Contrasting roles of the IL-1 and IL-18 receptors in MyD88-dependent contact hypersensitivity

Abstract

Contact hypersensitivity (CHS) requires activation of the innate immune system, and results in an adaptive immune response. Many cells of the innate immune system use Toll-like receptors (TLRs), which signal through the adaptor protein, MyD88, to initiate an immune response. MyD88 is also required for signaling downstream of the IL-1 and Il-18 receptors (IL-1R and IL-18R, respectively). Herein, we studied the MyD88 signaling pathway in the CHS response to DNFB. Mice deficient in MyD88 were unable to mount a CHS response to DNFB. In contrast, mice deficient in Toll/IL-1R-containing adaptor-inducing IFN-beta, TLR2, TLR4, TLR6, and TLR9 had no defect in their ability to respond to DNFB. Although both IL-1R and IL-18R-deficient mice showed a reduced CHS response to DNFB, in bone marrow chimera and adoptive transfer experiments, we found that MyD88 and the IL-18R were required in a radioresistant cell in the sensitization phase of the CHS response. In contrast, similar strategies revealed that the IL-1R was required in a radiosensitive cell in the sensitization phase of the CHS response. Taken together, these data indicate that the IL-1R and IL-18R/MyD88 pathways are required in distinctly different cells during the sensitization phase of CHS.

Figure 1. Contact hypersensitivity is MyD88-, IL-1R-, and IL-18R–dependent

(a) Hapten-specific ear swelling was measured 24 hours after challenge with DNFB in B6, MyD88- and TRIF-deficient mice. Results represent mean +/− SEM (n=4) from 1 of 2 or more separate experiments. Statistical significance is indicated versus B6 as n.s. = not significant, * p=0.0002. (b) Hapten-specific ear swelling was measured 24 hours after challenge with DNFB in B6, IL-1R-, IL-18R- and MyD88-deficient mice. Results represent mean +/− SEM (n=4) from 1 of 2 or more separate experiments. n.s. = not significant, * p <0.02.

Figure 2. MyD88 and the IL-18R are required in radioresistant, host-derived cells in the CHS response, while the IL-1R is required in radiosensitive, bone marrow-derived cells

(a) C57BL/6 mice congenic for Ly5.1 (5.1) were lethally irradiated and reconstituted with BM cells from mice that were either B6 or MyD88^−/− . After 6 weeks, the mice were sensitized and challenged with DNFB. The plots represent the hapten- specific ear swelling 24 hours after challenge with DNFB of each mouse relative to the response in B6 mice. Results represent the mean +/− SEM (n=4) of the response relative to B6 from 1 of 2 or more separate experiments. To determine the relative response, the hapten-specific response in B6 mice from each experiment was set to 100% and the hapten-specific response of each bone marrow transplant mouse was normalized to B6 and plotted as percent of B6. Statistical significance was determined from the individual experiments, prior to normalization to B6. n.s. = not significant, * p<0.01. (b) BM cells from WT Ly5.1 (5.1) mice were used to reconstitute lethally irradiated WT B6 or IL-1R^−/− mice (5.1>B6 or 5.1> IL-1R^−/−, respectively). Additionally, BM cells from mice that were either B6 or IL-1R^−/− were used to reconstitute lethally irradiated WT Ly5.1 (5.1) mice (B6>5.1 or IL-1R^−/−>5.1, respectively). (c) BM cells from mice that were from WT Ly5.1 (5.1) mice were used to reconstitute lethally irradiated WT B6 or IL-18R^−/− mice (5.1>B6 or 5.1>IL-18R^−/−, respectively). Additionally, B6 or IL-18R^−/− BM was used to reconstitute lethally irradiated WT Ly5.1 (5.1) mice (B6>5.1 or IL-18R^−/−>5.1, respectively). After 6 weeks the mice were sensitized and challenged with DNFB. The plots for b and c represent the hapten-specific ear swelling 24 hours after challenge with DNFB. Results represent mean +/− SEM (n=4) from 1 of 2 or more separate experiments. Statistical significance is indicated versus B6 as n.s. = not significant, * p<0.001.

Figure 3. MyD88, the IL-18R and the IL-1R are required in the sensitization phase of the CHS response

(a) WT mice were sensitized with DNFB (dWT) and 5 days later the lymphocytes from their skin-draining lymph nodes were injected into naïve WT (nWT) or naïve MyD88^−/− (MyD88^−/−) mice (dWT>WT or dWT>MyD88^−/−, respectively). In the converse experiment, lymphocytes from the skin-draining lymph nodes of DNFB-sensitized WT (dWT) or MyD88^−/− (dMyD88^−/−) mice were injected into naïve WT (WT) mice (dWT>WT or dMyD88^−/−>WT, respectively). After the injection of the lymphocytes the mice were challenged with DNFB and the hapten-specific response was measured 24 hours later. The results are depicted as the relative response to B6, as described in Figure 2A. The plots represent the mean +/− SEM (n=4) of the response relative to B6 from 1 of 2 or more separate experiments. Statistical significance was determined from the individual experiments, prior to normalization to B6. n.s. = not significant, * p<0.05. (b) WT or IL-18R^−/− mice were sensitized with DNFB (dWT or dIL-18R^−/−) and 5 days later the lymphocytes from their skin-draining lymph nodes were injected into either naïve WT (nWT) or naïve IL-18R^−/− (nIL-18R^−/−) mice. (c) In similar experiments, lymphocytes from the skin-draining lymph nodes of DNFB-sensitized WT (dWT) or IL-1R^−/− (dIL-1R^−/− ) mice were injected into either naïve WT (nWT) or naïve IL-1R^−/− (nIL-1R^−/− ) mice. After injection of the lymphocytes the mice were challenged with DNFB and the hapten-specific response was measured 24 hours later. The results are depicted as the mean +/− SEM (n=4) from 1 of 2 or more separate experiments. Statistical significance is indicated versus dWT>nWT as n.s. = not significant, and ** p<0.002. The difference between WT and dWT>nWT, dWT>nIL-18R^−/− , or dWT>nIL-1R^−/− was not significant (p>0.05).

Figure 4. CD86 expression on dendritic cells from skin-draining lymph nodes

(a) WT, MyD88^−/−(b) IL-1R^−/− and IL-18R^−/− mice were sensitized on the abdomen with a single dose of DNFB. Three days later the lymphocytes were removed from the skin-draining lymph nodes and analyzed by FACS. The dendritic cells were gated as CD11c⁺, MHC-II^high cells and plotted as a histogram showing CD86 expression. Dendritic cells from DNFB treated mice are plotted as solid lines and naïve mice of the same genotype are plotted as dashed lines. The irrelevant antibody is shown in the grey histogram. The results from vehicle treated mice are similar to those of the naïve mice and are omitted for clarity. The results are representative from 3 or more experiments.