Effects of short-wave ultraviolet radiation (UV-C) on delayed hypersensitivity in the guinea-pig

Guinea-pigs, previously sensitized to dinitrochlorobenzene (DNCB), were exposed to varying doses of UV-C radiation (254 nm) on one of the flanks for a period of 15 days. They were then patch-tested with DNCB immediately after UV treatment. The responses to DNCB were diminished both in the irradiated and non-irradiated skin compared with control animals which had not received UV-C radiation. This effect was dose dependent and could only be demonstrated for a limited period of time. Guinea-pig exposed to UV-C radiation did not show a decreased response to sodium lauryl sulphate, which is a potent irritant. The influence of UV-C on delayed hypersensitivity therefore seems to be an immunological effect.     

Cytochemical and Ultrastructural Studies of the Langerhans'' cells

In the guinea pig, experimental allergic contact dermatitis (ACD) and primary irritant contact dermatitis (PICD) were induced with different concentrations of dinitrochlorobenzene (DNCB). The epidermal Langerhans' cells (LCs) were observed sequentially by both adenosine triphosphatase (ATPase) and electron microscopy. Light microscopically, in ACD, the density and dendritic processes of LC decreased markedly within 12 h after antigen challenge. Almost no recognization LCs could be seen within 2 to 5 days. Later, LCs began to repopulate in the epidermis. Within 14 days, the density and shape of the LCs returned to normal. On the contrary, LCs changed more rapidly in PICD. The dendritic processes of LC decreased within 2 h and cell density decreased dramatically within 6 h after DNCB application. LCs also repopulated more rapidly in the epidermis. Electron microscopically, in ACD, we observed that lymphocyte-like cells apposed to LCs; LCs were activated and damaged; however, in PICD, we found neither the apposition of lymphocyte-like cells to LCs, nor the activation of LCs. LCs play an important role in the convalescence phase as well as in the early and later phases of contact allergic reaction.     

Cytochemical and Ultrastructural Studies of the Langerhans' Cells

Abstract: In the guinea pig, experimental allergic contact dermatitis (ACD) And primary irritant contact dermatitis (PICD) were induced with different concentrations of dinitrochlorobenzene (DNCB). The epidermal Langerhans' cells (LCs) were observed sequentially by both adenosine triphosphatase (ATPase) and electron microscopy. Light microscopically, in ACD, the density and dendritic processes of LC decreased markedly within 12 h after antigen challenge. Almost no recognization LCs could be seen within 2 to 5 days. Later, LCs began to repopulale in the epidermis. Within 14 days, the density and shape of the LCs returned to normal. On the contrary, LCs changed more rapidly in PICD. The dendritic processes of LC decreased within 2 h and cell density decreased dramatically within 6 h after DNCB application. LCs also repopulated more rapidly in the epidermis. Electron microscopically, in ACD, we observed that lymphocyte-like cells apposed to LCs; LCs were activated and damaged; however, in PICD, we found neither the apposition of lymphocyte-like cells to LCs, nor the activation of LCs. LCs play an important role in the convalescence phase as well as in the early and later phases of contact allergic reaction.     

Role of Langerhans cells in epidermotropism of T cells

Summary Certain T lymphocytes display a specific affinity for the epidermis (epidermotropism). Recent studies have suggested that Ia⁺ Langerhans cells (LCs) are possible targets for the epidermotropism. A variety of self-Ia-reactive cloned T cells were tested for their ability to migrate into the epidermis following intradermal inoculation into the footpads of syngeneic mice. Clone BB5 was chosen as representative of the epidermotropic T cells. We investigated whether the depletion of Ia⁺ LCs from the epidermis by tape-stripping could alter the migration of BB5 cells into the epidermis. The epidermal invasion of BB5 cells was markedly impaired in those mice whose LCs were depleted by 95% after repetitive tape-stripping. Because production of epidermal-derived thymocyte activating factor (ETAF) by the epidermal cells was augmented after repetitive tape-stripping, the diminished migration of BB5 cells into tape-stripped epidermis did not result from a decrease in ETAF production which is thought to attract T cells chemotactically. These results suggest that Ia⁺ LCs may play an inductive role in the preferential migration of T cells into the epidermis.     

Studies on the retest reaction in contact sensitivity to DNCB

DNCB-sensitized guinea pigs demonstrated an accelerated reactivity on retest of DNCB at the site of prior contact reaction, though presenting normal contact sensitivity at the virgin site. The retest reaction reached maximal at 9 h and waned at 24 h after antigenic challenge. Massive accumulation of eosinophils in either the epidermis or dermis was its distinguishing histologic feature. The reaction was induced at the site of delayed skin reaction to DNP-GPE in the animals sensitized with DNCB or DNP-GPE. A retest reaction in delayed sensitivity to DNP-GPE was also elicited at the site of contact reaction to DNCB in the animals. The significance of these findings is discussed.     

Effects of immunological responsiveness on Langerhans cell behavior in contact sensitization

Abstract The epicutaneous application of haptens results in a functional activation of the antigen-presenting Langerhans cells (LCs) which is necessary for the induction of contact sensitivity. In this ultrastructural study, We investigated the effects of the immune response on these cellular properties of the LCs by using 2 strains of guinea pigs with genetically determined high and non responsiveness, respectively, to the strong sensitizer 2,4-dinitrochlorobenzene (DNCB). After skin painting, both strains showed a similar cellular and endocytotic activation of the LCs and a similar intraepidermal localization of DNCB on immunoelectron microscopical visualization. There were however few LC-lymphoid cell interactions in the non responders, in contrast to numerous such appositions in the other strain. Intravenous tolerization with 2,4-dinitrobenzene-l-sulfonic acid, which is known to block the DNCB receptor of T cells, hampered the lymphoid cell interactions in the DNCB treated high responders, but it did not affect the LC activation. These in vivo observations demonstrate that the hapten-induced changes of the LC properties is the initial, T-cell independent event in contact sensitization.     

Quantitative study of the number of Langerhans cells per surface unit in allergic contact eczema

The key role of the Langerhans cell (LC) in the immune response and particularly in allergic contact dermatitis is well documented. At first, we determined in the epidermis of the forearm by a non-sensitized adult volunteer, the variations in the density of LC (number/mm2), following the application of various chemicals: nickel sulphate 5 p. 100; erythromycin base 2 p. 100, 2,4-dinitro-1-chlorobenzene (DNCB) 0.02 p. 100 in white petrolatum. Measurements were made at 6, 24 and 48 hours. In a second stage, the volunteer was sensitized to DNCB and a similar study was performed after application of DNCB 0.02 p. 100 at 6 and 24 hours. Normal skin was used as control. The epidermis was obtained by using the suction blister method: LC were identified by the enzymatic ATPase technique and by the immunocytological OKT6 technique. Surfaces were determined by means of a computerized digital tablet. Results obtained with the ATPase technique cannot be interpreted. The density of LC determined by the OKT6 is not significantly modified when the various chemicals are applied on the skin of the non-sensitized volunteer. After sensitization to DNCB, the number of LC is significantly increased after 6 hours of application of DNCB 0.02 p. 100 but not after 24 hours. Further studies are needed to confirm these preliminary observations.     

Impaired contact hypersensitivity in untreated psoriasis and the effects of photochemotherapy and dithranol/UV-B

Cell mediated immune reactivity was studied in eighty-five psoriasis patients and twenty-five healthy controls by an improved (quantitative) method for measuring contact hypersensitivity to 2,4 dinitrochlorobenzene (DNCB). Patients were sensitized with 500 μg DNCB, the lowest dose found to sensitize all healthy subjects. Responses to epicutaneous challenge with a series of concentrations of DNCB were measured as volumes calculated from diameter and thickness and the various groups were compared by the differences in the log dose-response curves. Patients with untreated psoriasis were less responsive than healthy controls; responses were less still in patients treated with dithranol/UV-B/tar and they were least of all in patients treated with photochemotherapy with 8-methoxypsoralen and UV-A (PUVA), particularly in those who pigmented least. Sensitization and challenge at different stages of treatment showed that both induction and elicitation of sensitization were impaired by PUVA. The possible relationship of these changes to DNCB metabolism, Langerhans cell damage and a decrease in circulating T cells is discussed. Although the clinical significance of the findings is unknown, treatment with dithranol/UV-B/tar has proved safe over many years of use.     

紫外线、补骨脂素加长波紫外线、境界线对皮肤接触过敏的影响

DNCB致敏豚鼠.分成3组,于背部一侧剃毛后皮肤分别应用治疗剂量UVR,GR照射、PUVA疗法,每周2-3次.共调.然后,于照射处和对称部位的非照射处皮肤进行DNCB激发试验和切取皮肤作Laagerhans细胞检查.结果表明:3种物理因子均可抑制DNCB引起的皮肤接触过敏反应.与此同时,Langerhans细胞的数量亦明显减少,试验的结果进一步证明皮肤接触过敏的发病与Langerhans细胞密切有关.同时提示了UVR,PUPA和GR可用于接触性过敏性皮炎的治疗.     

Topical application of cyclosporine on guinea pig allergic contact dermatitis

Topical cyclosporine applied to the test site substantially inhibited the elicitation reaction of contact sensitivity in the guinea pigs previously sensitized with 2,4-dinitrochlorobenzene (DNCB). This suppressive effect of the drug was short lived and reversible. Cyclosporine was not effective when given six hours or later after antigenic challenge to the test site. Cyclosporine had no effect on the toxic contact reaction in normal animals either to croton oil or to DNCB in high concentration. Cyclosporine applied topically to the challenge site also resulted in a reduction of retest and flare-up reactions of contact sensitivity to DNCB, but did not affect the production of generalized rash in the same animals. These results indicate that in the future local topical application of cyclosporine may make treatment of human cutaneous immune-mediated disorders a possibility without serious side effects.     

Psoriasis therapy. The effect of UV radiation on sensitization to mechlorethamine

A study was performed to determine the effect of UV-B radiation on the sensitization of patients to topically applied mechlorethamine hydrochloride. Patients with widespread psoriasis were randomized into two groups. One group was given six daily treatments of one minimal erythemal dose (MErD) prior to starting daily applications of a 0.02% mechlorethamine ointment and, thereafter, one MErD weekly; the other group was given daily applications of mechlorethamine but received no UV-B. The improvement of psoriasis was monitored by a severity score system and patients were treated for 30 days, or until contact dermatitis occurred. The addition of UV-B to topical applications of mechlorethamine reduced the incidence of allergic contact dermatitis to mechlorethamine from 64% to 22%. In those sensitized to mechlorethamine, the time required for sensitization to develop was increased from 14 to 23 days by UV-B therapy.     

Histological changes produced in skin by equally erythemogenic doses of UV-A, UV-B, UV-C and UV-A with psoralens

The sequential light microscopic histological changes produced in human skin by a single exposure of UV-A, UV-B, UV-C and oral 8-methoxypsoralen plus UV-A (PUVA) causing approximately equal degress of delayed erythema response, have been evaluated. UV-C and UV-B affect the epidermis to a greater degree than UV-A, while UV-A affects the dermis to a greater degree than UV-B and UV-C. PUVA has prominent effects on both epidermis and dermis, differing in degree from those changes induced by UV-A, UV-B, and UV-C and are longer lasting. The sequence of histological changes following UV exposure is completed more rapidly after exposure to shorter UV wavelengths.     

咪唑斯汀等几种抗组胺药对小鼠接触性皮炎作用的实验研究

目的：观察几种抗组胺药对小鼠接触性皮炎的疗效及对表皮朗格汉斯细胞(LCS)数目的影响。方法：制作小鼠接触性皮炎模型,测量用药各组鼠左耳中部厚度变化及应用ABC免疫组化法测定LCS的数目。结果：对诱发皮炎前、后24h鼠耳厚度差的比较,咪唑斯汀组作用较强;各实验组与对照组比较,OX4 LCs、OX3*LCs均明显减少。结论：几种抗组胺药对小鼠迟发型超敏反应均有抑制作用,咪唑斯汀的作用较强;几种抗组胺药可使表皮LCs数目呈不同程度的减少;抗组胺药对迟发型超敏反应的抑制作用可能与LCs的数目减少有关,LCs数目的减少可能是其抑制迟发型超敏反应的机制之一。     

A comparative study of allergic and primary irritant contact dermatitis with dinitrochlorobenzene (DNCB) in dogs.

Attempts were made to induce allergic contact dermatitis in dogs, a species generally considered poorly responsive to experimental allergic contact dermatitis. Yound Beagles were sensitized to 2,4 dinitrochlorobenzene (DNCB) by multiple intradermal injections. Two weeks after sensitization, these dogs were challenged topically with 0.1% DNCB by a standard closed-patch technique. Sensitization evidenced by various degrees of reaction following challenge was established in all of 14 pups used, while 7 nonsensitized control pups did not react to challenge. Primary irritant contact dermatitis was induced in the skin of nonsensitized Beagle pups by 1%, 5%, and 10% solutions of DNCB. In allergic contact dermatitis the sites of challenge were grossly indurated, erythematous, and edematous. Histologically at these sites there was an infiltration of mononuclear cells which reached maximum intensity at 3 to 4 days. Accumulations of lymphoid cells were marked around sweat galnds and hair follicles. Penetration of leukocytes into these cutaneous adnexa was associated with degenerative processes in their cellular structures. Mononuclear cell infiltration into the epidermis was mild. Spongiosis was observed in the epidermis, but vesicle formation was rare. In primary irritant contact dermatitis gross lesions were characterized by severe erythema, edema, and gangreen of the skin. Microscopically, the main lesions were necrosis of the epidermal cells, separation of the epidermis from the dermis, dermal edema, and massive infiltration of the dermis with polymorphonuclear cells.     

Biology of Langerhans cells: analysis by experiments to deplete Langerhans cells from human skin

In vivo studies have demonstrated that various treatments of skin, e.g., UV irradiation, topical corticosteroids, and tape-stripping, will temporarily deplete the epidermis of Langerhans cells (LC). Whether this loss represents simply a loss of cell surface markers unique to LC, or actual depletion of cells, is unknown. By design, normal human skin transplanted to the congenitally athymic (nude) mouse is a system devoid of circulating precursors for human LC. Because LC have been shown to be of bone marrow origin, any depletion of these cells in this system should be permanent. Treatments to deplete LC from human skin grafts on nude mice after grafting included: (a) large doses of UV radiation (400 mJ/cm2 every 48 h, X 3), (b) potent high-dose topical corticosteroids (2.5 mg betamethasone valerate/cm2 every day, X 5), (c) tape-stripping (X 20). Treatments before grafting included: (a) treating donor skin with 900 R of gamma irradiation, (b) complement fixing monoclonal antibody to Ia-like antigens of LC, followed by fresh complement, (c) monoclonal antibody conjugated to toxins. Quantitation of the number of LC was analyzed on control and treated epidermal sheets using immunodiagnostic reagents, anti-HLA-DR, and surface ectoenzymes , ATPase. Results show that both UV irradiation and topical corticosteroids reduce the number of LC by these analyses. However, within 3 weeks, recovery to pretreatment levels has occurred. X-irradiation and tape-stripping were without effect. Despite evidence that the monoclonal antibody, complement, and toxic systems were delivered to the LC within the epidermis, there is no evidence that these treatments resulted in a decrease in LC. It appears that LC are currently either long-lived or replaced locally from a proliferative pool and that certain cell membrane determinants of human LC are somewhat differentially sensitive to UV radiation and corticosteroids.     

Development of intravital intermittent confocal imaging system for studying Langerhans cell turnover

Although several studies have suggested relatively slow turnover of Langerhans cells (LCs), their actual lifespan remains elusive. Here we report the development of a new intravital imaging system for studying LC efflux and influx. Epidermal LCs expressing enhanced green fluorescent protein (EGFP) were visualized in anesthetized I-Abeta-EGFP knock-in mice by confocal microscopy. By overlaying two sets of EGFP+ LC images recorded in the same microscopic fields at time 0 and 24 hours later, we identified LC subpopulations that had disappeared from or newly emerged in the epidermis during that period. Of >10,000 LCs analyzed in this manner, an overwhelming majority (97.8+/-0.2%) of LCs showed no significant changes in the x-y locations, whereas 1.3+/-0.1% of the LCs that were found at time 0 became undetectable 24 hours later, representing LC efflux. Conversely, 0.9+/-0.1% of the LCs that were found at time 24 hours were not detectable at time 0, representing LC influx. From these frequencies, we estimated the half-life of epidermal LCs to range from 53 to 78 days, providing new insights into the immunobiology of LCs. Our intermittent imaging approach may be regarded as a technical breakthrough enabling direct visual assessment of LC turnover in living animals.     

Numerical, morphological and phenotypic changes in Langerhans cells in the course of murine graft-versus-host disease

BACKGROUND: In the course of graft-versus-host disease (GVHD) or diseases that histologically mimic GVHD (e.g. toxic epidermal necrolysis, Stevens-Johnson syndrome), it is known that epidermal Langerhans cells (LCs) are depleted from the epidermis. However, the mechanism and significance of LC depletion is not well known. OBJECTIVES: To investigate the numerical, morphological and phenotypic changes in LCs and apoptosis of LCs in the course of GVHD using a non-irradiated mouse GVHD model. METHODS: BALB/c nu/nu mice and C57BL/6 mice were used as recipients and donors, respectively. Recipient mice were injected with T-cell-enriched donor spleen cells. Skin samples were harvested at various times after the inoculation. The numerical and morphological changes were examined by an immunofluorescence study of epidermal sheets. Apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling method and flow cytometric analysis using annexin V. Phenotypic change was studied by flow cytometric analysis of epidermal cell suspensions. The mixed epidermal cell lymphocyte reaction (MELR) was performed to examine functional changes in the epidermal cells. RESULTS: Five days after inoculation, a graft-versus-host reaction occurred. Epidermal LCs began to decrease from the sixth day. On the fifth day, the LCs became larger and had prominent dendrites. Immediately before the LCs began to decrease, many LCs became round in shape, with scanty dendrites. LC apoptosis was not observed in the epidermis either on the fifth or seventh day. Phenotypically, the expression of CD40, CD80, CD86 and major histocompatibility complex class II antigen on the LCs was upregulated on the fifth and seventh day. Epidermal cells from GVHD mice showed an increased allostimulatory capacity in the secondary MELR. CONCLUSIONS: These results suggest that at early GVHD onset, most LCs may not undergo apoptosis in the epidermis but are phenotypically activated, resulting in further activation of alloreactive T cells and aggravation of the disease.     

Effects of PUVA on delayed hypersensitivity in the guinea-pig

Guinea-pigs previously sensitized to dinitrochlorobenzene (DNCB) were topically treated with 8-methoxypsoralen solution and longwave ultraviolet light (PUVA). The animals showed moderate or no clinical and microscopic responses to an elicitation dose of DNCB given immediately after the period of treatment. Exposure to ultraviolet light or psoralen alone did not affect the allergic response. Ten days after PUVA treatment there was a normal eczematous reaction after application of DNCB.     

Subtoxic concentrations of allergenic haptens induce LC migration and maturation in a human organotypic skin explant culture model: a novel method for identifying potential contact allergens

The accelerated migration of Langerhans cells (LCs) out of the epidermis and up-regulation of maturation markers, upon treatment with subtoxic concentrations of chemicals, were used as the criteria to determine the potential of allergenic chemicals capable of inducing a hapten-specific delayed-type hypersensitivity reaction. Here we report the findings of a study in which seven chemicals, coded and tested in a blind fashion, were classified as contact allergens or non-allergens using the human organotypic skin explant culture (hOSEC) model. All chemicals that were identified as a contact sensitizer on decoding induced a definite decrease in the number of CD1a and HLA-DR-positive epidermal LCs in the epidermis of the skin explants, as determined by both semiquantitative immunohistochemistry and quantitative flow cytometric analysis. A significant increase in the number of CD83(+) cells was accompanied by up-regulation of activation molecules in the epidermis of hOSEC exposed specifically to contact allergens. In contrast, there were only minor alterations in epidermal LC numbers, expression of CD83 and other activation markers by LCs when the biopsies were treated with non-toxic concentrations of non-allergenic irritants and vehicles. The data suggest that an increased epidermal LC migration and maturation accompanied by increased expression of activation markers could be used as end-point determinants to screen allergens in a non-animal alternative hOSEC model.     

The suppressive effect of tape-stripping treatment of guinea-pig skin on the induction of contact sensitivity by intradermal injection of haptenated epidermal cells

Summary Contact sensitivity (CS) to 2,4-dinitrochlorobenzene (DNCB) was produced in inbred JY1-strain guinea pigs by the intradermal injection of epidermal cells (ECs) prepared from DNCB-painted skin (DNP-ECs). When the site of DNP-EC-induced CS was pretreated by tape stripping, the rate and intensity of the challenge reactions to DNCB were diminished. The ability of DNP-ECs to induce CS returned to normal when normal peritoneal macrophages together with DNP-ECs were administered into the stripped skin. Normal ECs had a similar effect. Using either anti-Ia antiserum and complement or allogeneic ECs (strains 2 and 13), Ia-positive cells among the ECs (presumably Langerhans cells) were found to be essential for the recovery of CS. Tape-stripping treatment also resulted in the development of immunological tolerance, as assessed by subsequent painting with a sensitizing dose of DNCB. These findings suggest that the immunological function of the mononuclear-phagocyte system in the dermis may be impaired when the epidermal surface is markedly disturbed by tape-stripping treatment.