Alterations in cutaneous immune reactivity to dinitrofluorobenzene in graying C57BL/vi.vi mice

The fur of the C57BL/vi.vi mouse is black at 6 weeks of age. By 6 months of age the animals are white and there are no identifiable pigment cells within the epidermis or hair bulbs. Human subjects with vitiligo exhibit loss of epidermal pigment cells. The loss of pigment cells in human subjects with vitiligo has been associated with loss of cutaneous immune reactivity to contact allergens. Therefore, studies were performed to determine whether loss of pigment cells in these depigmenting mice also was associated with loss of the cutaneous immune response. The number of Ia-positive (Ia +) Langerhans cells (LC)/mm2 on the back and the ear, the sites of sensitization and challenge with dinitrofluorobenzene (DNFB), was quantified before, during, and after depigmentation. We observed that there were fewer LC/mm2 on the back and the ear before and after pigment loss in the graying mice than in the normal control C57BL/6 mice. The young pigmented C57BL/vi.vi mice were capable of developing moderate contact hypersensitivity; the older depigmented mice did not sensitize to DNFB. We conclude that the depigmented mice, like human subjects with vitiligo, have a loss of contact hypersensitivity associated with a loss of pigment cells within the epidermis. In the mouse, loss of melanocytes is associated with a decrease in the population density of Ia + cells.     

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.     

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.     

Telogen skin contains an inhibitor of hair growth

We have investigated whether C57B1-6 mouse skin with all its follicles in the telogen stage of the hair cycle contains a hair-growth inhibitory activity, as opposed to skin with anagen follicles. Crude aqueous extracts of whole telogen mouse skin (TE), anagen skin (AE) or vehicle alone (V) were injected intraperitoneally into mice in which anagen had previously been induced by plucking of telogen hair follicles. Injection of TE, but not AE or V, significantly retarded the development of anagen follicles, as measured by macroscopic and quantitative microscopic hair growth parameters (skin pigmentation and thickness, appearance of trichohyaline granules) and the incorporation of tritiated thymidine into mouse skin from animals previously treated with either TE or V (skin organ culture). This inhibitory activity seemed to be localized to the epidermis and was also present in rat epidermis. We suggest that this apparently non-species-specific inhibitor present in telogen skin may play a role in regulating the hair cycle in rodents.     

Pigmented guinea pig skin irradiated with Q-switched ruby laser pulses. Morphologic and histologic findings

Q-switched ruby laser pulses cause selective damage to cutaneous pigmented cells. Repair of this selective damage has not been well described. Therefore, using epilated pigmented and albino guinea pig skin, we studied the acute injury and tissue repair caused by 40-ns, Q-switched ruby laser pulses. Gross observation and light and electron microscopy were performed. No specific changes were evident in the albino guinea pigs. In pigmented animals, with radiant exposures of 0.4 J/cm2 or greater, white spots confined to the 2.5-mm exposure sites developed immediately and faded over 20 minutes. Delayed depigmentation occurred at seven to ten days, followed by full repigmentation by four to eight weeks. Regrowing hairs in sites irradiated at and above 0.4 J/cm2 remained white for at least four months. Histologically, vacuolation of pigment-laden cells was seen immediately in the epidermis and the follicular epithelium at exposures of 0.3 J/cm2 and greater. Melanosomal disruption was seen immediately by electron microscopy at and above 0.3 J/cm2. Over the next seven days, epidermal necrosis was followed by regeneration of a depigmented epidermis. By four months, melanosomes and melanin pigmentation had returned; however, hair follicles remained depigmented and devoid of melanocytes. This study demonstrates that selective melanosomal disruption caused by Q-switched ruby laser pulses leads to transient cutaneous depigmentation and persistent follicular depigmentation. Potential exists for selective treatment of pigmented epidermal and dermal lesions with this modality.     

Towards the development of a simplified long-term organ culture method for human scalp skin and its appendages under serum-free conditions

Organ culture of human scalp skin is usually performed with serum-containing medium, which limits its analytical usefulness. Here we report that intact human scalp skin can be grown at the air/liquid interface in supplemented, serum-free William's E medium for more than 2 weeks. Active hair shaft growth was visible until day 16 and was significantly enhanced compared with minimum essential medium (MEM) + 10% fetal bovine serum (FBS). Moreover, William's E medium protected better against cell death than MEM + 10% FBS before day 12. Using quantitative immunochemistry, proliferating (Ki-67+) cells could still be observed in the epithelium of hair follicles even on day 17 of serum-free skin organ culture. The number of apoptotic (TUNEL+) cells in the skin epithelium rose steadily after day 5. Giemsa stains revealed mature skin mast cells even after 13 days in culture. The percentage of surviving hair follicles (mostly with catagen- or telogen-like morphology) gradually increased over time displaying mostly catagen hair follicles after 17 days of culture. Although epidermis and hair follicle epithelium showed increasing atrophy and degeneration, and their pigmentation decreased gradually over time, some long-term-surviving epithelial islands were found in association with remnants of follicular structures as late as on day 88. These preliminary data suggest that a very simple serum-free organ culture method allows prolonged human skin and hair follicle survival as well as some limited hair follicle cycling in intact skin for more than 2 weeks under well-defined experimental conditions. This pragmatic assay invites multiple uses, and may become a valuable tool for both skin and hair research.     

Langerhans cell migration into ultraviolet light-induced squamous skin tumors is unrelated to anti-tumor immunity.

There has been much speculation as to the role of Langerhans cells (LC) in the induction of anti-tumor immunity. Whereas there is considerable circumstantial evidence that disruptions in the density and function of these cells during the early stages of ultraviolet (UV) light- and chemical carcinogen-induced carcinogenesis may be important for enabling developing neoplasms to escape immune destruction, the role of the large number of these cells found infiltrating developed skin tumors is less clear. To investigate this we have compared the LC density infiltrating transplanted non-immunogenic and immunogenic UV-induced murine tumors as well as LC in the epidermis overlying the tumors. Whereas two non-immunogenic tumor lines attracted large numbers of Ia+ dendritic cells, an immunogenic tumor line did not. Similar results were obtained whether the tumors were transplanted into syngeneic immunocompetent or athymic immunodeficient mice. Hence, there was no relationship between tumor immunogenicity or host immunocompetence and Ia+ dendritic cell density. Furthermore, there was no correlation with the pattern of T-cell infiltration of the tumors or CD4/CD8 cell ratio. Our results also indicate that whereas UV light decreased Ia+ cell density, both in the epidermis and the tumors, it did not inhibit the tumors from attracting Ia+ dendritic cells. Thus, the Ia+ dendritic cells infiltrating skin tumors are unlikely to indicate a host immune response to the tumor, but are more likely to be attracted by tumor-derived cytokines.     

Interleukin-1 decreases the number of Ia+ epidermal dendritic cells but increases their expression of Ia antigen

It is generally accepted that ETAF/IL-1 is produced in epidermis by both keratinocytes and Langerhans' cells. We have studied the density and morphology of Ia+ epidermal dendritic cells in mice after systemic or intracutaneous injection of recombinant IL-1 beta. We found that rIL-1 beta decreased the density of Ia+ dendritic cells in the time period 2-7 days after rIL-1 beta administration. However, the remaining dendritic cells were enlarged and more arborized with increased expression of Ia antigen 1-4 days after injection of rIL-1 beta. The implication of the results is that ETAF/IL-1 modulates the function of Langerhans' cells through autocrine and paracrine regulation.     

Immunohistochemistry of lymphocytes and Langerhans' cells in long-lasting allergic patch tests

A long-lasting allergic patch test is a "normal" allergic patch test that remains positive for weeks or months. An immunohistochemical study of immunocompetent cells in the skin in this rare type of patch tests was performed. Most inflammatory cells were T11 positive T-lymphocytes. The majority of these cells were of the helper/inducer phenotype (T4+), but a relative increase of T8+ cells as compared to the initial (1-2d) stages of allergic patch tests was observed. T6+ Langerhans' cells (LCs) were normal or increased in number in the epidermis, while very few dendritic cells displayed Ial antigen in the epidermis, indicating loss of Ial-staining of LCs. High to very high numbers of T6+ cells were found in the dermis. An inflammatory reaction of hair follicles with moderate numbers of T6+ cells in the peribulbar infiltrate was observed indicating that hair follicles might act as shunt pathways for allergens. A defect in down regulation of the contact hypersensitivity reaction and/or a constant antigen stimulation could be responsible for the long-lasting allergic patch tests.     

Variations in the number and morphology of Langerhans' cells in the epidermal component of squamous cell carcinomas

We investigated the number and morphology of Langerhans' cells in the epidermal component of squamous cell carcinomas located on the sun-exposed skin of 10 patients. Using adenosine triphosphatase-stained epidermis from the tumors, we compared the Langerhans' cells in squamous cell carcinoma with those in nontumorous skin specimens from the same patient. The nontumorous skin specimen was obtained from either sun-exposed perilesional or non-sun-exposed sites. In three patients a normal number and almost normal morphology of Langerhans' cells were observed within the epidermal component of the tumor. One patient showed a normal number but a profound alteration of the morphology of the cells. In the remaining six patients, a significant decrease in the number of Langerhans' cells was observed. Langerhans' cells within the epidermal component of the tumors of these patients exhibited morphologic alterations in that they were mainly round or oval rather than highly dendritic. In none of our patients was the number of Langerhans' cells in the tumor increased. We conclude that a decreased number and altered morphology of Langerhans' cells occur in some, but not all, squamous cell carcinomas of the skin, and that there is no apparent difference between the number of Langerhans' cells in sun-exposed vs unexposed skin from the same individual.     

p-Cresol: cause of ink-induced hair depigmentation in mice

p-Cresol has been identified as the active chemical in a laundry-ink responsible for the previously reported contact depigmentation of hair in agouti CBA/J mice. p-Cresol has been shown to induce depigmentation of both skin and hair in agouti as well as black mice. In the agouti mice there were bands and whorls, as well as plaques of white hair. However, the most striking finding was an occult hair depigmentation wherein the new hair shafts were white except for the tips. Such pigment-loss was not apparent on surface examination of the fur, becoming evident only on parting the hairs. All of these patterned hair pigment losses have been related to a unique sensitivity of the follicular melanocytes to the toxic effect of p-cresol during the earliest days of new hair formation, that is, during the initial anagen phase.     

Immunocompetent cells of fixed drug eruption

The positive provocation test reactions of the skin of six patients with fixed drug eruption (FDE) were studied from timed skin biopsies taken between 2 hours and 9 days after the appearance of FDE. Monoclonal antibodies to the following immunocompetent cell surface epitopes were used: T3, T4, T6, T8, T9, M1, Ia1, Drc, Leu7 and B cell. The dermal infiltrate comprised 60-80% of T lymphocytes at all the times studied. Cells with T4 and T8 epitopes were displayed in similar numbers. A transient decrease in the number of T6+ cells of the epidermis could be detected with a simultaneous and also transient increase of the T6+ cells in the dermis, which suggests a possible traffic of Langerhans' cells from the epidermis to the dermis. The epidermal Ia1+ cells showed changes similar to but less marked than the T6+ cells. The number of the dermal Ia1+ cells increased continuously. In the late biopsies these Ia1+ cells comprised up to 90% of the infiltrating cells. Except for the finding of a reduction of T6+ and Ia1+ epidermal cells, the cellular kinetics of FDE are similar to those seen in both cutaneous immunological and irritant reactions.     

Expression of cyclooxygenase isozymes during morphogenesis and cycling of pelage hair follicles in mouse skin: precocious onset of the first catagen phase and alopecia upon cyclooxygenase-2 overexpression

Cyclooxygenase (COX)-1 and -2 catalyze the key reaction in prostaglandin biosynthesis. Whereas COX-1 is found in most tissues, COX-2, with a few exceptions, is not expressed in normal tissues but becomes transiently induced in the course of inflammatory reactions. In many neoplastic epithelia, COX-2 is constitutively overexpressed. Here we show that COX isozymes are spatiotemporally expressed during morphogenesis of dorsal skin epithelium of NMRI mice. COX-1 and COX-2 mRNA and protein were detected in embryonic and postnatal epidermal tissue by RT-PCR, northern blot, and immunoblot analysis indicating that both isoforms may contribute to prostaglandin production. Being barely detectable in interfollicular epidermis and resting hair follicles of adult mice, COX-2 protein appeared in embryonic skin first in epidermal precursor cells and later on in the basal cells and the peridermal layer of the stratified epidermis. In the course of pelage hair follicle morphogenesis, COX-2 remained expressed in the basal interfollicular compartment and, in addition, became apparent in elongated hair germs and hair pegs and later on in the outer root sheath cells of the distal and proximal hair follicles as well as in basal sebaceous gland cells. During the subsequent synchronous phases of hair cycling, COX-2 expression declined in catagen, was barely detectable in telogen, and was reinduced in the basal outer root sheath and basal sebaceous gland cells of anagen hair follicles. COX-1 immunosignals were detected predominantly in the interfollicular spinous and granular layers of the developing, neonatal, and adult epidermis but not in follicular epithelial cells of developing or cycling hair follicles. Dendritic cells in the interfollicular epidermis and distal hair follicles were also COX-1-positive. Transgenic overexpression of COX-2 under the control of a keratin 5 promoter in basal cells of the interfollicular and follicular epidermis induced a precocious entry into the first catagen stage of postnatal hair follicle cycling and a subsequent disturbance of hair follicle phasing. Furthermore, transgenic mice developed an alopecia. Inhibition of transgenic COX-2 activity by feeding the specific COX-2 inhibitor valdecoxib suppressed the development of alopecia, indicating that COX-2-mediated prostaglandin synthesis is involved in hair follicle biology.     

中波紫外线诱导小鼠皮肤光损伤中miRNA141表达的研究

【摘要】 目的 探讨小室移植法重建小鼠毛囊,观察细胞成分对毛囊再生的影响。 方法 取0 ～ 2 d的C57BL/6乳鼠背部皮肤,胰酶消化后分离表真皮,再分离出毛囊上皮细胞。实验分表皮细胞混合毛囊胚芽组、真皮细胞组、表皮细胞混合毛囊胚芽 + 真皮细胞组、毛囊上皮细胞 + 真皮细胞组。用小室移植法接种于裸鼠背部,并于移植后1、2、4、8周观察变化,HE染色观察毛囊组织学形态。 结果 小鼠毛囊细胞移植1周时,背部小室开始脱落,伤口结痂;2周时除表皮细胞组外,另3组均长出了短小的毛发,镜下可见毛囊样结构;4周及8周时除表皮细胞组外,均长出了正常的毛发,表皮细胞与真皮细胞混合组及毛囊上皮细胞与真皮细胞混合组毛发生长情况良好,优于单独的真皮细胞组。 结论 毛囊细胞小室移植后可形成新的毛囊,上皮细胞及真皮细胞在毛囊重建中具有重要的作用。 【关键词】 毛囊; 毛囊重建; 鼠科     

RADIATION DEPIGMENTATION OF MOUSE HAIR: EFFECTS OF MOUSE STRAIN

SUMMARY. The radiation depigmentation response of 7 types of mice with various different coat colour genotypes has been studied by counting the number of follicles without pigmentation. The response appears to vary slightly between genotypes, Strong F (dilute brown chinchilla spotted) being the most sensitive. The sensitivity differences cannot be attributed to differences in the number of precursor cells in zigzag follicles. However, the overall precursor pool may vary due to changes in the relative porportions of the follicle types in different genotypes or strains. These small differences within 1 species are discussed in conjunction with the differences in the threshold doses quoted in the literature for depigmentation in various other species.     

Behavior of cutaneous Langerhans' cells and skin reactivity after gold sodium thiomalate treatment of pemphigus vulgaris

Skin biopsies from pemphigus vulgaris patients in the acute phase of disease and after successful GST therapy were studied in respect to their capacity to express OKT6, OKIa1 and HLA-DR antigens. In comparison to controls, the epidermis overlaying noninvolved skin contained significantly (p = 0.0001) fewer detectable Ia and T6 antigen-bearing Langerhans' cells. Lower epidermal Langerhans' cell density in comparison to controls was detected in both prednisolone-treated and untreated patients. A normalization of prior defective antigen expression was observed in patients in remittence after GST therapy. This correlated with a normal delayed type hypersensitivity reaction (Multitest Mérieux).     

The in vivo melanocytotoxicity and depigmenting potency of N-2,4-acetoxyphenyl thioethyl acetamide in the skin and hair

Summary It has been shown previously that N-acetyl-4-S-cysteaminylphenol (N-Ac-4-S-CAP) is a tyrosinase Substrate and a potent depigmenting agent of dark skin and black hair. The present study evaluated the depigmenting potency of an acetyl derivative of N-Ac-4-S-CAP. N-2,4-acetoxyphenyl thioethyl acetamide (NAP-TKA) in the skin and hair. We tested for (i) in vitro metabolites in the skin after topical application. and (ii) in vivo depigmenting potency in the skin and hair. We found that NAPTEA was stable in water. but was converted to N-Ac-4-S-CAP after topical application to human skin. Therefore. although NAP-TEA was not a tyrosinase substrate. it could react with tyrosinase after being converted to N-Ac-4-S-CAP by 0-deacetylation in vivo. NAP-TEA produced marked depigmentation of dark skin (Yucatan pig) after daily topical application. When given by intraper-itoneal injection. it resulted in complete loss of hair colour (white) grown at the epilated site in adnlt C57 black mice after daily administration for l0 days, and incomplete loss of coat colour (silver grey) in newborn C57 black mice after a single administration, The depigmentation of the skin and hair was reversible, Splil-dopa preparalion and eieclron microsnipy indicated thal lliis depigmentation is primarily related to (i) a marked decrease in the number of functioning melanocytes and melanized melanosomes, (ii) a decrease in the number of melanosomes transferred to keratinocytes, and (iii) selective degeneration/inactivation of melanocytes. and deposition of melanin-like malerial in the Golgi cisternae. coated vesicles and melanosomes. where tyrosinase is reported to be located. We propose the NAP-TEA is converted in vivo to N-Ac-4-.S-CAP which. via interaction with tyrosinase. causes reversible depigmentation of the skin and hair.     

An extended epidermal response heals cutaneous wounds in the absence of a hair follicle stem cell contribution

Hair follicles have been observed to provide a major cellular contribution to epidermal healing, with emigration of stem-derived cells from the follicles aiding in wound reepithelialization. However, the functional requirements for this hair follicle input are unknown. Here we have characterized the keratinocyte stem cell status of mutant mice that lack all hair follicle development on their tail, and analyzed the consequent alterations in epidermal wound healing rate and mechanisms. In analyzing stem cell behavior in embryonic skin we found that clonogenic keratinocytes are relatively frequent in the ectoderm prior to hair follicle formation. However, their frequency in the interfollicular epidermis drops sharply by birth, at which time the majority of stem cells are present within the hair follicles. We find that in the absence of hair follicles cutaneous wounds heal with an acute delay in reepithelialization. This delay is followed by expansion of the region of activated epidermis, beyond that seen in normal haired skin, followed by appropriate wound closure. JID Journal Club article: for questions, answers, and open discussion about this article please go to http://network.nature.com/group/jidclub.     

芳香烃受体在人表皮、毛囊和皮脂腺上的表达及其意义

目的 观察芳香烃受体(AhR)在人表皮、毛囊及皮脂腺细胞上的表达及在二(噁)英作用下的激活情况.方法 采用免疫荧光和免疫组化法观察AhR蛋白在人面部表皮、毛囊、皮脂腺及体外培养的HaCaT细胞和SZ95人皮脂腺细胞上的表达情况;Western印记法观察2,3,7,8-四氯二苯-p-二(噁)英(TCDD)作用HaCaT细胞和SZ95细胞3d后AhR蛋白表达的变化情况.结果 AhR蛋白在人面部皮肤表皮、毛囊、皮脂腺细胞上表达,并且AhR蛋白在表皮棘层和颗粒层的表达强于基底细胞层,在皮脂腺小叶的外周和中央表达强于皮脂腺小叶中间,在毛囊外毛根鞘和近毛小皮细胞上的表达强于内毛根鞘细胞.AhR蛋白在HaCaT细胞和SZ95细胞的细胞核和胞质中高表达.在TCDD作用下,HaCaT细胞和SZ95细胞内AhR蛋白表达明显下降,显示AhR在TCDD作用下被激活.结论 AhR在人表皮、毛囊、皮脂腺、HaCaT细胞及SZ95细胞上高表达,并在TCDD作用下激活,显示二(噁)英有可能通过AhR信号途径引起了人表皮、毛囊和皮脂腺的异常分化.