Interleukin-4 receptor alpha-deficient BALB/c mice show an unimpaired T helper 2 polarization in response to Leishmania major infection

Abstract

We recently generated interleukin-4 (IL-4) receptor alpha-deficient (IL-4Ralpha(-/-)) BALB/c mice and showed evidence for a protective role of IL-13-mediated functions in leishmaniasis. In this study, we investigated the IL-4 expression and T helper 2 (Th2) development in Leishmania major-infected IL-4Ralpha(-/-) mice. Here we show that the early burst of IL-4 expression observed in L. major-infected BALB/c mice is independent of IL-4Ralpha-mediated functions. Subsequently, we confirmed an impaired Th2 development in vitro. Unexpectedly, during L. major infection, isolated CD4(+) IL-4Ralpha(-/-) T cells expressed high IL-4- but low gamma interferon (IFN-gamma)-specific mRNA, comparable to Th2-polarized BALB/c CD4(+) cells and in contrast to Th1-polarized C57BL/6 CD4(+) cells. Since antigen-specific restimulated popliteal lymph node cells (PLN) of IL-4Ralpha(-/-) mice also responded with high IL-4 but low IFN-gamma production, comparable to Th2-polarized cells from wild-type BALB/c mice and in contrast to Th1-polarized C57BL/6 cells, these results suggested an unimpaired Th2 polarization during an established infection with L. major. To further define the observed IL-4 receptor-independent Th2 cell phenotype, we determined an independent Th2 marker, the IL-12 receptor beta-2 (IL-12Rbeta2)-specific transcript levels of CD4(+) T cells. Confirming Th2 polarization in L. major-infected IL-4Ralpha(-/-) mice, comparable IL-12Rbeta2 message levels between CD4(+) T cells from infected IL-4Ralpha(-/-) mice and Th2 cells from BALB/c mice were found, whereas Th1-polarized C57BL/6 cells showed strikingly increased IL-12Rbeta2 expression levels. These results indicate that signals mediated by the IL-4Ralpha are not necessary to induce and sustain an efficient IL-4 expression and Th2 polarization in L. major-infected BALB/c mice and suggest that IL-4Ralpha-independent mechanisms underlie the default Th2 development in L. major-infected BALB/c mice.

FIG. 1

L. major-induced early IL-4 expression. BALB/c IL-4Rα^−/−, BALB/c, and C57BL/6 mice (two per group) were intravenously injected with 200 μl of Hanks' balanced salt solution in the absence (−) or presence (+) of 2 × 10⁷ L. major promastigotes. Mice were sacrificed after 90 min, and total RNA was isolated from spleens. (a) Levels of IL-4-specific mRNAs were determined by RT-PCR after standardization of cDNAs for the constitutive housekeeping β2-microglobulin gene (β2mg) in a competitive RT-PCR. (b) Densitometric analysis of IL-4 PCR after normalization with corresponding β2-microglobulin cDNA PCR signal strength (pixels per square millimeter). The IL-4 densitometric values refer to PCR signal strength differences between uninfected (arbitrarily set as 1) and infected spleen cells of indicated mouse strains. Increased IL-4 expression levels (densitometric values, 1.4 and 1.8) were found in IL-4Rα^−/− and BALB/c mice, whereas a striking reduction (0.2) was found in spleen cells of infected C57BL/6 mice.

FIG. 2

Restimulation of PLN with anti-CD3 or L. major antigen. IL-4Rα^−/− (open bars), BALB/c (hatched bars), and C57BL/6 (filled bars) mice (four per group) were injected with 2 × 10⁷ L. major promastigotes into one hind footpad. At day 56 after infection, cells from the draining lymph nodes from each group were pooled and restimulated in 48-well plates with IL-2 (250 U/ml) in the presence of anti-CD3 (10 μg/ml) or L. major antigen (10⁶/ml) for 48 or 72 h, respectively. IFN-γ and IL-4 concentrations in the supernatants were determined by ELISA. Values are means and positive standard deviations from triplicate cultures. nd, not detected. Representative results from two independent experiments are shown.

FIG. 3

Competitive RT-PCR from CD4⁺ cells at day 49 after infection with L. major. IL-4Rα^−/− BALB/c, BALB/c, and C57BL/6 (four per group) were injected with 2 × 10⁶ L. major promastigotes into one hind footpad. At day 49 after infection, draining lymph nodes from groups were pooled, CD4⁺ T lymphocytes were purified by magnetic beads, and the isolated RNA was reverse transcribed. Levels of specific mRNAs were determined by competitive RT-PCR after standardization of cDNAs for the constitutive housekeeping β2-microglobulin gene (β2mg) at 3 × 10⁵ molecules. Arrows indicate equimolar amount of cDNA and competitor (comp.) PCR product, the latter used in fourfold dilutions with absolute numbers indicated. IL-12Rβ2-specific transcripts were measured semiquantitatively. (b) Transcript numbers of IFN-γ and IL-4 per 3 × 10⁵ molecules of β2-microglobulin of CD4⁺ T cells from indicated mouse strains. IL-4Rα and BALB/c CD4⁺ T cells had equal transcript numbers of IFN-γ (1.2 × 10³) and IL-4 (6.1 × 10²) but eightfold-reduced IFN-γ and at least eightfold-increased IL-4 transcripts in comparison to C57BL/6 cells (IFN-γ, 9.8 × 10⁴; IL-4, <75 molecules). (c) Densitometric analysis of IL-4Rα and IL-12Rβ2 PCR after normalization with corresponding β2-microglobulin cDNA PCR signal strength (pixels per square millimeter). The densitometric values refer to PCR signal strength differences (BALB/c values arbitrary set as 1) between indicated mouse strains. IL-4Rα expression levels between BALB/c and C57BL/6 were comparable. No significant difference was observed for IL-12Rβ2 expression levels between IL-4Rα^−/− and BALB/c mice, whereas C57BL/6 PLN showed a threefold increase after infection.

FIG. 4

Impaired in vitro Th2 differentiation. FACS-isolated CD4⁺ cells (>99% purity) of IL-4Rα^−/− (white bars) or wild-type (hatched bars) PLN (10⁶ cells/ml) were cultured with anti-CD3/CD28 in the presence of IL-2 (50 U/ml). T helper development was promoted by a combination of IL-12 (1 ng/ml) and anti-IL-4 (11B11; 10 mg/ml), to generate Th1 cells, or IL-4 (500 U/ml) or IL-13 (25 pg/ml) in the presence of anti-IFN-γ (R4-6A2; 10 mg/ml), to generate Th2 cells. Three days later, cells were washed and further incubated with IL-2 for 24 h. Finally, cells were transferred onto anti-CD3-coated plates without additional stimuli, and IFN-γ and IL-4 levels in the supernatants were determined 2 days later by ELISA. Values are means and standard deviations from triplicate cultures. PLN from IL-4Rα-derived cells showed no significant differences in the production of IFN-γ and IL-4 compared to corresponding BALB/c cells (P > 0.12 by Student's t test). The data are representative of two experiments.