Proanthocyanidins inhibit UV-induced immunosuppression through IL-12-dependent stimulation of CD8+ effector T cells and inactivation of CD4+ T cells

Abstract

The inhibition of UVB-induced immunosuppression by dietary grape seed proanthocyanidins (GSP) has been associated with the induction of interleukin (IL)-12 in mice, and we now confirm that GSPs do not inhibit UVB-induced immunosuppression in IL-12p40 knockout (IL-12 KO) mice and that treatment of these mice with recombinant IL-12 restores the inhibitory effect. To characterize the cell population responsible for the GSP-mediated inhibition of UVB-induced immunosuppression and the role of IL-12 in this process, we used an adoptive transfer approach. Splenocytes and draining lymph nodes were harvested from mice that had been administered dietary GSPs (0.5%-1.0%, w/w), exposed to UVB, and sensitized by the application of 2,4-dinitrofluorobenzene (DNFB) onto the UVB-exposed skin. CD8(+) and CD4(+) T cells were positively selected and transferred into naive mice that were subsequently challenged by application of DNFB on the ear skin. Naive recipients that received CD8(+) T cells from GSP-treated, UVB-irradiated donors exhibited full contact hypersensitivity (CHS) response. Naive mice that received CD4(+) suppressor T cells from GSP-treated, UVB-exposed mice could mount a CHS response after sensitization and subsequent challenge with DNFB. On culture, the CD8(+) T cells from GSP-treated, UVB-exposed mice secreted higher levels (5- to 8-fold) of Th1 cytokines than CD8(+) T cells from UVB-irradiated mice not treated with GSPs. CD4(+) T cells from GSP-treated, UVB-exposed mice secreted significantly lower levels (80%-100%) of Th2 cytokines than CD4(+) T cells from UVB-exposed mice not treated with GSPs. These data suggest that GSPs inhibit UVB-induced immunosuppression by stimulating CD8(+) effector T cells and diminishing regulatory CD4(+) T cells.

Figure 1

Prevention of UVB-induced suppression of the CHS response by GSPs requires IL-12. Dietary GSPs prevent UVB-induced suppression of the CHS response in wild-type mice but not in IL-12 KO mice. Wild-type (C3H/HeN) (Panel A) or IL-12 KO C3H/HeN (Panel B) mice that received either a standard diet or a diet supplemented with GSPs (0.5% or 1.0%, w/w) and exposed to UVB radiation (150 mJ/cm²) on four-consecutive days, sensitized to DNFB and the CHS response to application of DNFB on ear skin (challenge) assessed by measurement of ear swelling response 24 h later, as described in the Materials and Methods. Treatment group number 1 in each panel indicates that mice were not sensitized with DNFB but only challenged with DNFB in ear skin. One group of wild-type mice were injected with anti-IL-12 antibody (Panel A, 8^th bar), and one group of IL-12 KO mice (Panel B, 8^th and 9^th bar) were injected with 1,000 ng of IL-12, 3 h before DNFB sensitization. The change in ear thickness is reported in millimeter (mm ×10^-2) as the mean ± SD, with n=5 per group. The experiment was repeated once with similar results. *Significant sensitization versus UVB exposure in the absence of GSPs treatment (5^th bar), P<0.001; ^¶Significant inhibition versus the positive control of sensitization in the absence of UVB irradiation or GSPs treatment (2^nd bar), P<0.005.

Figure 2

GSPs protect against UVB-induced immunosuppression and does so even after cessation of treatment. Mice (C3H/HeN) that received a standard diet or a GSPs-supplemented diet were UVB-irradiated, sensitized through the UVB-irradiated skin, challenged by application of DNFB on the right ear (primary challenge), and the ear skin swelling measured 24 h later. The CHS response after primary challenge of the UVB-exposed mice that did not receive GSPs was significantly lower than the CHS response of the mice that were not UVB-irradiated, whereas mice that received GSPs in the diet before and during the CHS protocol mounted a CHS response to the primary DNFB challenge that was comparable to the response in the mice that were not UVB-irradiated. When the mice were rested for 4 weeks after primary challenge, then further challenged with DNFB (secondary challenge) on the left ear similar results were obtained. The change in ear swelling response in each group is reported as mean ± SD, n=5 per group. The experiment was repeated once with similar observations. *Significant increase versus UVB exposure in the absence of GSPs treatment, P<0.001; ^¶Significant increase versus UVB in the absence of GSPs treatment, P<0.005; ^†Significant inhibition versus the positive control (unirradiated mice) (2^nd bar from the top), P<0.001.

Figure 3

GSPs prevent transferable UVB-induced suppression through activation of T cells. A, Donor mice (C3H/HeN) that received either a standard diet or a diet supplemented with GSPs were sensitized, UVB-irradiated, sacrificed 5 days later and single-cell suspensions prepared from the regional lymph nodes and spleens, as detailed in the Materials and Methods. Syngeneic Recipient mice were injected i.v. with 5× 10⁷ spleen and lymph node cells obtained from syngeneic donor mice. Recipient mice were sensitized with DNFB 24 h after transfer, ear challenge was performed 5 days later, and ear skin thickness was measured before and 24 h after challenge. B, The donor mice were treated as described in Panel A, except that CD8⁺ T cells were positively selected from the spleen and lymph nodes cell preparations. The CD8⁺ T cells (8× 10⁶) were injected i.v. into naïve mice, the recipient mice were challenged immediately and the ear swelling response was measured 24 h later. In one group of mice, the donor mice were administered recombinant IL-12 (1000 ng/mouse) i.p. 3 h before sensitization. In another group, donor mice received an i.p. injection of anti-IL-12 (500 ng/mouse) 24 and 3 h before DNFB sensitization. Control mice received rat IgG1 (isotype control of anti-IL-12). The change in ear thickness is reported as the mean of millimeters (mm × 10^-2) ±SD, n=5 per group. *Significantly greater CHS response versus UVB irradiation in the absence of GSPs treatment, P<0.001; ^¶Significantly greater CHS response versus recipient of T cells from UVB+ DNFB treated mice, P<0.01; ^†Significantly lower CHS response versus the positive control (DNFB-sensitized) group, P<0.001

Figure 4

Treatment of mice with GSPs or rIL-12 enhances the levels of production of IL-2 and IFNγ by CD8⁺ T cells. Mice were treated and CD8⁺ T-cells isolated as described under Figure 3. The CD8⁺ T-cells were then co-cultured with DNSB-labeled BMDC for 48 h, as detailed in the Materials and Methods. The concentrations of cytokines in the cell supernatants were estimated by ELISA and are presented as the mean± SD in terms of pg or ng per 2 million cells. n=5/group. *Significant increase versus UVB+DNFB group, P<0.001

Figure 5

(A) GSPs prevent transferable suppression by UVB through the inactivation of UVB-induced CD4⁺ suppresser T cells. Mice were treated as described in Figure 3 and CD4⁺ T cells purified from the splenocytes and lymphocytes by positive selection. Twenty-four hours after i.v. injection of CD4⁺ T cells into naïve mice, the mice were sensitized with DNFB, and ear challenged 5 days after sensitization, as detailed in the Materials and Methods. Those naïve mice that received CD4⁺ T cells from UVB-exposed donor mice that were GSPs treated showed a greater CHS response than UVB-exposed mice that were not GSPs treated. Those naïve mice that received CD4⁺ T cells from UVB-exposed rIL-12-injected donor mice showed a significantly higher CHS response than UVB-exposed mice that were not injected with rIL-12. The change in the ear swelling response in each group is reported as mean ± SD (n=5 per group). Significantly greater CHS response versus UVB irradiation in the absence of GSPS or rIL-12 treatment, ^†P<0.001; Significantly lower CHS response versus positive control group (2^nd bar), ^¶P<0.01. (B) Treatment of mice with GSPs or rIL-12 decreases the production of IL-4 and IL-10 by CD4⁺ T cells. Mice were sensitized to DNFB after UVB-irradiation described in Figure 3, the CD4⁺ T cells isolated by positive selection and then co-cultured with DNSB-labeled BMDC for 48 h, as detailed in the Materials and Methods. The concentrations of cytokines in the cell supernatants were estimated by ELISA and are presented as the mean ±SD in terms of pg/2 million cells. Experiment was repeated once, n=5. Significant decrease versus UVB+DNFB group, ^†P<0.001; Significant decrease versus positive control (DNFB alone), ^¶P<0.001; Significant increase versus positive control (DNFB alone), *P<0.001