Interleukin 1alpha promotes Th1 differentiation and inhibits disease progression in Leishmania major-susceptible BALB/c mice

Abstract

Protective immunity against pathogens such as Leishmania major is mediated by interleukin (IL)-12-dependent Th1-immunity. We have shown previously that skin-dendritic cells (DCs) from both resistant C57BL/6 and susceptible BALB/c mice release IL-12 when infected with L. major, and infected BALB/c DCs effectively vaccinate against leishmaniasis. To determine if cytokines other than IL-12 might influence disease outcome, we surveyed DCs from both strains for production of a variety of cytokines. Skin-DCs produced significantly less IL-1alpha in response to lipopolysaccharide/interferon gamma or L. major when expanded from BALB/c as compared with C57BL/6 mice. In addition, IL-1alpha mRNA accumulation in lymph nodes of L. major-infected BALB/c mice was approximately 3-fold lower than that in C57BL/6 mice. Local injections of IL-1alpha during the first 3 d after infection led to dramatic, persistent reductions in lesion sizes. In L. major-infected BALB/c mice, IL-1alpha administration resulted in increased Th1- and strikingly decreased Th2-cytokine production. IL-1alpha and IL-12 treatments were similarly effective, and IL-1alpha efficacy was strictly IL-12 dependent. These data indicate that transient local administration of IL-1alpha acts in conjunction with IL-12 to influence Th-development in cutaneous leishmaniasis and prevents disease progression in susceptible BALB/c mice, perhaps by enhancing DC-induced Th1-education. Differential production of IL-1 by C57BL/6 and BALB/c mice may provide a partial explanation for the disparate outcomes of infection in these mouse strains.

Figure 1.

Langerhans cell-like dendritic cells of Leishmania-susceptible BALB/c mice produce less IL-1α than dendritic cells of resistant C57BL/6 mice. (A) FSDDCs were stimulated with LPS/IFN-γ (100 ng/1,000 U/ml) or L. major amastigotes (3:1 organisms/cell) for 18 h and expression of various cytokines was determined using RNase protection assays. Relative intensities of bands corresponding to IL-1α mRNA and mean fold differences between C57BL/6 and BALB/c DC of three independent experiments are shown. (B) FSDDCs (n ≥ 5), BMDCs (n = 6), peritoneal MΦ (P-MΦ, n = 5), and skin-MΦ (n ≥ 4) were stimulated as indicated. After 18 h, protein levels in cell lysates were determined by ELISA (ng/ 10⁶ cells, mean ± SEM). *P < 0.05 compared with untreated control, differences between mouse strains are marked with bars above columns (**P < 0.05). (C) IL-1α mRNA accumulation in lymph nodes of uninfected mice or, in a separate experiment, mice infected with 2 × 10⁵ L. major was assessed by semi-quantitative RT-PCR at the time points indicated. Results shown are representative of two independent experiments.

Figure 2.

Improvement of disease outcome in Leishmania-susceptible and -resistant mice after systemic treatment with IL-1α. Groups of ≥5 BALB/c (A) or C57BL/6 (B) mice were infected with 2 × 10⁵ L. major and treated locally with 50 ng IL-1α or PBS on days 1, 2, and 3 after infection. Lesion development was assessed weekly (mean ± SEM). Results are representative of ≥3 experiments. (C and D) BALB/c mice were infected as described above and treated with different doses of IL-1α as indicated. Lesion volumes were measured after 3 wk and the number of parasites/lesion was determined in limiting dilution assays. Dots represent number of parasites in one ear, bars show the arithmetic mean of all mice/group. (E) Groups of three BALB/c mice were infected with L. major promastigotes and treated with 50 ng IL-1α or PBS on days 1–3 after infection. Treatment was delivered locally, into the contralateral ear or intradermally into the retroauricular region of the infected side (local LN). 3 wk later, lesion sizes were assessed (mean ± SEM). (F) IL-1α treatment prevents progressive disease in low dose cutaneous leishmaniasis. Groups of >5 BALB/c mice were infected with 1,000 L. major promastigotes and treated locally with 50 ng IL-1α or PBS on days 11–14 after infection. Development of lesions was expressed as mean ± SEM. (A–F) Statistical differences between treatment groups at different time points are indicated as *P < 0.05, **P < 0.005, and ***P < 0.002 compared with PBS-treated control mice.

Figure 3.

IL-1α treatment of Leishmania-susceptible BALB/c results in Th₁-predominant immune responses. BALB/c mice were treated with 50 ng IL-1α or PBS on days 1–3 after infection with 2 × 10⁵ L. major. (A) After 1 and 3 wk, LN cells of groups of three mice were harvested and plated at 10⁶ cells/200 μl. Cells were stimulated with soluble Leishmania antigen (SLA) for 48 h and antigen-specific cytokine release was determined using ELISAs specific for murine IFN-γ and IL-4 (mean ± SEM). For each mouse, the ratio between IFN-γ and IL-4 was calculated (see boxes) and statistical significance was determined (*P < 0.05 compared with PBS-treated controls). (B) After 1 wk, LN cells were isolated and T cell populations isolated using magnetic beads. Total LN cells (10⁶) or CD4⁺ and CD8⁺ T cells (5 × 10⁵) were plated into 96-well plates (200 μl) and BMDCs and SLA added. Cytokine responses were studied after 48 h as described above. One out of two experiments with similar results is shown.

Figure 4.

IL-1α is as efficient as IL-12 for treatment of cutaneous leishmaniasis in susceptible BALB/c mice. BALB/c mice (five/group) were inoculated intradermally in the ears with 2 × 10⁵ metacyclic L. major promastigotes. Fifty ng of IL-12, IL-1α, IL-1β, IL-18, or IL-6 was injected locally on days 1–3. After 3 wk, lesion volumes were determined (A) and expressed as mean ± SEM. Organisms were cultured from challenge sites and enumerated as described (B). Each data point represents the number of organisms from one ear, bars indicate arithmetic means. Cytokine profiles of LN cells were determined by in vitro restimulation with soluble Leishmania antigen (SLA). Supernatants were assayed for the presence of IFN-γ (C) and IL-4 (D) after 48 h (mean ± SEM, boxes represent IFN-γ/IL-4 ratios, *P < 0.05 compared with PBS-treated controls). Data of one experiment out of three with similar results is shown.

Figure 5.

Effective treatment of cutaneous leishmaniasis in Leishmania-susceptible BALB/c mice with IL-1α requires IL-12. BALB/c or IL-12p40 ko BALB/c mice were infected with high dose inocula into ear skin. IL-1α or PBS was applied locally on days 1–3 after infection. Three weeks later, lesions sizes (A) and parasite burdens in lesional ear skin (B) were determined. Cytokine profiles of lymph node cells restimulated with antigen were assayed as described in Materials and Methods using ELISAs specific for IFN-γ (C) and IL-4 (D). All data are expressed as mean ± SEM (*P < 0.05, ***P < 0.002) and the results shown are pooled from two experiments (six mice/group).