Gary M. Halliday

Quantitative Assessment of Langerhans Cells in Actinic Keratosis, Bowen's Disease, Keratoacanthoma, Squamous Cell Carcinoma and Basal Cell Carcinoma

&NA; The quantitative distribution of Langerhans cells (LC) was studied in a range of pre‐neopiastic, in‐situ and invasive neoplastic skin lesions using an antibody to S100 protein and the indirect immunoperoxidase technique. LC numbers were increased within the lesions of actinic keratosis, Bowens disease, keratoacan‐thoma, squamous cell carcinoma and basal cell carcinoma. In all lesions except actinic keratosis the LC density was also significantly increased in the adjacent non‐neoplastic epithelium. The increased LC density in neoplastic epithelium suggests either that LC are being retained within the abnormal epithelium for longer periods of time than normal or that increased numbers of LC are being actively attracted by factors produced by the neoplastic epithelium. While reduction of intraepithelial LC density may allow the initiation of neoplasia the increased density observed in this study suggests that at later stages of tumour growth LC may have a functional role in the host response to cutaneous neoplasia.

Induction of tolerance via skin depleted of langerhans cells by a chemical carcinogen

Since treatment of mouse skin with the chemical carcinogen 7,12-dimethylbenz[alpha]anthracene (DMBA) substantially decreases the density of cutaneous Langerhans cells (LC), the immune status of mice sensitized to 2,4-dinitrofluorobenzene (DNFB) through DMBA-treated skin was investigated. Mice did not develop contact sensitivity to DNFB when applied to DMBA-treated dorsal trunk skin, whereas sensitization resulted when DNFB was applied to untreated abdominal wall skin. Mice immunized with DNFB via DMBA-treated skin did not respond to subsequent immunization through untreated dorsal trunk skin, demonstrating the generation of suppressor cells which could inhibit the activation of effector lymphocytes. Adoptively transferred spleen cells from mice immunized with DNFB through DMBA-treated skin inhibited the response of sensitized hosts, indicating the presence of efferently acting suppressor cells which could inhibit the function of effector lymphocytes. This study has demonstrated that sensitization via skin depleted of LC by chemical carcinogen treatment induces an active state of tolerance rather than immunity.

Sensitization through carcinogen-induced Langerhans cell-deficient skin activates specific long-lived suppressor cells for both cellular and humoral immunity

Application of 2,4-dinitrofluorobenzene (DNFB) to BALB/c mouse skin depleted of epidermal Langerhans cells (LC) by the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) activated cells which suppress both contact sensitivity and antibody production when transferred into naive host mice. Tolerance was induced by a concentration of DNFB optimal for inducing contact sensitivity in solvent-treated control mice. The cellular and humoral responses of hosts to a second antigen, 2,4,6-trinitrochlorobenzene (TNCB), were unaffected by these suppressor cells, demonstrating specificity for DNFB. Suppressor cells for cellular and humoral immunity could still be demonstrated 6 months following activation, by which time some mice had died, presumably of old age. The dose responses to sensitizer for generation of cells which suppressed contact sensitivity and antibody production differed, indicating that separate populations of suppressor cells probably inhibit these responses. Hence, during cutaneous chemical carcinogenesis, depletion of LC may allow activation of specific long-lived suppressor cells capable of inhibiting cellular or humoral antitumor immune responses.

Effects of low or high doses of short wavelength ultraviolet light (UVB) on Langerhans cells and skin allograft survival

Since Langerhans cells (LC) are normally the only cells within the epidermis to express the class II major histocompatibility complex (MHC) transplantation antigens, depletion of LC could be expected to prolong skin allograft survival by reducing the antigenic disparity between host and recipient. To assess this hypothesis, donor C57BL mouse shaved dorsal trunk or tail skin was exposed to high (200 mJ/cm2) or low (40 mJ/cm2) doses of short wavelength ultraviolet light (UVB) before grafting on to the thorax of BALB/c mouse recipients of the same sex. These strains have different major and minor transplantation antigens. The effects of UVB treatments on LC were determined by electronmicroscopy. Skin grafted 1–14 days following a single high dose of UVB irradiation was ultrastructurally depleted of LC and survived significantly longer than unirradiated skin before being rejected. After a 21‐day interval between exposure and grafting when LC were again present in the epidermis there was no significant difference between treated and control graft survival. Exposure to low dose UVB irradiation only significantly increased graft survival for skin transplanted 1–3 days after irradiation; skin grafted 4 days following irradiation survived for a similar period to unirradiated control skin grafts. Electronmicroscopy showed that the low UVB dose did not deplete LC from the epidermis. We conclude that after low dose UVB treatment the class II MHC antigens on the LC plasma membrane were lost temporarily, thus prolonging graft survival, but when the plasma membrane antigens were re‐expressed graft survival returned to normal. In contrast, high‐dose UVB irradiation prolonged graft survival by depleting LC from the epidermis, with graft survival only returning to control values as LC repopulated the epidermis.

Antigen presented in the local lymph node by cells from dimethylbenzanthracene-treated murine epidermis activates suppressor cells

Application to skin depleted of LC by treatment with the chemical carcinogen DMBA of a dose of contact sensitizer optimal for inducing contact sensitivity activates transferrable suppressor cells. Excision of solvent- or DMBA-treated skin at various times following application of the contact sensitizer DNFB indicated that the fraction of antigen which leaves the skin within the first few hours induces tolerance. An initial signal inducing unresponsiveness, observed within 1/2 hr, was overturned 3-6 hr later. A more permanent tolerogenic signal in the DMBA- but not solvent-treated lymph node resulted from an epidermal cell from DMBA-treated skin presenting antigen to suppressor cells. Therefore it is likely that suppressor cells are activated in DMBA-treated mice by an epidermal cell which migrates to the local lymph node. Local lymph node cells from DMBA-treated mice also have a diminished ability to present antigen in vivo but they do not activate suppressor cells.

Langerhans Cell Presentation of Sheep Red Blood Cells Induces Antibody Production

Mouse epidermal Langerhans cells (LC) were found to be capable of presenting sheep red blood cells (SRBC) to immune spleen lymphocytes, resulting in antibody production as asessed by a direct plaque cell assay. Spleen lymphocytes, prepared by removal of adherent cells, were unable to respond to SRBC in a 5‐day culture; however, when co‐cultured with either epidermal cells or splenic adherent cells, a response to SRBC occurred, as demonstrated by an increase in the number of antibody‐producing cells. The LC were shown to be the antigen‐presenting cells in these cultures, as depletion of LC via their Fc receptors abrogated the response. A similar reduction in the number of epidermal LC added to cultures likewise failed to induce antibody production. These experiments demonstrate that LC are able to present complex cellular antigens to the immune system, resulting in a B lymphocyte response.

Langerhans cell depletion in gliotoxin-treated murine epidermis

&NA; Langerhans cells (LC) are dendritic antigen presenting cells of bone marrow origin which reside in the suprabasal layer of the epidermis. They express high concentrations of Class II MHC glycoproteins on their plasma membrane and transport cutaneous antigen to local lymph nodes for presentation to helper T cells. They are thus essential for the induction of cutaneous immunity. Gliotoxin is a member of the epipolythiodioxopiperazine (ETP) group of fungal metabolites, derived from the human pathogen Aspergillus fumigatus. It has been shown to have immunomodulating properties in vivo and in vitro, and has been proposed as a potential immunosuppressant for transplantation therapy. Epicutaneous application of gliotoxin reduced the numbers of epidermal LC by 30–35 per cent with an associated morphological change from highly dendritic to a more rounded form. Electron microscopic studies showed selective damage to LC at very low (nM) concentrations of gliotoxin, with no obvious effect on adjacent keratinocytes. LC numbers remained depleted for 13 weeks after initial treatment, suggesting that systemic suppression or prolonged retention of gliotoxin within the skin may play a role in its mechanism of action.

Acceptance of class II major histocompatibility complex disparate skin grafts associated with suppressor cells and elevated langerhans cell numbers

Summary Class II major histocompatibility complex (MHC) molecules are only present on Langerhans cells (LC) in normal murine epidermis. Depletion of this antigen with the chemical carcinogen dimethylbenzanthracene (DMBA) causes i‐E disparate B10.A(2R) congenic tail skin to be accepted permanently when grafted onto B10.A(4R) recipients. Adoptive transfer of spleen cells from these recipients into naive syngeneic hosts inhibited the ability of the host mice to reject untreated B10.A(2R) tail skin grafts. Hence Dmba‐treated LC depleted l‐E disparate skin grafts activate suppressor cells which did not inhibit Balb/c mice from rejecting a B10.A(2R) tail skin graft. In contrast, the tobacco derived carcinogen benzo(a)pyrene (BP) increased the number of epidermal LC but had no effect on either class I or class II Mhc disparate skin graft survival time. This confirms that the number of class II MHC‐positive LC is critical for the initiation of skin graft rejection; when the threshold level is attained graft rejection proceeds at a maximal rate that cannot be enhanced by raising the number of LC. The tolerant skin grafts had increased numbers of LC; this was not observed in syngeneic grafts and therefore may be related to the active suppression of immunity.