Suppressor cell activation and enhanced skin allograft survival after tumor promotor but not initiator induced depletion of cutaneous Langerhans cells

During chemical carcinogenesis Langerhans cells (LC) are depleted from the epidermis, disrupting the normal immunological functions of the skin. Tumor promotors but not initiators, have been shown to deplete adenosine triphosphatase (ATPase)-positive LC from the skin and therefore the cutaneous immune system may be impaired during tumor promotion but not initiation. The present study shows that the tumor promotor 12-O-tetradecanoylphorbol 13-acetate (TPA) but not the initiator urethane depletes Ia-positive LC from BALB/c murine ear epidermis, and beta-glucuronidase-positive LC from C57BL mouse tail skin. Sensitization with 2,4-dinitrofluorobenzene (DNFB) through urethane-treated skin resulted in a normal contact sensitivity response when the mice were challenged 5 days later. In contrast, tolerance resulted from sensitization through TPA-treated skin as a result of the generation of suppressor cells. In addition, TPA but not urethane-treated C57BL mouse tail skin survived for an extended time when grafted onto histoincompatible BALB/c mice. Therefore, impairment of the normal immunological functions of skin resulted from treatment with the tumor promotor TPA but not the tumor initiator urethane, which suggests that a loss of LC during tumor promotion may impair immunological protection against skin tumors.     

A mouse model for vitiligo

As the result of a long search for a depigmenting mouse that could serve as a model for the study of vitiligo, we have located a strain that arose from the C57BL/6J. Its provisional genetic designation is C57BL/6J Ler-vit/vit. This vitiligo mouse has congenital dorsal and ventral white spots (piebaldism) as well as progressive replacement of pigmented hairs by white hairs with each spontaneous molt or after plucking. The lack of pigment is due to the absence of melanocytes from the amelanotic hair follicles and epidermis. As in human beings and the Smyth chicken model, there is also diminution of ocular pigment. Reciprocal skin transplants between C57BL/6J and vitiligo mice, and transplants into nude mice, suggest a programmed pigment cell death in the vitiligo mice. Like human beings with vitiligo, maximally depigmented vitiligo mice have a decreased contact sensitivity response in comparison to age-matched C57BL/6J controls. The resistance to injected B16 melanomas is lowered. Vitiligo mice show no signs of premature aging. Already at this early stage in the study of this new animal model, there are findings that open a range of new approaches to the study and treatment of patients with vitiligo and melanomas.     

Effects of ultraviolet irradiation on surface marker expression by epidermal immunocompetent cells and contact sensitization to dinitrofluorobenzene in mice

We studied the effects of ultraviolet (UV) irradiation on murine epidermal Ia-positive Langerhans cells (Ia + LC) and Thy-I-positive dendritic epidermal cells (Thy-I + dEC). We also studied contact hypersensitivity to dinitrofluorobenzene (DNFB) introduced through UV-treated epidermis. C3H/HeN mice were exposed to UVB or 8-methoxypsoralen plus UVA (PUVA). UVB and PUVA treatment led to a dramatic reduction in surface marker expression of both Ia + LC and Thy-I + dEC. High-dose UVB irradiation (360 J/m2) interfered with contact hypersensitivity to DNFB; the density of Ia + LC may thus be related to the sensitizing potential. In contrast, low-dose UVB (120 J/m2) and PUVA treatment had little effect on contact hypersensitivity despite a marked reduction in Ia + LC. The density of Thy-I + dEC appeared not to be associated with contact hypersensitivity. These results suggest that there may be a Langerhans cell density-independent mechanism for the induction of contact hypersensitivity.     

Langerhans' cells in hair follicles of the depigmenting C57Bl/Ler-vit.vit mouse. A model for human vitiligo

The C57Bl/Ler-vit.vit mouse grows a black pelage after birth. During successive hair molts, the fur loses its pigmentation. By 6 months of age, most of the fur of the animal is white. The epidermis of the ears and tail also loses its pigmentation. Histologic studies confirm that in the epidermis and hair follicles there is an absence of pigment cells identifiable by various histochemical or electron microscopic techniques. This mouse may be an excellent model in which to study the role of Langerhans' cells and the immune response in the pathogenesis of vitiligo, a study not easily done in humans. From results of prior studies, we postulated that if Langerhans' cells were involved in the destruction of melanocytes, they would be abnormal (either more or less numerous) in number during the active phase of depigmentation and normal in number after depigmentation was complete. To determine whether the Langerhans cell (Ia+/adenosine triphosphatase dendritic epidermal cell) might be involved in destruction of pigment cells, we quantified the number of Ia+ and adenosine triphosphatase dendritic cells in the hair follicles in skin from the ear, abdomen, back, and tail from male C57Bl/Ler-vit.vit mice while the fur and skin were depigmenting and after depigmentation was almost completed. We found that Langerhans' cells were normal in number during depigmentation and were most numerous after depigmentation. Previous studies indicate that Langerhans' cells in these mice are functionally defective and respond poorly to some contact allergens. From these morphologic and functional data, we conclude that Langerhans' cells probably are uninvolved in causing depigmentation in these mice. We also observed that the epithelium of hair follicles has a significantly higher (up to 1600/mm2) population density of Langerhans' cells than interfollicular skin.     

Cutaneous dermal Ia+ cells are capable of initiating delayed type hypersensitivity responses

The presence of Langerhans cells (LC) within the epidermis has been shown to be critical for inducing T-cell-mediated immune responses in the skin. The purpose of this study was to assess whether cells in the dermis can initiate T-cell-mediated delayed-type hypersensitivity responses in vivo. Initially, back skins from C3H mice were trypsinized to remove the epidermis. The dermis was enzymatically dispersed and filtered to obtain a cell suspension. However, dermal cells exposed to trypsin were contaminated with numerous disaggregated hair follicles. These hair follicles contained Ia+ cells (presumably LC), and upon haptenation in vitro with trinitrophenyl, initiated contact hypersensitivity reactions in vivo. We therefore used dispase in place of trypsin to prevent follicular disaggregation and to allow preparation of dermal cell suspensions free of hair follicles. These hair follicle-free dermal cells were haptenated with trinitrophenyl and injected intradermally. Elicitation of contact hypersensitivity by epicutaneous painting 6 d later revealed the mean +/- SEM incremental ear-swelling response to be 53 +/- 8 mm X 10(-3). In contrast, mice sensitized by injection with dermal cells depleted of Ia+ cells demonstrated only 10 +/- 1 mm X 10(-3) of ear swelling. Thus, like dendritic LC of the epidermis, perivascular dendritic Ia+ cells of the dermis are capable of initiating T-cell-mediated contact hypersensitivity in vivo and may be highly relevant for presentation of antigen to T cells trafficking through the dermis.     

Skin contact irritation conditions the development and severity of allergic contact dermatitis

Irritant contact dermatitis (ICD) is a frequent inflammatory skin disease induced by skin contact with low molecular weight chemicals such as haptens endowed with proinflammatory properties. Allergic contact dermatitis (ACD) is a frequent complication of ICD and is mediated by hapten-specific T cells primed in lymph nodes by skin emigrating dendritic cells. The aim of this study was to analyze the relationship between ICD and ACD to 2,4-dinitrofluorobenezene (DNFB) in C57BL/6 and BALB/C mice, which develop a severe and a moderate skin inflammation, respectively. Upon a single skin painting with DNFB, C57BL/6 developed within hours a more severe dose-dependent ICD response as compared to BALB/C mice, which was associated with enhanced upregulation of IL-1beta, IL-6, and IL-10. Skin exposure to a low dose of DNFB resulted, in both strains, in a low ICD that resolved in a few hours. Alternatively, skin painting with either an intermediate or a high DNFB concentration induced an ICD that subsequently gave rise to an ACD reaction whose intensity was proportional to the magnitude of the ICD response and was more severe in C57BL/6 mice than in BALB/C mice. In conclusion, the hapten-induced skin contact irritation conditions the development and the severity of ACD.     

Deleterious effects of cis-urocanic acid and UVB radiation on Langerhans cells and on induction of contact hypersensitivity are mediated by tumor necrosis factor-alpha.

Ultraviolet B (UVB) light disrupts epidermal Langerhans cells (LC) universally and impairs the induction of contact hypersensitivity (CH) to epicutaneously applied haptens in certain strains of mice. Similar effects are observed when tumor necrosis factor-alpha (TNF alpha) is injected intradermally (ID) in mice. Trans-urocanic acid (UCA), a photoreceptor for UVB radiation, is known to be immunosuppressive. To determine whether cis-UCA is important in the process by which UVB and/or TNF alpha act in the skin, cis-UCA was injected ID into C57BL/6, C3H/HeN, BALB/c, and C3H/HeJ mice. Whole mounts of epidermis were removed 5 h later and stained immunochemically with anti-Ia antibodies. Microscopy revealed that Ia-bearing LC had lost their dendrites, had rounded up, and were reduced in number in all strains examined. Moreover, when dinitrofluorobenzene (DNFB) was applied epicutaneously to the injected site, induction of CH was grossly impaired. When neutralizing anti-TNF alpha antibodies were administered intraperitoneally 2 h prior to ID injection of cis-UCA, the deleterious effects on LC and CH induction were largely reversed. These results indicate that the actions of cis-UCA on LC and on CH induction are very similar to those achieved by ID injections of TNF alpha and by cutaneous exposure to low-dose UVB. Because the effects of UVB radiation and cis-UCA are reversed by anti-TNF alpha antibodies, we propose that UVB radiation impairs the induction of CH in mice by converting trans-UCA to cis-UCA within the epidermis; cis-UCA in turn causes the local release of TNF alpha, which thwarts sensitization by its ability to alter the functional program of epidermal Langerhans cells, thereby preventing the induction of CH.     

Strain variation in the induction of tolerance by epicutaneous application of trinitrochlorobenzene

It has been postulated that a relationship exists between the density of epidermal Langerhans cells and the capacity of the epidermis to promote the induction of contact sensitization. This postulate was developed, in part, because (1) mouse tail epidermis contains fewer ATPase-positive (presumably Langerhans) cells than does abdominal epidermis, and (2) when tails of C57Bl/6 mice were painted with dinitrofluorobenzene (DNFB), the mice were less sensitive than those painted on the abdomen. In addition, tail-painted mice were shown to be tolerant to subsequent attempts at sensitization with DNFB. In this study we found that by painting the tails of mice with the hapten trinitrochlorobenzene (TNCB), sensitization was induced in certain mouse strains (BALB/c, A/J, and CBA--haplotypes H-2d, H-2a, H-2k, respectively), but tolerance resulted from painting the tails of other strains (C57Bl/6, C57Bl/10, and AB.Y--haplotype H-2b). The ability to become sensitive or tolerant is not related to Langerhans cell density as detected by ATPase staining. While the mechanism for this strain difference in the induction of tolerance is unknown, tolerance induced in C57Bl/6 mice is mediated in part by the generation of suppressor cells.     

Immediate contact hypersensitivity induced by repeated hapten challenge in mice

An immediate reaction was investigated during repeated challenge testing for contact hypersensitivity to dinitrofluorobenzene (DNFB) in BALB/c mice. The mice were sensitized to DNFB on back skin and repeatedly challenged with the same hapten on the left ear at 1 week intervals. The ear after the 5th challenge showed biphasic responses which consisted of an immediate and a delayed-type reaction. The reactions were hapten specific. Mast cell-deficient WBB6F1 W/WV mice did not show any immediate reaction, while congenic normal mice showed both immediate and delayed-type reactions. Histologically, numerous dermal mast cells were found in the left ear of repeatedly challenged BALB/c and WBB6F1 normal mice, while there were few mast cells in the ear of WBB6F1 W/WV mice. Anti-DNP IgE antibodies were detected in BALB/c, WBB6F1 normal and W/WV mice after repeated challenge with DNFB. Intradermal injection of anti-IgE antibodies in the repeatedly DNFB-challenged ear elicited an immediate reaction. These results suggest that immediate contact hypersensitivity develops through the production of anti-DNP IgE antibodies and an increase in dermal mast cells after repeated challenge with DNFB.     

Sex differences in the densities of epidermal Langerhans cells of the mouse

Cutaneous immune reactions are known to show sexual dimorphism. Langerhans cells (LCs) are bone marrow-derived immune cells in the epidermis and are essential to immune reactions in the skin. In the present research, a study was made of the differences in LC density of male and female mice. Epidermal sheets were separated from the skin of the glabrous part of hind limbs and ears of specific pathogen-free (SPF) mice by ethylenediaminetetraacetic acid (EDTA) treatment and stained for adenosine triphosphatase (ATPase) activity. The density of LCs of hind limb epidermis in male C57BL/6 (823 +/- 20/mm2) and BALB/c (1689 +/- 66/mm2) mice was significantly less than that in females (1363 +/- 52/mm2, p less than 0.001; 2249 +/- 105/mm2, p less than 0.001, respectively). Langerhans cell density in the ears of male C57BL/6 (465 +/- 24/mm2) mice was also significantly less than that in females (542 +/- 17/mm2, p less than 0.02). Although ovariectomy failed to bring about any change in the LC density of hind limb epidermis in female C57BL/6 mice, the LC density in male C57BL/6 mice increased significantly at 4 weeks following orchiectomy (sham operation, 564 +/- 27/mm2; castration, 1179 +/- 49/mm2, p less than 0.001). These results indicate that mouse epidermal LC density depends on sex, i.e., male mice have fewer LCs than female mice. The reduction in LC density in males may possibly be caused by the testis.     

Autoimmunity as an aetiological factor in vitiligo

Vitiligo is a common dermatological disorder characterized by the presence on the skin of depigmented macules resulting from the destruction of cutaneous melanocytes. Autoimmunity is an important hypothesis with regard to vitiligo aetiology and the evidence for autoimmune responses being involved in the pathogenesis of this disorder will be discussed in the present review. All immune system compartments, including innate and adaptive immunity have been implicated in vitiligo development. Particularly relevant are autoantibodies and autoreactive T cells in vitiligo patients that have cytotoxic effects upon pigment cells. Furthermore, predisposition to vitiligo appears to be associated with certain alleles of the major histocompatibility complex class II antigens as well as with other autoimmune-susceptibility genes. Moreover, the association of vitiligo with autoimmune disorders, the animal models of the disease, and the positive response to immunosuppressive therapeutic agents emphasize the role of autoimmunity in the development of this disorder.     

Rapid induction of Thy-1 antigenic markers on keratinocytes and epidermal immune cells in the skin of mice following topical treatment with common preservatives used in topical medications and in foods

Earlier experiments from our laboratory revealed that the medication most commonly used for depigmenting patients with vitiligo, monobenzyl ether of hydroquinone (MBEH), when applied to the skin of DBA/2 mice caused an increase in the population density (cells/mm2) of identifiable Ia+ and ATPase+ Langerhans cells. Further, this increase in Langerhans cell density could be correlated with an increase of contact hypersensitivity (CHS) reactivity to dinitrofluorobenzene (DNFB). The current experiments demonstrated that other compounds chemically similar to MBEH, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), which are used as preservatives/antioxidants in many topical medications, cosmetics, food, and rubber products, can in five days significantly increase the population density of Thy-1+ dendritic epidermal cells. These compounds had no effects on Ia+ cells. This observation suggests that the Thy-1+ DEC cells may be more mobile and/or their surface markers may be readily expressed and are not a slowly mobile (trafficking) population of cells as suggested by the results of previous work. In addition, these parasubstituted phenolic compounds behaved like pertussis toxin and induced Thy-1 and Ia expression on keratinocytes. These changes in Thy-1 immune markers were not accompanied by functional alterations in the immune response to contact allergens as measured by the ear swelling technique.     

Thy-1阳性树枝状表皮细胞及郎格罕细胞的比较观察

观察了青、老年小鼠及大鼠不同部位皮肤内的Thy-1阳性树枝状表皮细胞(Thy-1⁺dEC)及郎格罕细胞(LC).于C57BL/6Ss小鼠,Thy-1⁺dEC通常比LC大;Thy-1⁺dEC的Thy-1抗原主要存在于细胞膜,而LC的Ia抗原较均匀分布,Thy-1⁺dEC突起常比LC短,有的甚至并不明显,正常角肮细胞表面也有少量Thy一抗原,但无Ia抗原.二种细胞于老年动物都少于青年动物.于Lewis/Ss及BN/Ss大鼠,未见到Thy-1⁺dEC.我们认为Thy-1⁺dEC可能具有免疫学功能.对人的Thy-1⁺dEC相应物的探索,也许能在皮肤病发病机理的研究中展开一条新的途径.     

小鼠毛囊周期中表皮朗格汉斯细胞及树突状表皮T细胞的变化

目的:观察拔毛诱导的小鼠毛发周期中毛囊间表皮LC及DETC的密度及形态变化,探讨二者与毛囊周期的关系。方法:选用自然休止期C57BL/6小鼠,拔毛诱导毛发进入生长期,应用ABC免疫组化法连续观察毛囊间表皮LC及DETC的密度及形态变化。结果:(1)密度变化:拔毛后1天LC及DETC与拔毛前无明显变化。拔毛后第2天开始二者密度较拔毛前升高(P<0.05),拔毛后第4~8天二者密度最高(P<0.01),以后逐渐下降,至拔毛后第17~20天二者密度基本恢复到拔毛前数值。(2)形态变化:拔毛前后2天内,多数LC及DETC胞体小,树突短小不明显;第4~8天,二者多数细胞体大,树突多且粗大,分枝明显;9~16天,胞体大小不一,树突变细;17~20天,胞体较小,树突短小。结论:拔毛诱导的小鼠毛发周期中毛囊间表皮LC及DETC的密度及形态变化与毛囊周期具有相关性。这种变化在皮肤免疫应答中可能起重要作用。     

Role of Keratinocytes in the Development of Vitiligo

Vitiligo is an acquired depigmentary disorder of the skin that results from the loss of functioning epidermal melanocytes. Most studies on vitiligo have concentrated on the abnormality of melanocytes rather than the abnormality of keratinocytes; however, epidermal melanocytes form a functional and structural unit with neighboring keratinocytes. In fact, direct cell-to cell contact stimulates in vitro proliferation of melanocytes, and growth factors produced by adjacent keratinocytes regulate the proliferation and differentiation of melanocytes. The potential role of keratinocyte-derived cytokines has also been presented. We focused on the structural changes in vitiliginous keratinocytes, which may result in loss of melanocytes, to examine the pathomechanism of vitiligo. The results of a comparison between depigmented and normally pigmented epidermis in patients with vitiligo showed that the keratinocytes in the depigmented epidermis were more vulnerable to apoptosis. Impaired Phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (Akt) activation followed by reduced nuclear factor-κB activation under increased tumor necrosis factor-α levels was demonstrated as a mechanism for keratinocyte apoptosis. The role of aquaporin 3 in keratinocyte apoptosis was addressed based on the relationship between the PI3K/AKT pathway and the E-cadherin-catenin complex. Apoptotic keratinocytes induced a lower expression of keratinocyte-derived factors, including stem cell factor, in depigmented epidermis, resulting in passive melanocyte death.     

Hermansky-Pudlak HPS1/pale ear gene regulates epidermal and dermal melanocyte development

The pale ear (ep) mouse strain is a model for the Hermansky-Pudlak syndrome type 1 (HPS-1), an autosomal recessive disorder causing pigmentary dilution, visual disturbances, bleeding diatheses, pulmonary fibrosis, and granulomatous colitis. The ep mice have a coat color very similar to the black-colored parental strain, C57BL/6. However, the ears and tails of ep mice are significantly hypopigmented compared with the control animals, suggesting that the gene mutation in ep mice reveals a differential regulation of melanocyte function in dorsal back skin melanocytes versus tail or ear skin. In this study, we analyzed the mutant phenotype in detail and determined that in the tail, the defective gene causes delayed onset of interfollicular epidermal melanocyte tyrosinase activity, decreased numbers of melanocytes in the interfollicular epidermis and dermis, and severe immaturity of tail epidermal melanosomes, findings not observed in dorsal back follicular melanocytes. These results highlight differences between follicular and interfollicular melanocyte biology and demonstrate that defects in the ep protein not only affect melanosome biogenesis, but also play a developmental role in determining interfollicular epidermal and dermal melanocyte function. The implications of these findings for the mechanisms governing physiologic variation in human pigmentation and for the pathogenesis of vitiligo are discussed.     

Thy-1 antigen-bearing dendritic cells populate murine epidermis

Two distinct cell populations, melanocytes and Langerhans cells (LC), have been recognized previously to possess dendritic configuration in normal mammalian epidermis. Employing immunofluorescence microscopy with monoclonal antibodies against Thy-1.2 antigen to identify cells in whole mounts of murine epidermis, we have identified a third dendritic cell population which differs from both LC and melanocytes. Thy-1 antigen-bearing (Thy-1+) epidermal cells are primarily dendritic, although round and angular forms may be found. They are distributed relatively evenly across skin surfaces, although densities vary greatly from site to site and from strain to strain. Densities were highest in ear epidermis from the pigmented strain B10.A (580 cells/mm2), a value approaching that of epidermal LC, and were lowest in ear epidermis from the albino strain BALB/c (5 cells/mm2). Thy-1+ epidermal cells possess neither Ia antigens nor substantial amounts of melanin, and their surface distributions are disparate from those of both LC and mature melanocytes. We propose that at least some of these cells are T lymphocytes whose malignant counterparts account for cutaneous T-cell lymphomas.     

Langerhans' cell population studies with OKT6 and HLA-DR monoclonal antibodies in vitiligo patients treated with oral phenylalanine loading and UVA irradiation

In vitiligo patients, treated with oral phenylalanine loading combined with UVA irradiation (Phe-UVA), Langerhans' cells (LC) were counted in pigmented and depigmented skin. The LC, which were labelled with OKT6 and HLA-DR monoclonal antibodies, were expressed per linear mm epidermis. Before treatment the number of OKT6(+) cells was significantly increased in vitiliginous skin especially in the basal layer. Under treatment the number went down and was comparable to normal skin. When using HLA-DR labelling the number of LC increased in vitiliginous skin which had been treated with Phe-UVA. The influence of Phe-UVA on the shift of LC subpopulations is discussed.