity and control of intracellular tachyzoite replication, rIL-4 decreased the rIFN-γ-induced INDO expression by 53.3 %. A similar inhibitory effect of rIL-4 on INDO mRNA expression was observed in human lung fibroblasts exposed to 100 IU / ml IFN-γ (data not shown). Together, these results suggest that rIL-4 inhibits INDO activity at the transcriptional level. In contrast to rIL-4, rIL-10 was unable to alter the rIFN-γ-induced INDO expression (Fig. 1 C and D).

2.4 Inhibition of rIFN-γ-induced CD2 and HLA-DR expression by rIL-4

To evaluate the ability of rIL-4 to regulate the expression of other genes induced by IFN-γ, we measured the expression of stably transfected CD2 and endogenous HLA-DR 26, 27 in the 2C4 cell line. The fibroblasts were cultured in the presence or absence of rIFN-γ with or without rIL-4, and the surface expression of such molecules was analyzed be immunostaining with PE-conjugated anti-CD2 and FITC-conjugated anti-HLA-DR mAb. Fig. 2 shows the histograms of a FACS analysis and the percentage of stained 2C4 cells. After treatment with rIFN-γ 59.0 and 16.0 % of the cells became positive for CD2 and HLA-DR antigens, respectively. rIL-4 treatment did not change considerably the percentage of cells expressing CD2 and HLA-DR. In the presence of rIL-4, the rIFN-γ-mediated increase of CD2 and HLA-DR expression was blocked by 17.7 % and 43.8 % (p < 0.05), respectively. In contrast to rIL-4, the addition of rIL-10 showed no effect either on CD2 or HLA-DR expression in the human fibroblast cell line activated with rIFN-γ (data not show).

2.5 Detection of IL-4, IL-10 and IL-13 receptor mRNA by RT-PCR

IL-4R and IL-13R are multimeric and share at least one common chain called IL-4Rα. Specific IL-13R are formed by the association between either IL-4-Rα and IL-13Rα2 or between IL-13Rα1 and IL-13Rα2. IL-4R, in non-hematopoietic cells, is a heterodimer composed of IL-4Rα and IL-13Rα1 chains 28. To characterize the expression of IL-4R and IL-10R mRNA in human fibroblasts, reverse transcription (RT)-PCR analysis was conducted using sets of primers described in Sect. 4.4. Specific transcripts for IL-4Rα, IL-13Rα1 and IL-13Rα2 chains were detected in 2C4 fibrosarcoma, as well in the cell lines LL-24 and MRC-5 of human lung fibroblasts stimulated or not with different cytokines (Fig. 3 A). Our data show that human fibroblast cells constitutively express the mRNA for different chains of IL-4R and IL-13R. As can be seen in Fig. 3 B, the specific transcript for IL-10R was detected in human monocytic THP-1 cells, but not in the human fibroblast cell lines. The transcript for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH); used as internal control) was equally expressed in all samples. Together with the functional data, the RT-PCR results suggest that human fibroblasts express specific receptors for IL-4 and IL-13, but not for IL-10.

2.6 Inhibition of rIFN-γ-induced INDO activity and microbiostasis by IL-13

Since all the fibroblast cell lines were shown to express mRNA for the different chains that compose the IL-13R (Fig. 3 A), we tested the activity of rIL-13 in human fibroblasts. The results shown in Table 5 show that on its own rIL-13 had no effect on INDO activity or parasite replication in the 2C4 cell line. In contrast, rIL-13 inhibited the trytophan degradation (33.0 %), kynurenine formation (41.5 %) and anti-Toxoplasma activity (75.7 %) in IFN-γ-activated fibroblasts. Pre-incubation of IFN-γ-activated fibroblasts with rIL-4, rIL-13 or rIL-4 plus rIL-13 resulted in similar levels of inhibition of INDO activity and microbiostasis.

4.1 Cell culture

The human fibrosarcoma 2C4 26, the human lung fibroblast LL-24 (ATCC, CCL-171) and MRC-5 (ATCC, CCL-151) and the human monocytic THP-1 (ATCC, 45502) cell lines were used. The cells were grown in DMEM (Gibco, Grand Island, NY) or RPMI 1640 medium (Gibco) supplemented with 10 % heat-inactivated FBS (Gibco), 5 μM L-glutamine (Sigma, St. Louis, MO), 40 μg / ml gentamicin and 25 mM Hepes buffer, pH 7.3 (Sigma). The tryptophan concentration in the RPMI and the DMEM media was 5 mg / l (24.5 μM) and 16 mg / l (78.4 μM), respectively. The cells were cultured at 37 °C in humidified air containing 5 % CO₂. Their viability after trypsin treatment was assessed by trypan blue exclusion.

4.2 Cytokines

Human rIFN-γ, rIFN-β and rTNF-α were provided by Genentech, Inc. (San Francisco, CA) and human rIL-4 and rIL-13 was purchased from R&D Systems, Inc. (Minneapolis, MN). The concentrations used in the assays were 100 and 500 IU / ml for rIFN-γ, 1,000 IU / ml for rIFN-β, 60 IU / ml for rTNF-α and 10 ng / ml for rIL-4, rIL-10 and rIL-13. The appropriate concentrations of each cytokine employed in our study were determined after tritration experiments.

4.3 T. gondii and growth assay

The RH strain of T. gondii was used in the parasite growth assays 4, 11. The parasites were maintained by serial passages in 2C4 human fibroblast monolayer cultures. T. gondii tachyzoite forms were obtained on the second day of the cell culture. Confluent cells were trypsinized (0.034 % trypsin in 0.1 % EDTA, both from Sigma), added to eight-well tissue culture chamber slides (Nunc, Inc.) in duplicates at a final concentration of 3 × 10⁴ cells / well in 430 μl RPMI in the presence or absence of cytokines, and incubated at 37 °C in 5 % CO₂ at 95 % humidity for 24 h. The medium was changed after 24 h, and T. gondii was added at a ratio of three parasites per cell in a volume of 200 μl, with or without the cytokines. After 3 h of infection, the parasites that had not infected the cells were removed by washing, replaced with 430 μl fresh medium, and further incubated with or without the cytokines for 24 h. After the incubation, the chambers were washed with PBS, fixed with methanol, stained with May-Grünwald-Giemsa, and mounted on slides. The results were expressed as infection index, i. e. the average number of parasites per 100 infected cells obtained from three independent experiments performed in duplicate.

4.4 mRNA expression assay

To verify the mRNA expression, 24-well culture plates containing 3 × 10⁴ cells / well were used 11. Briefly, the cells were incubated in the presence or absence of cytokines for 14 – 16 h. The medium was discarded, and the cells were washed with PBS. Total RNA was extracted with TRIzol (Gibco) according to the manufacturer's instructions. The cDNA synthesis was obtained in a final volume of 30 μl containing 200 U Moloney murine leukemia virus reverse transcriptase (Pharmacia Biotech, Uppsala, Sweden), 200 mM of each deoxynucleoside triphosphate (Promega, Madison, WI), 2.5 μl buffer (0.1 M MgCl₂, 0.5 M Tris-HCl, 1 mM DTT, 2 mg BSA per ml; pH 7.2 at 20 °C, Boehringer Mannheim, Germany), 240 pmol / μl of oligo-dT₁₀ (Boehringer), 0.1 M DTT, 1 U / μl of the RNase inhibitor Rnasin (Promega), and 0.4 μg total RNA. The samples were incubated at 37 °C for 30 min and the cDNA were stored at − 20 °C.

The expression of mRNA was detected in the host cells RT-PCR assays. The human sets of primers were as follows: INDO: forward, 5′-AGT TGA AGT TAA ACA TGC-3prime;; reverse, 5′-TGA TCG TGG ATT TGG TGA-3prime;, amplification product, 487 bp (55 °C, 30 cycles); IL4Rα: forward, 5′-GAC CTG GAG CAA CCC GTA TC-3prime;; reverse, 5′-CAT AGC ACA ACA GGC AGA CG-3prime;, amplification product, 335 bp (60 °C, 30 cycles); IL-13Rα1: forward, 5′-AGG ATG ACA AAC TCT GGA-3prime;; reverse, 5′-CTC AAG GTC ACA GTG AAG G-3prime;, amplification product, 358 bp (55 °C, 30 cycles); IL-13 Rα2: forward 5′-TGG GAC CTA TTC CAG CAA-3prime;; reverse, 5′-TTT TTG GGT AGG TGT TTG GC-3prime;; amplification product, 332 bp (55 °C, 30 cycles); IL-10R: forward 5′-CCA TCT TGC TGA CAA CTT CC-3prime;; reverse, 5′-GTG TCT GAT ACT GTC TTG-GC-3prime;; amplification product, 460 bp (60 °C, 30 cycles); GAPDH: forward, 5′-GTG GTG AAG CAG GCG TCG-3prime;; reverse, 5′-GAC TGA GTG TGG CAG GGA-3prime; amplification product, 311 bp (55 °C, 30 cycles). The amplification was carried out in a thermocycler (PTC 100; MJ Research, Inc., Waltham, MA) in a final volume of 10 μl containing 0.5 U Taq DNA polymerase (Cenbiot, Porto Alegre, RS, Brazil), 200 μM of each deoxynucleoside triphosphate, 1.5 mM MgCl₂, 50 mM KCl, and 10 mM Tris-HCl (pH 8.5), together with 10.0 and 1.0 pmol of the others and GAPDH primers, respectively. The expression of the genes was detected by electrophoresis through 6 % nondenaturing polyacrylamide gels and silver staining as previously described 61. For semi-quantitative RT-PCR, amplified products of INDO were quantified using specific bands of the housekeeping gene GAPDH transcripts as reference on densitometric scanning.

4.5 Assay of INDO activity

INDO activity was assessed by both reduction in tryptophan and increase in kynurenine concentration in the supernatants of the culture of cells measured by HPLC 62 2C4 human fibroblasts were plated at a density of 3 × 10⁴ cells / well in 24-well culture plates in 500 μl DMEM. At 24 h, the medium was replaced with 300 μl RPMI 1640 containing 24.5 μM tryptophan, or 300 μl RPMI 1640 plus 1 μCi / ml [5-³H] tryptophan (Specific activity 24 Ci / mmol, Amersham Corp.) in presence or absence of the cytokines. The cells were pre-incubated with rIL-4 and rIL-10 for 3 h and then incubated with rIFN-γ, rIFN-β and rTNF-α. After 24 h of incubation the supernatants were removed and deproteinized with 30 % (w / v) trichloroacetic acid (TCA). The tubes were incubated at 50 °C for 30 min to hydrolyze N-formylkynurenine to form kynurenine. After removal of the protein precipitate by centrifugation (2 min, 10,000 × g), samples were stored at − 20 °C. The levels of tryptophan and kynurenine in the acid-soluble supernatants were analyzed directly by HPLC. In the experiments employing radioactive tryptophan, the radioactivity was detected by both continuous monitoring of effluent by UV spectrophotometry and by collecting fractions corresponding to tryptophan and kynurenine and determining the cpm per fraction by scintillation spectrometry (Beckman LS6000Sc).

4.6 HPLC determination of tryptophan and kynurenine

Total free tryptophan and kynurenine were quantitated by HPLC 63. The chromatography was performed in a Shimadzu LC-10A liquid chromatograph. A Nova-Pak C18 reversed-phase column (4 μm particle size, 3.9 × 150 mm I.D.) was purchased prepacked (Waters, Milford, Massachusetts); a guard-Pak Nova-pak C18 column (Waters) was attached between the injector and the analytical column to extend to life of the analytical column. The column was eluted isocratically at a flow rate of 1.0 ml / min with 1 mM potassium phosphate buffer, pH 4.0, containing 5 % (v / v) of methanol. The absorbance of the column effluent was monitored at 265 nm. For determinations, 100 μl protein-free supernatant was injected onto the column. Kynurenine and tryptophan concentrations in the supernatants were determined based on their peak heights relative to standard solutions obtained in the chromatograms. The standard solutions of kynurenine and tryptophan, purchased from Sigma, were prepared fresh each day at concentrations ranging from 5 to 25 μM of kynurenine and tryptophan in RPMI and submitted to precipitation with 30 % TCA and incubation at 50 °C for 30 min. Chromatography of the standards and the samples was performed under the same conditions.

4.7 Flow cytometric analysis

Expression of HLA-DR and CD2 was assayed by immunofluorescent flow cell cytometry as previously reported 27. Briefly, 2.5 × 10⁵ cells were seeded in a 4-cm petri dish (Nunc, Inc.) and after an overnight incubation, they were prestimulated with rIL-4 for 3 h and then treated with rIFN-γ for 48 h. After incubation the fibroblasts were harvested and stained by successive 30 min incubation at 4 °C with PE-conjugated anti-CD2 and FITC-conjugated anti-HLA-DR mAb (Becton Dickinson). Cells incubated under similar conditions either without or with an isotype-matched control antibody (PE-conjugated anti-IgG1, Diatec, Oslo, Norway) were used as controls. After washing, the cells were pelleted and fixed in 1 % paraformaldehyde. Flow cytometric measurements were performed on a Becton Dickinson FACScan (Becton Dickinson, Mountain View, CA). Data from about 1.0 × 10⁵ cells were obtained and analyzed using a Cell-Quest program (Becton Dickinson). The relative FITC or PE fluorescence intensity was analyzed using a single histogram representation.

4.8 Statistical analysis

Differences between groups were assessed with the Student's t-test. p values of < 0.05 were considered statistically significant.