Figure 1.
Generation of LckcreIL-4Rα−/lox Mice
(A) Mouse breeding strategy. IL-4Rαlox/lox BALB/c mice were intercrossed with transgenic BALB/c mice expressing Cre-recombinase under control of the Lck promoter and IL-4Rα−/− BALB/c mice to generate LckcreIL-4Rα−/lox mice. The “loxed” IL-4Rα allele, gray arrows; deleted allele, black arrows.
(B) Genotyping of LckcreIL-4Rα−/lox mice. The deleted IL-4Rα PCR yields a product of 471 bp, LoxP, 188 bp (loxed), and 94 bp (WT), and Cre-specific a 450-bp product.
(C) Phenotypic analysis. WT (solid line), IL-4Rα−/− (gray line), and LckcreIL-4Rα−/lox BALB/c mice (dashed line) LN cells were stained for expression of IL-4Rα. T cell subsets were identified using anti-CD3, anti-CD4/CD8, or δ-TCR. B cells, anti-CD19. DCs, CD11c/I-Ad. Macrophages, F4/80/I-Ad.
(D) Efficiency of IL-4Rα deletion. The ratio of IL-4Rα exon 5 and exon 8 alleles was determined by real-time PCR from genomic DNA purified from CD4+ or CD19+ cells. PCR products of amplified genomic DNA from real-time PCR reactions (75 cycles) were visualized on agarose gel. Data is representative of 2 independent experiments with triplicate values ± SD.
Figure 2.
Functional Analysis of LckcreIL-4Rα−/lox Mice
(A) Impaired proliferation in response to IL-4. [3H] thymidine incorporation by CD4+ T cells stimulated by serial dilutions of rIL-4 (left panel) or IL-2 (right panel). One of three representative experiments is shown with means of triplicate values ± SD.
(B) Impaired Th2 differentiation of CD4+ T cells. CD4+ T cells were cultured in Th1 or Th2 polarizing conditions and IL-4 or IFN-γ secretion was measured by ELISA. A representative of one of three experiments is shown with means of triplicate values ± SD.
(C) IL-4 and IL-13 suppress macrophage NO secretion in LckcreIL-4Rα−/lox mice. Peritoneal exudate cells were incubated with IL-4, IL-13, or IL-10 in combination with LPS/IFN-γ, LPS/IFN-γ alone, or medium alone. Nitrite levels were measured by Griess reaction. One of three experiments is shown with means of triplicate values ± SD. (**p < 0.01 or ***p < 0.001, LPS/IFN-γ versus LPS/IFN-γ + IL-4 or IL-13)
(D) IgE production. Total IgE was measured in sera taken at 3 wk after infection and boosted with OVA (three mice per group). One representative experiment of two is shown.
Figure 3.
LckcreIL-4Rα−/lox Mice Control Footpad Swelling and Parasite Burden during Acute and Chronic L. major Infection
(A) Lesion development. Footpad swelling was measured at weekly intervals in mice (five per group) infected with 2 × 106 stationary phase L. major LV39 (MRHO/SV/59/P) metacyclic promastigotes into the hind footpad. Asterisk indicates ulceration or necrosis/mouse. A representative of one of five experiments is shown with mean values ± SD.
(B) Week six footpad parasite load. Parasite load was determined by limiting dilution of single-cell suspensions from homogenized footpads at 6 wk after infection.
(C) Week six LN parasite load. Parasite load was determined by limiting dilution of single-cell suspensions from the draining LNs at 6 wk after infection.
(D) L. major parasite detection using real-time PCR at 36 wk after infection. Kinetoplast DNA was quantified from footpads at week 36 after infection. One of two representative experiments is shown, with values representing mean parasite number ± SD.
(E) LckcreIL-4Rα−/lox BALB/c mice are resistant to reinfection. At 6 wk after infection with L. major, mice were reinfected in the contralateral hind footpad, and footpad swelling was monitored for 18 wk. Data are representative of two independent experiments.
(F) LckcreIL-4Rα−/lox BALB/c mice are resistant to L. major (MHOM/IL/81/FEBNI). Lesion development: mice (four per group) were infected with 2 × 106 stationary phase L. major (MHOM/IL/81/FEBNI) metacyclic promastigotes into the hind footpad. Asterisk indicates ulceration or necrosis per mouse. Footpad swelling was measured at weekly intervals up to week 14 and every 2 wk thereafter. A representative of one of two experiments is shown with mean values ± SD.
Figure 4.
IL-10 Inhibits Protective Responses
(A and B) Increased numbers of IL-10–secreting cells in IL-4Rα−/lox mice. (A) CD4+ IL-10–secreting cells were identified by intracellular FACS in LN cells restimulated with SLA for 24 h in vitro from L. major–infected mice 6 wk after infection. Data represent one of two independent experiments (pool of eight popliteal LNs/group). (B) Total numbers of CD4+ IL-10–secreting cells per draining LN.
(C–E) Susceptible mice exhibit poor DTH responses controlled by IL-10. At 6–8 wk after infection with L. major, mice (five mice per group) were injected in the contralateral hind footpad with (C) 10 μg SLA subcutaneously, (D) 10 μg SLA and 0.5 μg IL-10 subcutaneously, and (E) 10 μg SLA and 1.5 μg anti–IL-10R subcutaneously. Footpad swelling was monitored every 24 h for 5 d. (***p < 0.001, **p < 0.01 LckcreIL-4Rα−/lox versus WT). The data represent one of two independent experiments.
(F) Increased IL-12p70 in DTH footpads of resistant mice. Lysates of footpads (four per group) taken 24 h after induction of DTH responses were analyzed for IL-12p70. The data represent the pool of two independent experiments (*p < 0.05, LckcreIL-4Rα−/lox versus WT).
(G) Increased IFN-γ in DTH footpads of resistant mice. Lysates of footpads (four per group) taken 24 h after induction of DTH responses were analyzed for IFN-γ. The data represent the pool of two independent experiments (**p < 0.01, LckcreIL-4Rα−/lox versus WT).
Figure 5.
Type 1 Immunity Is Enhanced in LckcreIL-4Rα−/lox Mice in Response to L. major Infection
(A and B) IL-12p35 mRNA expression is increased in resistant mice. IL-12p35 expression was determined by real-time RT-PCR from RNA prepared from pooled popliteal LN cells from week 3 L. major–infected mice (eight mice per group). Data are expressed as IL-12p35 copy numbers relative to GAPDH (A) or as fold increase in IL-12p35 mRNA from 3 wk versus 1 wk after infection (B). Mean ± SEM of three runs on the same sample. Data are representative of two independent experiments (*p < 0.05, LckcreIL-4Rα−/lox versus WT).
(C and D) Increased IFN-γ production in resistant mice. (C) IFN-γ secretion by CD4 T cells cultured with fixed APCs and SLA. (**p < 0.01, LckcreIL-4Rα−/lox versus WT) and in (D) footpad homogenates (*p < 0.05, LckcreIL-4Rα−/lox versus WT) (homogenate data represent the pool of two independent experiments) from week 10 L. major–infected mice.
(E and F) Maintained IL-4 production in resistant mice. (E) IL-4 secretion by CD4 T cells cultured with fixed APCs and SLA and in (F) footpad homogenates from week-ten L. major–infected mice.
(G) iNOS production. iNOS mRNA copy number was calculated from footpad mRNA 6 wk after infection with L. major. At the same time, parasite DNA copy number was quantitated by PCR detecting L. major kinetoplast DNA. (*p < 0.05 LckcreIL-4Rα−/lox versus WT). The data represent the means of two individual experiments ± SEM.