Correlation between keratinocyte expression of Ia and the intensity and duration of contact hypersensitivity responses in mice

Langerhans cells are the only cells within the epidermis that normally express immune response-associated antigens (referred to as Ia in mice and HLA-D in humans). However, in the epidermis of patients with allergic contact dermatitis or individuals undergoing a delayed-type hypersensitivity response, the keratinocytes at the reaction site are induced to express HLA-DR. In this study the inducible expression of Ia by the keratinocytes of mice was found to be directly correlated with the intensity and duration of experimentally induced contact hypersensitivity (CH) responses. During a CH response in animals that were sensitized on the belly and challenged on the ear with the contact-sensitizing (CS) agent oxazolone, the keratinocytes in the challenged, but not the unchallenged, ear were induced to express Ia. In comparison with animals that were sensitized and challenged at different sites, an intensified expression of Ia by the keratinocytes was associated with a twofold increase in ear swelling in mice that were sensitized and challenged with oxazolone at the same site. Curiously, the challenge of oxazolone-sensitized ears with dinitrofluorobenzene (an unrelated CS agent), croton oil (a nonspecific inflammatory agent), or acetone/olive oil (a noninflammatory agent) also induced both a marked keratinocyte expression of Ia and an enhanced CH response. These results suggest that residual antigen at the original sensitization site may be mobilized to function as the challenge stimulus to elicit a CH response, in association with keratinocyte expression of Ia, when CS-sensitized skin is perturbed with a nonspecific agent. Further evidence of an association between CH responsiveness and keratinocyte expression of Ia came from the following observations. First, the magnitude and duration of a CH response was markedly increased in pertussis toxin (PT)-treated mice. These enhanced responses were associated with intense Ia expression by the keratinocytes in the epidermis at the reaction site. Because PT is known to have an adjuvant effect on delayed-type hypersensitivity reactions, as well as to alter the normal regulatory mechanisms associated with this type of response, it is possible that Ia+ keratinocytes play a synergistic role in the enhanced CH responses that are observed in PT-treated animals. Second, a direct correlation between keratinocyte expression of Ia and CH responsiveness was observed in athymic nude mice that were challenged with oxazolone after receiving an adoptive transfer of lymphoid cells from oxazolone-primed normal syngeneic donors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.     

Dose response of Langerhans cells in mouse footpad epidermis after X irradiation

Effects of a range (2-50 Gy) of single doses of 250 kV X rays on epidermal Langerhans cells in vivo were quantified in groups of CBA/CaH mice. Animals were sacrificed and compared with controls on the 10th day after local irradiation of their hind feet, when Langerhans cell numbers were at a minimum. ATPase-positive Langerhans cells in sheets of footpad epidermis were counted by light microscopy and cells with Birbeck granules were enumerated by electron microscopy. Both methods revealed a dose-dependent loss of Langerhans cells after ionizing radiation. Fifty percent of the ATPase-positive cells were lost after 14.4 +/- 1.3 Gy, and 50% of Birbeck granule-containing cells were lost after 17.9 +/- 4.2 Gy, suggesting that differentiated epidermal Langerhans cells are radioresistant. Loss of equivalent proportions of ATPase-positive and ultrastructurally identifiable cells after a range of doses indicates that X rays do not merely alter Langerhans cell surface markers but actually deplete the epidermal population of these cells.     

Inhibited differentiation of Langerhans cells in the rat epidermis upon systemic treatment with cyclosporin A

The immunosuppressant drug cyclosporin A (CsA) is known to cause reduction in number, DNA synthesis and function of Langerhans cells (LC). Since also the differentiation of LC is known to be hampered in conditions of acquired immunodeficiency not due to drugs, we investigated whether this occurs with CsA. Rats were injected subcutaneously with CsA (5, 10 and 50 mgxkg(-1) x d(-1)) for three weeks; the skin was analyzed by Ia immunohistochemistry and by electron microscopy. Epidermal immunolabeled cells were 15+/-3.5 (mean +/- SEM) per 100 basal keratinocytes in untreated controls and 8.75+/-1.3, 4.75+/-1.0 and 1.7+/-1.2 upon increasing doses of CsA (p<0.01). By electron microscopy, monocytoid cells with deep invaginations of the plasma membrane and roundish LC poor in Birbeck granules appeared in the epidermis upon treatment. The results suggest that CsA inhibits the differentiation of LC precursors in the epidermis and that this can in part explain the selective increase in the risk of skin viral disease and cancer in chronically treated patients.     

Langerhans cells are strongly reduced in the skin of transgenic mice overexpressing follistatin in the epidermis

Activins are members of the transforming growth factor-beta (TGF-beta) family and are important for skin morphogenesis and wound healing. TGF-beta1 is necessary for the population of the epidermis with Langerhans cells (LC). However, a role for activin in LC biology is not known. To address this question, we analyzed skin from transgenic mice overexpressing the activin antagonist follistatin in the epidermis. Using immunofluorescence, we observed a striking decrease in the number of LC in the epidermis of transgenic mice in comparison to wild-type mice. Nevertheless, these LC expressed normal levels of major histocompatibility complex (MHC)-class II and Langerin/ CD207 in situ. In explant cultures of whole ear skin the number of dendritic cells (DC), which migrated into the culture medium, was reduced. This reduction was even more pronounced in cultures of epidermal sheets. Virtually all emigrated cutaneous DC displayed typical morphology with cytoplasmic "veils", showed translocation of MHC-class II to the surface membrane, and expressed the maturation marker 2A1. Thus, cutaneous DC from transgenic mice seemed to mature normally. These results demonstrate that overexpression of follistatin in the epidermis affects LC trafficking but not maturation and suggest a novel role of the follistatin-binding partner activin in LC biology.     

The effects of ultraviolet light and certain drugs on Ia-bearing Langerhans cells in murine epidermis

Langerhans cells and indeterminate cells are immune macrophages of the epidermis and have Ia markers on their surface. Because of their position in the epidermis, they are subject to many environmental toxins like ultraviolet light. Also medications like cortisone applied topically to the skin could have important effects on these cells. We have used an anti-Ia serum and an indirect immunofluorescent technique to study Langerhans cells in epidermal sheets. We found that shortwave ultraviolet light (250–320 nm) and ultraviolet B (280–320nm) increased the density of Ia-bearing cells (Langerhans cells) in the skin. Psoralens and ultraviolet A (PUVA) (320–400 nm) depleted the skin of Ia-bearing cells, an effect which takes 2 weeks to produce but which persists for several weeks after stopping treatment. Triamcinolone acetonide administered topically or intraperitoneally also depletes the skin of Ia-bearing cells. These agents, light and steroids, either destroy the Ia-bearing cells or remove the Ia markers from the cellular surface.     

Relationship between expression of matrix metalloproteinases and migration of epidermal and in vitro generated Langerhans cells

Langerhans cells (LC) are dendritic cells that capture foreign antigens and migrate with them to the regional lymph nodes where they are presented to naive T cells. The possible role of matrix metalloproteinase-9 (MMP-9) in migration was suggested following experiments in a mouse model and in human skin explants. Using in vitro generated LC (iLC) derived from CD34+ cord blood cells and epidermal LC (eLC), we investigated the correlation between MMP-9 and other MMPs production and cell migration. Cells were activated by Bandrowski's base (BB), a chemical allergen, or by recombinant birch pollen allergen 1 (rBetv 1). Contact with allergens triggered migration of these cells, with a maximum rate being reached after 24 h. Migration was preceded by production of MMP-2 and MMP-9; part of the molecules were recovered as pro-MMPs in cell culture supernatant and part were associated with cell membrane proteins. At the cellular level, membrane-type 1 (MT1) and MT3-MMP were also identified. Addition of tumor necrosis factor-alpha (TNF-alpha) initiated pro-MMP-2 and pro-MMP-9 production followed by cell migration in a dose-dependent manner. These data imply that TNF-alpha is a key molecule for MMP production and cell migration. Furthermore, activation of iLC with BB or rBet v 1 induced synthesis of TNF-a and expression of TNF RII on the cell membrane, suggesting an autocrine loop. In conclusion, membrane-associated MMP-2 and-9 rather than soluble MMPs appear to be involved in cell migration.     

Correlation between the 1.6 A crystal structure and mutational analysis of keratinocyte growth factor

A comprehensive deletion, mutational, and structural analysis of the native recombinant keratinocyte growth factor (KGF) polypeptide has resulted in the identification of the amino acids responsible for its biological activity. One of these KGF mutants (delta23KGF-R144Q) has biological activity comparable to the native protein, and its crystal structure was determined by the multiple isomorphous replacement plus anomalous scattering method (MIRAS). The structure of KGF reveals that it folds into a beta-trefoil motif similar to other members of fibroblast growth factor (FGF) family whose structures have been resolved. This fold consists of 12 anti-parallel beta-strands in which three pairs of the strands form a six-stranded beta-barrel structure and the other three pairs of beta-strands cap the barrel with hairpin triplets forming a triangular array. KGF has 10 well-defined beta strands, which form five double-stranded anti-parallel beta-sheets. A sixth poorly defined beta-strand pair is in the loop between residues 133 and 144, and is defined by only a single hydrogen bond between the two strands. The KGF mutant has 10 additional ordered amino terminus residues (24-33) compared to the other FGF structures, which are important for biological activity. Based on mutagenesis, thermal stability, and structural data we postulate that residues TRP125, THR126, and His127 predominantly confer receptor binding specificity to KGF. Additionally, residues GLN152, GLN138, and THR42 are implicated in heparin binding. The increased thermal stability of delta23KGF-R144Q can structurally be explained by the additional formation of hydrogen bonds between the GLN side chain and a main-chain carbonyl on an adjoining loop. The correlation of the structure and biochemistry of KGF provides a framework for a rational design of this potentially important human therapeutic.     

Nitric oxide-mediated depletion of Langerhans cells from the epidermis may be involved in UVA radiation-induced immunosuppression.

Ultraviolet A (UVA) irradiation of the dorsal skin of mice reduced the contact hypersensitivity (CHS) response and the density of epidermal Langerhans cells (LC). The roles of nitric oxide (NO) and reactive oxygen species (ROS) in these biological effects of UVA were investigated. Topical application of N(G)-monomethyl-L-arginine acetate, an inhibitor of NO production, 2,2'-dipyridyl, an iron chelater, or 4-hydroxy-tempo, a superoxide dismutase mimicking agent, inhibited UVA-induced suppression of the CHS response. N(G)-monomethyl-L-arginine acetate but not the ROS inhibitors prevented UVA from reducing LC numbers in the epidermis. This suggests that NO but not ROS produced in response to UVA mediates a depletion of LC from the epidermis, probably by signaling these cells to migrate from the skin. This could be responsible for UVA-induced immunosuppression. UVA-induced ROS can also cause immunosuppression, but by a different mechanism. Agents that inhibit or modulate NO or ROS production may be useful for preventing damage caused by the UVA component of sunlight to the skin immune system.     

Keratin polypeptide expression in mouse epidermis and cultured epidermal cells

Adult mouse epidermis contains up to 11 distinct keratin polypeptides, as resolved by two-dimensional gel electrophoresis. These include both basic (Type II; 67-, 65-, 63-, 62-, and 60-kDa) and acidic (Type I; 61- to 59-, 54-, 52-, 49-, and 48-kDa) keratins that exhibit multiple isoelectric forms. Several, but not all, of these keratins, identified by immunoblotting, were found to be actively synthesized in the skin when assayed in short-term pulse-labeling experiments. When compared to the adult, newborn mouse epidermis expresses fewer keratin subunits. However, greater amounts of keratins associated with differentiated suprabasal cells and stratum corneum, which is more pronounced morphologically in the newborn, were identified. We also observed strain-specific differences in the expression of a Type I acidic keratin. This 61-kDa (pI, approx. 5.3) keratin was produced exclusively by the CF-1 mouse and, based on peptide mapping, appeared to be related to the acidic 59-kDa keratin that was identified in this strain as well as all other mouse strains. The 61-kDa keratin was not expressed in vitamin A-deficient animals, suggesting that its appearance may be related to a retinoid-dependent posttranslational modification. In comparison to keratin expression in vivo, primary mouse keratinocyte monolayer cultures maintained in low Ca2+ (less than 0.08 mM) did not express the terminal differentiation keratins of 67-kDa (basic) or 59-kDa (acidic), although enhanced synthesis of the 60-kDa (basic) and the 52-kDa and 59-kDa (acidic) keratins associated with proliferation were observed. In addition, a subpopulation of nonadherent cells was continuously produced by the primary keratinocyte cultures that expressed the 67-kDa (basic) keratin specific for terminal differentiation. When the keratinocyte cultures were induced to terminally differentiate with Ca2+, the overall pattern of keratin expression was not changed significantly. Taken together, these results provide further evidence for the variable nature of keratin expression in mouse epidermal keratinocytes under different growth conditions.     

Development of ATPase-positive,immature Langerhans cells in the fetal mouse epidermis and their maturation during the early postnatal period

Summary The development and maturation of Langerhans cells during the differentiation of skin was studied in mice from fetal day 13 to adult using 3 indices: (1) ATPase activity; (2) ultrastructure; and (3) quantitative evaluation of the cell population.ATPase-positive Langerhans cells appeared in the epidermis at first at fetal day 16, and they increased in number in the differentiating epidermis during the late fetal period. The earliest appearance of Birbeck granules was at postnatal day 4. Cored tubules were also formed in the Langerhans cells in the dermis at around the same age. The cells containing Birbeck granules or cored tubules are considered to be mature Langerhans cells. In the Langerhans-cell lineage, those cells in the epidermis at stages earlier than postnatal day 4 and not yet containing specific organelles are considered to be immature Langerhans cells. These immature Langerhans cells can be identified ultrastructurally in the epidermis at fetal day 16, coinciding with the appearance of ATPase-positive cells. The increase in the number of immature Langerhans cells during the perinatal period was shown by quantitative analysis of nuclear density and relative Langerhans-cell area on the electron micrographs.It is concluded that ATPase is a marker of the Langerhans-cell lineage from the early development stages, while Birbeck granules and cored tubules are markers that identify mature Langerhans cells in electron micrographs.     

Correlation between expression of selectins and migration of eosinophils into the bovine ovary during the periovulatory period

Leukocytes enter specific ovarian areas at a precisely defined moment, influencing cyclically changing structures such as follicles and corpora lutea. As yet, no studies have been published on the trafficking mechanisms involving the interaction between adhesion molecules on endothelial cells (ECs) and those on leukocytes. First, antibodies against human adhesion molecules were examined by flow cytometry with the aim of identifying the same bovine antigen. Western blot analysis and immunoprecipitation revealed that the molecules had the same molecular weight as their human counterparts. Afterwards, we investigated the distribution of these antigens in various ovarian stages using immunohistology. Among the molecules, P-selectin (CD62P) and L-selectin (CD62L) showed stage-dependent expression, and were thus examined further. In the preovulatory follicle, microvascular ECs were negative for CD62P. Few of the leukocytes expressed CD62L. In a freshly ruptured follicle, CD62P expression was found in the dilated vessels of the former thecal layer. Simultaneously, a large proportion of the rapidly increased numbers of leukocytes, mainly eosinophils, located around the microvessels of the outer thecal layer expressed CD62L. In the early corpus luteum development stage, CD62L showed peak expression with 70%-80% positive cells compared to leukocytes. In the secretory stage, the septal venules showed a consistent, but now weak, staining for CD62P. Few leukocytes expressed CD62L. During regression, the total number of leukocytes, now representing macrophages, increased significantly, but the proportion of CD62L-positive cells remained constant. In summary, we found a strong correlation of CD62P expression on activated ECs and the appearance of CD62L-positive leukocytes in the early corpus luteum development stage, suggesting the participation of both selectins in the migration of eosinophils under physiological conditions.     

HaCaT细胞中FUT4表达与其启动子区甲基化的相关性分析

岩藻糖基转移酶Ⅳ(Fucosyltransferase Ⅳ,FUT4)在正常细胞中表达量很低,但其低表达的调控机制以及是否受其启动子甲基化调控并不十分清楚。文章采用Western blot、免疫荧光和Real-time PCR的方法检测正常人永生化表皮细胞系HaCaT细胞FUT4的表达,观察DNA甲基转移酶抑制剂5-aza-dC处理对FUT4表达的影响。应用甲基化特异性PCR方法分析HaCaT细胞中FUT4启动子甲基化状态。结果表明,HaCaT细胞中FUT4的表达水平明显低于人表皮鳞癌细胞A431和SCC12。5 μmol/L的5-aza-dC处理72 h的HaCaT细胞,其FUT4 mRNA水平明显升高,并且与未经5-aza-dC处理的对照组相比,U引物扩增检测到的产物量增加,M 引物扩增检测到的产物量明显减少。这些结果表明,HaCaT细胞中FUT4的低表达可能与其启动子区CpG岛甲基化有关。