Exposure of murine skin to low doses of ultraviolet‐B (UVB) radiation before sensitization with hapten reduces the ability of antigen presenting cells (APC) in the draining lymph nodes to initiate contact hypersensitivity responses in vivo and results in the induction of hapten‐specific suppressor T cells. In the present study, we tested the hypothesis that exposure of skin to UVB radiation suppresses T cell responses to hapten in vivo by altering the functions of APC, resulting in decreased stimulation of Th1 lymphocytes, which mediate contact hypersensitivity responses, and preferential activation of Th2 cells. C3H/HeN mice were exposed to either a single 2 kJ/m2 dose of UVB or to 400 J/m2 of UVB daily from FS40 sunlamps for four consecutive days and sensitized with fluorescein isothiocyanate on UV‐irradiated skin. Draining lymph node cells were collected 18 h after sensitization and co‐cultured with nylon wool‐purified T cells from naive or fluorescein‐immunized mice. Unseparated lymph node cells or sorter‐purified fluorescein‐bearing APC from UV‐irradiated mice induced less T cell proliferation than APC from non‐UV‐exposed mice. Lymph node cells produced less Th1 and Th2‐associated cytokines, interferon‐gamma and interleukin‐4, respectively, in response to APC from UV‐irradiated animals compared with APC from unirradiated, fluorescein‐sensitized mice. Thus, low doses of UV radiation do not result in preferential stimulation of Th2 response in lymph nodes, and results from cloned cell lines may incompletely reflect T cell responses in vivo.

Ear/footpad swelling following sensitization and challenge with 2,4-dinitrofluorobenzene (DNFB)/allogenic splenocytes (AS) were used to monitor the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on contact hypersensitivity (CHS) and delayed hypersensitivity (DHS) reactions, respectively. Topical treatment of dorsal or ventral SENCAR mouse skin 4x with 2 micrograms of TPA prior to sensitization of dorsal skin with DNFB suppressed attempts to induce CHS by subsequent challenge with DNFB. The adoptive transfer of splenocytes isolated from mice pretreated on the dorsum with TPA prior to dorsal sensitization with DNFB inhibited the development of CHS to DNFB in recipient mice. Conversely, topical treatment with TPA prior to s.c. sensitization with AS neither suppressed subsequent attempts to induce DHS, nor resulted in the generation of a splenocyte population capable of suppressing DHS reactions in adoptive transfer studies. Thus, promoting doses of topically applied TPA has differential effects on CHS and DHS reactions.

Reduced/oxidized glutathione (GSH/GSSG) and the activities of catalase (CAT), Superoxide dismutase (SOD), glutathione peroxidase (GPX) and xanthine oxidase (XO) were quantitated in initiated SENCAR mice being promoted with 1 μg of 12-O-tetradecanoylphorbol-13-acetate (TPA). Within 24 hr of 4 or 10 applications of TPA epidermal SOD, CAT, GPX and XO specific activities were∼56, 39, 76 and 350

Several structurally unrelated classes of chemicals defined as promoters in the murine skin multistage carcinogenesis protocol were surveyed for their abilities to modify contact hypersensitivity (CHS) responses in SENCAR mice. Sensitization of dorsal skin with 2,4-dinitrofluorobenzene (DNFB) and subsequent challenge of ears 5 days later with DNFB resulted within 24 h in ear swelling. Pretreatment of dorsal skin with multiple applications (2 x/week for 2 weeks) of promoting doses of 12-O-tetradecanoylphorbol-13-acetate (TPA), anthralin, butylated hydroxytoluene hydroperoxide, n-dodecane and ethyl phenylpropionate (EPP) prior to sensitization with DNFB inhibited, to a comparable extent, the subsequent induction of CHS by DNFB challenge. Pretreatment of dorsal skin with promoting doses of benzoyl peroxide resulted in reproducible, but diminished suppression of CHS, relative to that mediated by the other chemical promoters. Application of promoting doses of TPA, anthralin and EPP, but not the other chemicals, to ventral skin prior to DNFB sensitization of dorsal skin also significantly inhibited DNFB-induced CHS. However, suppression of CHS mediated by ventral application of these three chemicals was quantitatively less than that occurring when the chemicals were applied to the site of DNFB sensitization. Collectively, these studies demonstrate that various classes of structurally unrelated tumor promoters have in common the ability to suppress CHS, a cell-mediated immune response. Furthermore, some tumor promoters exert their suppressive effects through both local and systemic processes.

Tissue glutathione (GSH) and glutathione disulfide (GSSG) contents were quantitated in the skins of female SENCAR mice following the topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), and in the skin tumors generated by an initiation-promotion protocol. Total epidermal GSHt (GSH + GSSG) and GSSG contents were not reproducibly and significantly altered 0.5, 4 or 24 h after one or four topical applications of 1 microgram TPA, relative to the values obtained in age-matched, solvent-treated mice. Similar findings held for dermal GSHt at all times of analyses, and for dermal GSSG contents 0.5 and 4 h after TPA application. However, dermal GSSG contents were slightly elevated 24 h after TPA application. The GSHt and GSSG contents of skins initiated with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and harvested 17, 29 and 37 days after the cessation of chronic treatment with acetone (14 weeks, twice a week) were comparable to the values measured in age-matched, non-treated skins. In contrast, GSHt contents of papillomas harvested 17, 29 and 37 days after the cessation of chronic treatment with 1 microgram TPA (14 weeks, twice a week) were 2- to 4-fold greater than the values measured in non-treated mice, and DMBA-initiated, acetone-promoted mice, and the non-tumorous tissue adjacent to the papillomas. Comparable changes did not occur in papilloma GSSG contents. GSHt contents in squamous cell carcinomas (SCC) were twice the values measured in papillomas and 5- to 8-fold greater than the values measured in non-treated skins, and the non-tumorous tissue adjacent to SCC. Similarly, GSSG contents in SCC were elevated multifold relative to papillomas, non-treated skin and the non-tumorous tissue adjacent to SCC. Epidermal cell suspensions prepared by the trypsin-flotation procedure retained less than 2% of their original GSHt content and had reduced GSHt/GSSG ratios. Collectively these studies suggest that (i) if promoting doses of TPA induce oxidative stress in murine epidermis, it cannot be detected by measurements of GSH/GSSG; (ii) the antioxidant capacity of epidermal cells prepared by the trypsin-flotation procedure is severely compromised; and (iii) GSHt contents progressively increase during skin tumor ontogeny.

A procedure was developed for the per cell estimation of catalase activities in suspensions and cultures of murine epidermal keratinocytes (MEKs). Per cell catalase activity in MEKs cultured in low Ca2+ medium was relatively constant during the proliferation phase of culturing, but increased approximately 100% within 24 h of cessation of cell division. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment of proliferating MEKs cultured in low Ca2+ medium resulted in (i) an initial suppression of proliferation, (ii) the accelerated detachment and differentiation of detached MEKs and (iii) a suppression of catalase induction in the detached population. Induction of MEK differentiation by raising the medium Ca2+ concentration resulted in rapid inhibition of cell division and approximately 200% increases in per cell catalase activities. Addition of TPA immediately prior to Ca2+ shift completely suppressed the Ca2(+)-dependent increases in activity. However, the addition of TPA 48 h after the induction of differentiation by Ca2+ shift had no effects on the elevated, pre-existing catalase activities. Per cell catalase activities varied in vivo with the stage of MEK differentiation. Specifically, the lowest and highest per cell activities (approximately 4-fold difference) were measured in enriched basal cell and spinous cell populations respectively. Catalase activity in the more differentiated MEKs was reduced approximately 33% within 24 h of topical treatment of dorsal skin with a promoting dose of TPA. However, catalase activity in enriched basal cell preparations was unaffected. Collectively, these studies demonstrate that per cell catalase activities increase as MEKs differentiate, and that TPA suppresses the increases in catalase activities that normally occur during differentiation.