In Vivo Suppression of Heat Shock Protein (HSP)27 and HSP70 Accelerates DMBA-Induced Skin Carcinogenesis by Inducing Antigenic Unresponsiveness to the Initiating Carcinogenic Chemical

Abstract

Heat shock proteins (HSPs) are constitutively expressed in murine skin. HSP27 is present in the epidermis, and HSP70 can be found in both the epidermis and dermis. The purpose of this study was to investigate the role of these proteins in cutaneous chemical carcinogenesis and to determine whether their effects on cell-mediated immune function were a contributing factor. In vivo inhibition of HSP27 and HSP70 produced a reduction in the T cell-mediated immune response to 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene in C3H/HeN mice and resulted in a state of Ag-specific tolerance. When mice were pretreated with anti-HSP27 and anti-HSP70 Abs in vivo prior to subjecting them to a standard two-stage DMBA/12-O-tetradecanoylphorbol-13-acetate cutaneous carcinogenesis protocol, the percentage of mice with tumors was much greater (p < 0.05) in anti-HSP27- and HSP70-pretreated animals compared with mice pretreated with control Ab. Similar results were obtained when the data were evaluated as the cumulative number of tumors per group. Mice pretreated with HSP27 and HSP70 Abs developed more H-ras mutations and fewer DMBA-specific cytotoxic T lymphocytes. These findings indicate that in mice HSP27 and HSP70 play a key role in the induction of cell-mediated immunity to carcinogenic polyaromatic hydrocarbons. Bolstering the immune response to carcinogenic polyaromatic hydrocarbons may be an effective method for prevention of the tumors that they produce.

Figure 1

Contact hypersensitivity to DMBA is inhibited by pretreatment of skin with antibodies to heat shock proteins 27 (HSP27) and 70 (HSP70). (A) The application of neutralizing antibodies to HSP27 or HSP70 to C3H/HeN mice (n=5 per group) significantly inhibited the CHS response and treatment with antibodies to HSP27 and HSP70 gave an additive effect (*,p<0.05). (B) Treatment with isotype control antibodies did not diminish the CHS response to DMBA (*, p<0.05). Results are expressed as the change in auricular thickness ± SEM.

Figure 2

Effect of HSP27 and -70 on cytokine production by draining lymph node T-cells after DMBA sensitization in C3H/HeN mice. Sensitized T-cells were stimulated with bone marrow dendritic cells (BMDC). Lymph node cells from mice pre-treated with anti-HSP antibodies secreted significantly lower levels (*,p<0.05) of IL-17(A) and IFN-γ (B); and higher levels of IL-4 (C) and IL-10 (D) than control antibody treated mice.

Figure 3

Tolerance to DMBA follows HSP antibody treatment and is antigen specific. C3H/HeN mice (n=5 per group) were treated with anti-HSP antibodies on the abdomen after which 0.1% DMBA was applied to the same site. After 14 days, the mice were re-sensitized on the shaved back with either (A) 0.1% DMBA or (B) 0.1% B(a)P. Mice were ear challenged 5 days later with the same hapten that had been applied to the back. Ear swelling responses indicate that inhibition of CHS produced by anti-HSP antibodies was present in the DMBA sensitized mice (*,p<0.05), but not in the B(a)P sensitized mice (*, p>0.05). In another set of experiments, C3H/HeN mice (n=5 per group) were treated with anti-HSP antibodies on the abdomen after which 0.1% B(a)P was applied to the same site. After 14 days, the mice were re-sensitized on the shaved back with either (C) 0.1% B(a)P or (D) 0.1% DMBA. Mice were ear challenged 5 days later with the same hapten that had been applied to the back. Ear swelling responses indicate that inhibition of CHS produced by anti-HSP antibodies was present in the B(a)P sensitized mice (*,p<0.05), but not in the DMBA sensitized mice (*, p>0.05).Results are expressed as change in auricular thickness ± SEM.

Figure 4

Effect of HSP27 and HSP70 antibodies on DMBA/TPA induced cutaneous carcinogenesis in C3H/HeN mice (n=10 per group). There was a significantly higher (*, p<0.05) number (A), percentage of tumors (B), and tumor volume (C) in the group of mice which was treated with anti-HSP antibodies. (D) Groups of mice with tumors after 25 weeks of DMBA/TPA treatment.

Figure 5

Increased expression of mutant H-ras in DMBA/TPA induced tumors from anti-HSP antibody treated mice after 25 weeks of DMBA/TPA treatment by RT-PCR. (A) Mutant H-ras is indicated by cleaved products (lanes 5&6) after digestion with XbaI. Lanes 1 and 2, naïve skin; lane 3, control antibody; lane 4, anti-HSP antibody; lane 5, control antibody; lane 6, anti-HSP antibody (after digestion with XbaI). (B) Densitometric analysis of mutant H-ras expression from gel, (C) qPCR for mutant H-ras expression in tumors. The frequency of the H-ras mutation was significantly greater (*, p<0.05) in tumors from the anti-HSP antibody treated mice than in mice treated with the control antibody.

Figure 6

HSP antibody pretreatment inhibits development of DMBA specific CTLs. A. In vivo CTL assay of DMBA-pulsed CFSE low target spleen cells. Mice were pretreated with antibodies then sensitized, as indicated. After 8 days following treatment, mice (n=3 per group) received a 1:1 mixture of DMBA-pulsed CFSE low and unpulsed CFSE hi stained splenocytes, by i.v. After 16 hours, splenocytes were harvested and analyzed by flow cytometry. The % CFSE low population of total CFSE labeled cells is shown in each histogram. The % CFSE low population in naïve mice provided an input baseline control of 53% (confirming the 50:50 input mix). B. Bar graph of the mean ± SEM % DMBA specific cytotoxicity is shown for each group. Arrow indicates % Inhibition. *, p<0.05 by Student’s T test.