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.

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.

Profiles of cytokine mRNAs in the skin and lymph nodes of SENCAR mice treated epicutaneously with dibenzo[a,l]pyrene or dimethylbenz[a]anthracene reveal a direct correlation between carcinogen-induced contact hypersensitivity and epidermal hyperplasia.

The potent carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P) in mouse skin is associated with an inflammatory response and a striking epidermal hyperplasia. The mechanism of these tissue responses is not known. However, a recent study has shown DB[a,l]P to be a contact sensitizer. In view of the programmed expression of cytokines during induction of contact hypersensitivity (CHS) and elicitation of CHS reactions, we analyzed cytokine mRNAs in treated skin and draining lymph nodes of SENCAR mice, at selected times after a single, epicutaneous application of DB[a,l]P or dimethylbenz[a]anthracene (DMBA), a substantially weaker carcinogen and a weaker contact sensitizer than DB[a,l]P. Cytokine mRNAs were quantified by first-strand DNA synthesis with reverse transcriptase (RT) and DNA amplification by the polymerase chain reaction (PCR). Histopathology of treated skin was determined in the same experiments. Time-response profiles of interferon (IFN) gamma and interleukin (IL) 2 in the DLN and IL1beta, IL10, tumor necrosis factor (TNF) alpha, and IL4 mRNAs in the skin of mice treated with 200 nmol of DB[a,l]P were in remarkable agreement with established profiles in mice treated with conventional contact sensitizers, e.g., oxazolone or dinitrochlorobenzene. Strong upregulation of DLN IFNgamma mRNA coupled with little change in IL 2 mRNA suggested a CD8(+) T-cell response characteristic of CHS induction. Coordinate expression of epidermal IL1beta, TNFalpha, and IL10 mRNAs, 24 h after DB[a,l]P treatment, was also characteristic of CHS induction. IL1beta and IL10 are upregulated by allergens and not by chemical irritants. Time-response profiles of epidermal IL1beta, TNFalpha, IL10, and IL4 mRNAs, 3-14 d after DB[a,l]P treatment, corresponded with expression of these cytokines during elicitation of CHS reactions. Epidermal IL4 is specifically upregulated during CHS reactions. Cytokine mRNA responses were dose-dependent (50, 100, and 200 nmol of DB[a,l]P) and markedly weaker in animals treated with 400 nmol of DMBA. Significantly, the intensity of epidermal hyperplasia correlated with the strength of the cytokine mRNA signals in DLN and skin. In conclusion, our data support carcinogen-specific CHS as a mechanism by which the very potent carcinogen DB[a,l]P induces epidermal hyperplasia, a requirement for tumor promotion in mouse skin.

Susceptibility to the biological effects of polyaromatic hydrocarbons is influenced by genes of the major histocompatibility complex.

Polyaromatic hydrocarbons are ubiquitous environmental chemicals that are important mutagens and carcinogens. The purpose of this study was to determine whether genes within the major histocompatibility complex (MHC) influence their biological activities. Cell-mediated immunity to dimethylbenz(a)anthracene (DMBA) was investigated in congenic strains of mice. On three different backgrounds, H-2(k) and H-2(a) haplotype mice developed significantly greater contact-hypersensitivity responses to DMBA than H-2(b), H-2(d), and H-2(s) mice. In B10.A(R1) mice, which are Kk and Id, a vigorous contact-hypersensitivity response was present, indicating that the response was governed by class I, rather than class II, MHC genes. C3H/HeN (H-2(k)) and C3H.SW (H-2(s)) strains were also compared for the development of skin tumors and the persistence of DMBA-DNA adducts. When subjected to a DMBA initiation, phorbol 12-tetradecanoate 13-acetate (TPA)-promotion skin-tumorigenesis protocol, C3H/HeN mice, (which develop cell-mediated immunity to DMBA) were found to have significantly fewer tumors than C3H.SW mice (a strain that failed to develop a cell-mediated immune response to DMBA). DMBA-DNA adducts were removed more rapidly in C3H/HeN than in C3H.SW mice. The results indicate that genes within the MHC play an important role in several of the biological activities of carcinogenic polyaromatic hydrocarbons. The observations are consistent with the hypothesis that cell-mediated immunity to chemical carcinogens serves to protect individuals by removing mutant cells before they can evolve into clinically apparent neoplasms.

Down-regulation of an established immune response via chemical carcinogen or UVB-altered skin.

The ability to produce antigen-specific down-regulation of an established immune response was investigated in 2,4,6-trinitrochlorobenzene (TNCB)-immune mice by delivery of antigen through chemical carcinogen- or ultraviolet B (UVB)-treated skin. When TNCB-immune mice were treated on the dorsal trunk skin with 7,12-dimethylbenz(a)anthracene (DMBA) followed by TNCB there was an antigen-specific reduction in both contact sensitivity and antibody production. Further, immune mice that received spleen cells from naive syngeneic donors treated with DMBA followed by TNCB also exhibited a reduction in both contact sensitivity and antibody production. In contrast, mice treated with UVB irradiation followed by TNCB had a reduction in contact sensitivity but not antibody production. These results provide evidence that an ongoing immune response can be manipulated by immunization through a modified skin immune system. This may provide a beneficial approach for the treatment of autoimmune disease.

Persistent suppression of humoral and cell-mediated immunity in mice following exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene.

Carcinogen-induced immunosuppression has been implicated as an epigenetic mechanism in promoting the outgrowth and metastasis of neoplastic cells. It has previously been reported that the complete carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) suppresses both humoral immunity (HI) and cell-mediated immunity (CMI) 3-5 days following exposure. Since persistent systemic immunosuppression may be more relevant in tumor outgrowth, assays quantitating HI and CMI, including those functions involved in tumor resistance, were performed 4 and 8 weeks following exposure to tumorigenic doses of DMBA. Adult B6C3F1 female mice were administered DMBA dissolved in corn oil subcutaneously at 5, 50 and 100 micrograms/g body weight in ten equal doses over 2 weeks (corn oil = vehicle control). The number of splenocytes producing IgM antibody to the T-dependent antigen, sheep erythrocytes, was suppressed up to 95% and 98% at 4 and 8 weeks, respectively. The IgG response was similarly depressed 75% and 98% at 4 and 8 weeks, respectively. Lymphoproliferation of splenocytes in response to the mitogens LPS, PHA and Con A were depressed up to 88%, 78% and 83% at 4 weeks and 63%, 63% and 67% respectively, at 8 weeks. In addition, alloantigen-induced proliferation of splenocytes in a one-way mixed lymphocyte culture was suppressed up to 90% at 8 weeks. The ability to generate cytotoxic T-lymphocytes (CTL) in vitro against P815 tumor cells was depressed at both time periods (88% and 60%, respectively) as was natural killer (NK) cell cytolysis of YAC-1 tumor targets (84% and 55%, respectively). The immunosuppression noted in these parameters was similar to that observed within 3-5 days following DMBA dosing. The persistent immunosuppression induced by the PAH carcinogen DMBA, including CTL and NK cell tumoricidal functions, may represent an important epigenetic mechanism contributing to tumor outgrowth or metastasis by this class of agents.

Immunosuppression following 7,12-dimethylbenz[a]anthracene exposure in B6C3F1 mice. I. Effects on humoral immunity and host resistance.

It has previously been demonstrated that the polycyclic aromatic hydrocarbon (PAH), benzo(a)pyrene (B[a]P), suppresses the terminal step in B-cell differentiation, resulting in a decrease in antibody production to T-dependent and B-2 T-independent antigens. The purpose of this study was to ascertain if this effect was common to carcinogenic PAHs or specific for B[a]P. The PAH 7,12-dimethylbenz[a]anthracene (DMBA) was administered to B6C3F1 female mice by ten sc injections of 0.5, 5, or 10 micrograms/g over a 2-week period (i.e., total dose of 5, 50, and 100 micrograms/g). Immune function and host resistance assays were performed 3 to 5 days following the last injection. The 10 micrograms/g dosage resulted in a marked decrease in spleen weights and spleen and bone marrow cellularity, while thymus and body weights were not significantly altered. The ability to generate B-lymphocyte colonies in vitro from spleen precursor cells was also suppressed at the 10 micrograms/g dose. Exposure to DMBA at 5 micrograms/g or greater resulted in a reduction of up to 97% in the number of IgM plaque-forming cells in response to the T-dependent antigen sheep red blood cells (SRBC). The IgG response to SRBC was similarly depressed. The IgM response to the hapten-conjugated T-independent antigens trinitrophenyl-lipopolysaccharide (TNP-LPS) (specific for B-1 cells) and trinitrophenyl (TNP)-Ficoll (specific for B-2 cells) was also depressed (88 and 97%, respectively) at 10 micrograms/g. DMBA exposure resulted in an increased susceptibility to challenge with the PYB6 transplantable sarcoma and the bacterium Listeria monocytogenes, in contrast to B[a]P exposure, which had no effect on host resistance assays. Thus, DMBA, a more potent carcinogen than B[a]P, produces a more extensive B-cell suppression than B[a]P as well as alters host resistance to tumor and bacterial challenge.

Protection of mice against 7,12-dimethylbenz(a)anthracene-induced skin tumors by immunization with a fluorinated analog of the carcinogen.

5-Fluoro-12-methylbenzanthryl-7-acetic acid (5-FMBAAA) is an analog of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) with little or no carcinogenic activity. CD-1 mice immunized with 5-FMBAAA conjugated to bovine serum albumin (BSA) developed serum antibodies capable of binding DMBA. As a means of testing whether this immunization protected against DMBA-induced tumors, a low-dose carcinogenesis model system was developed, entailing the repeated skin application of 25 ng DMBA in dodecane alternating with applications of the tumor promoter, phorbol myristate acetate. Mice immunized with the 5-FMBAAA:BSA conjugate and subsequently exposed to this low-dose regimen for 40 weeks developed significantly fewer skin tumors (0.23 papilloma/mouse) than did unimmunized mice, mice immunized with BSA, or mice immunized with an unconjugated mixture of BSA and 5-FMBAAA (0.47 to 0.54 papilloma/mouse). Immunization did not reduce tumor incidence in mice treated with phorbol myristate acetate alone. The results suggest that, when mice are exposed to a carcinogen at doses low enough to approach environmental levels, immunization against the carcinogen can provide specific protection.

The induction of immunologic tolerance in guinea pigs infused with dimethylbenz(a)anthracene.

The infusion of 7,12 dimethylbenz(a)anthracene (DMBA) dissolved in DMSO:glycofurol into adult guinea pigs rendered most recipients unresponsive to the induction of contact sensitization with DMBA, either by injection in adjuvant or topical application. The results were similar when guinea pigs infused with DMBA dissolved in Upjohn fat emulsion, were challenged with DMBA in adjuvant, but studies attempting topical sensitization to demonstrate unresponsiveness were inconclusive. Mammary tumors appeared after a latent period of 8 to 11 mo in 4 guinea pigs infused with DMBA in fat emulsion in which topical sensitization with DMBA was attempted.

Benzo[alpha]pyrene antibody inhibition of benzo[alpha]pyrene-induced mutageneis.

An antibody to benzo[alpha]pyrene (BP) was prepared. The isolated antibody showed a specificity for BP and a low reactivity with another carcinogenic hydrocarbon, 7,12-dimethylbenz[alpha]anthracene (DMBA). The BP-antibody inhibited the in vitro cytotoxic and mutagenic activity of BP in both a rat embryo fibroblast- and a rat lung cell-mediated mutagenesis system. A possible correlation of these in vitro findings to the in vivo carcinogenesis situation is discussed.

Reduction of respiratory tract binding of benzo[a]pyrene in mice by immunization.

Male inbred A/J mice immunized by combined ip and intranasal administration of a bovine serum albumin conjugate of 5-fluoro-12-methylbenzanthryl-7-acetic acid developed tracheal antibodies capable of binding the carcinogen benzo[a]pyrene (BP). Immunized mice administered 92 ng of [3H]BP intranasally exhibited a one-third reduction in BP content in respiratory tract tissues (nose and trachea) when compared with control mice 20 hours after BP administration.

Expression of an early, nonstructural antigen of herpes simplex virus in cell transformed in vitro by herpes simplex virus.

Hyperimmune rabbit antiserum to an early, nonstructural herpes simplex virus type 2 (HSV-2)-induced polypeptide (VP143) reacted in immunofluorescence tests with a variety of cell lines transformed by HSV-2. Cytoplasmic fluorescence was observed in 10 to 50% of HSV-2-transformed cells, whereas no fluorescence was observed in cells transformed by other oncogenic DNA viruses or by a chemical carcinogen. VP143-specific reactivity could be absorbed from anti-VP143 serum with HSV-2-transformed cells but not with cells transformed by other agents. When HSV-2-transformed cells were synchronized in mitosis and examined at various times postmitosis for VP143-specific fluorescence, the expression of VP143 was shown to be cell cycle dependent.

Initiation-promotion skin carcinogenesis and immunological competence.

The immune competence of mice during initiation-promotion skin carcinogenesis was determined by skin allograft rejection and lymphocyte mitogenesis. The carcinogen 7, 12-dimethylbenzanthracene inhibited the cellular immune competence of mice while lymphocytes from croton oil treated mice had enhanced PWM response. Chlorphenesin, a stimulator of cellular immunity, was found to inhibit tumorigenesis in initiation-promotion skin carcinogenesis when injected during promotion.