IL-12 deficiency exacerbates inflammatory responses in UV-irradiated skin and skin tumors

Abstract

IL-12 deficiency has been shown to promote photocarcinogenesis in mice. As UVB-induced inflammation is an important tumor-promoting event in the development of skin tumors, we determined the effects of IL-12-deficiency on UVB-induced inflammatory responses in mice. For this purpose, IL-12-knockout (IL-12 KO) and their wild-type counterparts were subjected to a photocarcinogenesis protocol; skin and tumor samples were collected at the termination of the experiment, and analyzed for biomarkers of inflammation and their mediators. We found that the levels of infiltrating leukocytes, myeloperoxidase, proliferating cell-nuclear antigen (PCNA), COX-2, PGE2, and the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6 were higher in the UVB-exposed skin of IL-12 KO than in that of wild-type mice. In a short-term experiment, pretreatment of IL-12 KO mice with rIL-12 (50 ng per mouse) before each exposure to UVB increased the repair rate of UVB-induced cyclobutane pyrimidine dimers, while inhibiting UVB-induced increases in myeloperoxidase, COX-2, PGE2, PCNA, TNF-alpha, and IL-1beta in the skin as compared with non-rIL-12-treated IL-12 KO mice. Similarly, tumors of IL-12 KO mice expressed higher levels of inflammatory responses than those of wild-type mice. Together, our data suggest that IL-12 KO mice are more susceptible to both UVB-induced inflammation and photocarcinogenesis because of the deficiency in the repair of UVB-induced DNA damage.

Figure 1

a, IL-12 KO mice develop higher number of UVB-induced skin tumors than wild-types. Mice were exposed to UVB three times/week for 35 weeks and tumor data were recorded on weekly basis, n=20/group. Significant difference versus wild-types, ^¶p<0.001 at the termination of the tumor experiment. b, Chronic exposure of IL-12 KO mice to UVB results in greater leukocyte infiltration than observed in UVB-irradiated wild-type (C3H/HeN) mice skin. The paraffin-embedded skin samples (5 μm thick) were processed for routine H & E staining following a standard protocol. Representative examples of micrographs of H & E staining are shown from experiments conducted in skin samples and that had identical patterns (n=10). c, MPO was determined as a marker of UVB-induced tissue infiltration. The levels of UVB-induced MPO were greater in the skin samples of IL-12 KO mice compared to their wild-type counterparts. Data were reported as mean ± SD, n=10. Significant differences; *p<0.01; ^¶p<0.001. Bar = 50 μm.

Figure 2

IL-12-deficiency enhances the proliferation index potential of epidermal cells and COX-2 expression in UV-exposed skin. a, Immunohistochemical analysis of PCNA+ cells in chronic UV-exposed IL-12 KO and WT mouse skin. The PCNA staining was more intense in IL-12 KO mice than WT counterparts. b, The levels of PCNA were determined employing western blot analysis and real-time PCR. The mRNA expression of PCNA is presented after normalization to β-actin using the C_t method. c, Immunohistochemical detection and localization of COX-2 expression in skin samples. The UVB-irradiated skin expressed higher levels of epidermal COX-2 compared to normal skin, and the skin of IL-12 KO mice expressed higher and intense staining pattern of COX-2 after UVB exposure than WT counterparts. d, Epidermal COX-2 expression analysis using western blotting, as described in Materials and Methods. In western blot analysis, a representative blot is shown from three independent experiments with identical observations. In each experiment, epidermis was pooled from 2-3 mice for preparing lysate samples, and equivalent protein loading was confirmed by probing stripped blots for β-actin as shown. e, PGE₂ is determined in the epidermal homogenate samples using an enzyme-linked immunosorbent assay. The levels of PGE₂ were higher in the skin samples of IL-12 KO mice compared with their WT mice. The concentration of PGE₂ is expressed in terms of pg/mg protein as a mean ± SD, n = 10. Significant difference versus wild-types, *p<0.01. Significant difference versus normal skin, ^¶p<0.001. Bar = 50 μm.

Figure 3

Chronic exposure of UVB increases relatively higher levels of pro-inflammatory cytokines in the skin of IL-12 KO mice than WT counterparts. At the termination of the experiment, epidermal homogenates were prepared for the analysis of the levels of TNF-α, IL-1β, IL-6, and IL-10 using ELISA. Significant increases in the levels of these cytokines were observed after UVB irradiation compared to non-UVB-exposed animals. The skin samples of IL-12 KO mice were found to have higher levels of these cytokines over their WT mice. The concentration of each cytokine is reported in terms of pg/mg protein as a mean ± SD, n = 8-10. Significant increases in IL-1β (^†p< 0.05), TNF-α (*p<0.01), IL-6 (*p<0.01), and IL-10 (^†p< 0.05) in UVB-exposed IL-12 KO versus UVB-exposed wild-types were seen. Significant difference versus non-UVB-irradiated control skin samples, ^¶p<0.001; *p<0.01.

Figure 4

In vivo subcutaneous treatment of IL-12 KO mice with rIL-12 inhibits multiple UVB exposure-induced leukocyte infiltration, the levels of MPO, COX-2, PGE₂ and PCNA compared to non-rIL-12-treated IL-12 KO mice. Mice were UVB-irradiated (180 mJ/cm²) on alternate days for total ten days. One group of mice was s.c. injected with murine IL-12 (50 ng/mouse) 3 h before each exposure of UVB. Mice were sacrificed at different time points (6, 12, 24, 48 and 72 h) after the last UVB exposure, and skin samples were obtained and analyzed for MPO, infiltration, PCNA, COX-2, and PGE₂ as described in Materials and Methods. Data were analyzed using biochemical assays for MPO and PGE₂ (Panels a & d), immunohistochemistry for infiltration, PCNA and COX-2 (Panel b) and western blot analysis for PCNA (Panel c). Epidermal lysates were used for western blot analysis, and equivalent protein loading was confirmed by probing stripped blots for β-actin as shown. A representative blot is shown from three independent experiments with identical observations. n=3 at each time point studied. Panel d, PGE₂ was determined in the skin samples using an enzyme-linked immunosorbent assay. Significant inhibition in rIL-12-treated mice versus non-rIL-12-treated but UV-exposed mice, *p<0.01; ^¶p<0.001. Bar = 50 μm.

Figure 5

UV-induced CPDs removes or repairs rapidly in WT mice than in IL-12 KO mice, and treatment of s.c. rIL-12 to IL-12 KO mice before UV-irradiation removes or repairs UV-induced CPDs rapidly than IL-12 KO mice which were not treated with rIL-12. Mice were exposed to either acute (Panels a-c) or multiple UV (180 mJ/cm²) exposure (Panels d-f). In multiple UV exposure, mice were exposed to UV on alternate days for 10 days. One group of mice was s.c. injected with murine rIL-12 (50 ng/mouse) 3 h before exposure of UV. Mice were sacrificed at different time points (1/2, 48 and 72 h) after the last UVB exposure, and skin samples were obtained and analyzed for CPDs using immunohistochemistry and dot-blot analysis. Frozen sections (5 μm thick) were subjected to immunoperoxidase staining to detect CPD+ cells that are dark brown. CPDs were not detected in non-UV-exposed skin whether treated or not treated with rIL-12. Epidermal PGE₂ was determined as a marker of inflammation using immunoassay kit (Panels c and f), as describes in Materials and methods. Panel e, Epidermal genomic DNA was subjected to Southwestern dot blot analysis to detect UV-induced damaged DNA using an antibody specific to CPD. Panel f, Inflammatory cytokines TNF-α and IL-1β were determined in epidermal homogenates using ELISA kits. Experiments were conducted and repeated separately in 5-6 animals in each group with identical results. Results are expressed as mean ± SD. Significantly less than IL-12 KO mice, *p<0.01; ^¶p<0.001.

Figure 6

The UV-induced tumors developed in IL-12 KO mice expressed higher levels of inflammatory mediators, such as, PCNA, cyclin D1 (Panel a), COX-2 and PGE₂ (Panel b), and inflammatory cytokines, TNF-α and IL-1β (Panel c) compared to WT mice. The expression levels of PCNA and COX-2 were determined using immunostaining, western blot analysis and/or real-time-PCR. Representative example of micrographs of staining for PCNA and COX-2 was presented from at least six mice which showed identical patterns. Bar = 50 μm. The representative blots are shown from three independent experiments, and in each experiment the tumor samples were pooled from at least three mice. The results of mRNA expression of PCNA and cyclin D1 are presented after normalization to β-actin using the C_t method. The levels of PGE₂, TNF-α and IL-1β were determined in tumor homogenates using ELISA kits and data are presented as mean± SD in terms of pg/mg protein. Experiments were repeated in tumor samples from at least 6 mice with identical results. *Significant difference versus WT, p<0.01.