Skin Explant Cultures Research Articles

Matrix metalloproteinases 9 and 2 are necessary for the migration of Langerhans cells and dermal dendritic cells from human and murine skin.

Dendritic cells migrate from the skin to the draining lymph nodes. They transport immunogenic MHC-peptide complexes, present them to Ag-specific T cells in the T areas, and thus generate immunity. Migrating dendritic cells encounter physical obstacles, such as basement membranes and collagen meshwork. Prior work has revealed that matrix metalloproteinase-9 (MMP-9) contributes to mouse Langerhans cell migration. In this study, we use mouse and human skin explant culture models to further study the role of MMPs in the migration and maturation of skin dendritic cells. We found that MMP-2 and MMP-9 are expressed on the surface of dendritic cells from the skin, but not from other sources. They are also expressed in migrating Langerhans cells in situ. The migration of both Langerhans cells and dermal dendritic cells is inhibited by a broad spectrum inhibitor of MMPs (BB-3103), by Abs to MMP-9 and -2, and by the natural tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-2. Inhibition by anti-MMP-2 and TIMP-2 define a functional role for MMP-2 in addition to the previously described function of MMP-9. The importance of MMP-9 was emphasized using MMP-9-deficient mice in which Langerhans cell migration from skin explants was strikingly reduced. However, MMP-9 was only required for Langerhans cell migration and not maturation, since nonmigrating Langerhans cells isolated from the epidermis matured normally with regard to morphology, phenotype, and T cell stimulatory function. These data underscore the importance of MMPs, and they may be of relevance for therapeutically regulating dendritic cell migration in clinical vaccination approaches.

A Close-Up View of Migrating Langerhans Cells in the Skin

Langerhans cells of the epidermis and dermal dendritic cells screen the skin for invading antigens. They initiate primary immune responses after migrating from sites of antigen uptake to lymphoid organs. The skin is a feasible model to study the morphology and regulation of dendritic cell migration. We therefore used murine skin explant cultures for tracking the pathways of dendritic cell migration by electron microscopy. Several novel observations are reported. (i) In 48 h cultures of epidermal sheets numerous Langerhans cells migrated out between keratinocytes extending long and thin cytoplasmic processes ("veils"). (ii) Langerhans cells in transition from epidermis to dermis were observed by transmission electron microscopy. Where Langerhans cells penetrated the basement membrane, the lamina densa was focally absent. (iii) This was highlighted by scanning electron microscopy, which presented the basement membrane as a tightly packed and dense network of fibrils. (iv) Scanning electron microscopy of the dermis revealed dendritic cells extending their cytoplasmic processes and clinging to collagen fibrils. (v) Entry of dendritic cells into dermal lymphatics was observed by transmission electron microscopy. It occurred by transmigration through intercellular spaces of adjacent endothelial cells. Entry through wide gaps between endothelial cells also seemed to take place. (vi) Dendritic cells inside the afferent lymphatics frequently carried material such as melanosomes and apoptotic bodies. These observations visualize the cumbersome pathway that dendritic cells have to take when they generate immunity.

Increased levels of keratin 16 alter epithelialization potential of mouse skin keratinocytes in vivo and ex vivo.

The process of wound repair in adult skin is complex, involving dermal contraction and epithelial migration to repair the lesion and restore the skin's barrier properties. At the wound edge, keratinocytes undergo many changes that engender an epithelialization behavior. The type II keratin 6 and type I keratins 16 and 17 are induced well before cell migration begins, but the role of these proteins is not understood. Forced expression of human K16 in skin epithelia of transgenic mice has been shown to cause dose-dependent skin lesions concomitant with alterations in keratin filament organization and in cell adhesion. Here we show, with the use of a quantitative assay, that these transgenic mice show a delay in the closure of full-thickness skin wounds in situ compared with wild-type and low-expressing K16 transgenic mice. We adapted and validated an ex vivo skin explant culture system to better assess epithelialization in a wound-like environment. Transgenic K16 explants exhibit a significant reduction of keratinocyte outgrowth in this setting. This delay is transgene dose-dependent, and is more severe when K16 is expressed in mitotic compared with post-mitotic keratinocytes. Various lines of evidence suggest that the mechanism(s) involved is complex and not strictly cell autonomous. These findings have important implications for the function of K16 in vivo.

Interleukin-16 Supports the Migration of Langerhans Cells, Partly in a CD4-Independent Way

Migration of cutaneous dendritic cells is essential for the induction of primary immune responses. Chemotaxis plays an important part in guiding migrating cells through the skin. Therefore, we investigated the influence of interleukin-16, a potent chemoattractant, on the migratory properties of cutaneous dendritic cells. Interleukin-16 added to murine and human skin explant cultures, enhanced emigration of Langerhans cells as well as dermal dendritic cells out of the skin. In contrast to tumor necrosis factor-alpha, intradermally injected interleukin-16 did not reduce the density of Langerhans cells suggesting a chemotactic rather than a mechanistic migration-inducing effect of interleukin-16. In support of these findings, the known migration-promoting effect of tumor necrosis factor-alpha in skin explant cultures could be neutralized by anti-interleukin-16 antibody and vice versa, indicating different but cooperative ways of action for both cytokines. In whole skin explant cultures blocking of the interleukin-16 effect was also achieved with a monoclonal antibody against CD4, the receptor for interleukin-16. In contrast, in cultures of murine epidermis alone no blocking by anti-CD4 became obvious and in CD4-deficient mice Langerhans cell migration in response to interleukin-16 was maintained. This suggests that another receptor for interleukin-16 might be operative for Langerhans cells in the mouse epidermis. Finally, we detected interleukin-16-positive cells in the dermis of skin explants, tumor necrosis factor-alpha-treated and contact allergen-treated skin. Taken together, it seems likely that locally secreted interleukin-16 might serve to enhance the migration of cutaneous dendritic cells and optimize the response to foreign antigen encountering the skin.

An automated method for the quantification of immunostained human Langerhans cells

Allergic contact dermatitis is a frequent and increasing health problem. For ethical reasons, the current animal tests used to screen for contact sensitizers should be replaced by in vitro alternatives. Contact sensitizers have been shown to accelerate Langerhans cell (LC) migration from human organotypic skin explant cultures (hOSECs) more rapidly than non-sensitizers and it has been proposed that the hOSEC model could be used to screen for sensitizers. However, chemically induced decreases in epidermal LC numbers need to be accurately quantified if the alterations in epidermal LC numbers are to form the basis of an alternative system for screening contact sensitizers in vitro. As manual counting of LCs is labour intensive and subject to intra- and inter-personal variation we developed an image analysis routine, using the Leica QWin image analysis software, to quantify LCs in situ using immunohistochemically stained skin sections. LCs can be identified using antibodies against the membrane molecule CD1a or the Lag antibody, which recognises cytoplasmic Birbeck granules. Quantification of epidermal LC number using the image analysis software had a much lower inter-person variation than when the same specimens were counted manually, using both the anti-Lag and CD1a antibodies. The software-aided quantification of epidermal LCs provides an accurate method for measuring chemically-induced changes in LC numbers.

Methyl green-pyronine staining of porcine organotypic skin explant cultures: an alternative model for screening for skin irritants.

We describe a new alternative method for screening for skin irritants by using fresh intact porcine skin biopsies. Test chemicals were applied to the epidermis of the biopsies, which were then incubated for different times in tissue culture medium at 37°C and with 5% carbon dioxide. A decrease in epidermal keratinocyte RNA staining, visualised in frozen sections by using a modified methyl-green pyronine (MGP) staining procedure, was employed as a marker of irritancy. If a decrease in epidermal RNA was observed after incubation for 4 hours (strong irritant), the chemical had an MGP score of 3; if after incubation for 24 hours (moderate irritant), the MGP score was 2; and if after incubation for 48 hours (weak irritant), the MGP score was 1. If no keratinocyte cytotoxicity was observed after incubation for 48 hours, the chemical was classified as non-irritant (MGP score = 0). At least three ears were used per chemical. The average MGP score was used to classify the chemical. Based on the MGP score for 20% sodium dodecyl sulphate, chemicals classified as strong or moderate irritants by using the MGP test were grouped together as category R38 chemicals. Weak irritants or non-irritants were not classified (NC). The MGP staining correctly identified 23 of 25 skin irritants for which reference data were available.

Inhibition of contact sensitizer-induced migration of human Langerhans cells by matrix metalloproteinase inhibitors.

Emigration of Langerhans cells (LC) from the epidermis upon exposure to contact sensitizers is regarded as an essential event in the development of contact sensitization. Since migration of several types of cells depends on the activity of matrix metalloproteinases (MMPs), in the present study we tested whether MMP inhibitors (BB94, BB2116 and CT1166) can prevent the emigration of LC in cultured skin explants, which were exposed to a contact sensitizer (NiSO4). Epicutaneous application of NiSO4 significantly reduced the number of LC within the epidermis and the remaining LC were localized along the epidermal-dermal junction indicating the emigration of LC. In the presence of each of the MMP inhibitors tested, NiSO4-induced migration of LC was strongly decreased. Since after the epicutaneous application of contact sensitizer, its presentation by skin LC is essential for the development of contact sensitization instead of the development of antigen-specific tolerance, our results suggest that the use of MMP inhibitors may be beneficial for the prevention of contact sensitization of the host.

Influences of keratinocyte–fibroblast interaction on the expression of epimorphin by fibroblasts in vitro

Epimorphin was demonstrated to be a mesenchymal signal factor modulating epithelial morphogenesis of skin, lung and liver in vitro. Most of the previous studies were performed biochemically and functionally. In the present study, expression of epimorphin was immunohistochemically compared between cultured fibroblasts and cocultured fibroblasts with keratinocytes obtained from normal skin. Cultured fibroblasts revealed a low level of epimorphin expression. In contrast, the expression by fibroblasts was greatly enhanced in skin explant culture where both fibroblasts and keratinocytes were present. In three-dimensional coculture of fibroblasts and keratinocytes, the expression of epimorphin was enhanced. The staining pattern of epimorphin in three-dimensional coculture was similar to that in human skin. These results suggest that dermal fibroblasts are manufacturers of epimorphin, and keratinocyte–fibroblast interaction may play important roles in the expression of epimorphin in vitro.

Immunization through dermal delivery of protein-encoding DNA: a role for migratory dendritic cells.

The early mechanisms by which DNA-dependent immunization occurs remain poorly understood. We determined whether intradermal injection of a cytomegalovirus (CMV) promoter-driven plasmid encoding hen egg lysozyme (pCMV:HEL) induced sensitization against the encoded protein, and whether cutaneous dendritic cells (DC) were involved in this sensitization. Both humoral and cellular responses to HEL were observed. DC that migrated from skin explant culture 3 days after injection of pCMV:HEL DNA contained mRNA encoding HEL. They induced a 3.5-7-fold increase in [3H]thymidine incorporation by HEL protein-primed CD4+ T cells compared to that induced by DC from mice injected with control plasmid. DC emigrating from skin explants recovered from pCMV:HEL injected mice also sensitized naive mice after adoptive transfer and induced the generation of CTL. Thus following DNA delivery within the dermis, DC can induce primary and secondary immune responses.

Relationship between telomerase activity, proliferative activity and apoptosis in placenta of normal and abnormal prengancy

Tissue hypoxia is a characteristic feature of malignant tumors and healing wounds, conditions that are associated with angiogenesis and with increased expression of vascular permeability factor (VPF; also called vascular endothelial growth factor, VEGF), a selective endothelial cell mitogen inducing microvascular hyperpermeability in vivo. We investigated the regulation of VPF/VEGF and its receptors by tissue hypoxia in normal human skin explants and in cultured skin cells in vitro. VPF/VEGF mRNA expression was dramatically upregulated in epidermal keratinocytes, dermal fihroblasts, and dermal microvessels after 24 h of skin organ culture. Hypoxia also enhanced the expression of VPF/VEGF in cultured epidermal keratinocytes and dermal microvascular endothelial cells (predominantly VPF/VEGF121 and V-PF/VEGF165) and in dermal fibroblasts (additional upregulation of VPF/VEGF189). The expression of the VPF/VEGF receptor Fit-1 was selectively induced on dermal microvessels in skin explant cultures and in dermal endothelial cell monolayer cultures under hypoxic conditions. In contrast, the KDR receptor was downregulated by hypoxia. These results suggest that hypoxia likely regulates cutaneous angiogenesis and mierovascular permeability by two distinct mechanisms: (i) Induction of VPF/VEGF in epithelial and mesenchymal cells, including endothelial cells. (ii) Differential modulation of VPF/VEGF receptor expression by microvascular endothelial cells. These mechanisms may be of importance in the pathogenesis of healing wounds and some malignant tumors that are commonly characterized by hypoxia and overexpression of VPF/VEGF.

Heparin-binding ligands mediate autocrine epidermal growth factor receptor activation In skin organ culture.

Exogenous EGF and TGF-alpha accelerate wound healing, but treatment effects are often modest. Using short-term human skin organ culture, we found that autocrine EGF receptor activation could account for this observation. Amphiregulin and heparin-binding EGF-like growth factor (HB-EGF) transcripts were rapidly and markedly induced, whereas EGF and TGF-alpha mRNAs were undetectable or only slightly increased. Vascular permeability factor and keratin 6 transcripts were also strongly induced, albeit with a >/= 3 h delay relative to HB-EGF and amphiregulin. All four transcripts were upregulated in actual healing skin wounds, HB-EGF and keratin 6 being the most prominent. The highly EGF receptor-specific tyrosine kinase inhibitor PD153035 strongly inhibited induction of all four transcripts in organ culture, as well as release of immunoreactive HB-EGF into the medium. These effects were confirmed using the anti-EGF receptor mAb 225 IgG. Neither PD153035 nor 225 IgG was toxic to keratinocytes, as judged by calcein-AM uptake. PD153035 completely abrogated the proliferative phase of keratinocyte outgrowth in skin explant cultures, whereas it had no effect on the antecedent migratory phase. Based on these results, we conclude that EGF receptor activation by highly inducible, keratinocyte-derived heparin-binding ligands is an important mechanism for amplification and transmission of the cutaneous wound healing signal.

Hypoxia Regulates the Expression of Vascular Permeability Factor/Vascular Endothelial Growth Factor (VPF/VEGF) and its Receptors in Human Skin

Tissue hypoxia is a characteristic feature of malignant tumors and healing wounds, conditions that are associated with angiogenesis and with increased expression of vascular permeability factor (VPF; also called vascular endothelial growth factor, VEGF), a selective endothelial cell mitogen inducing microvascular hyperpermeability in vivo. We investigated the regulation of VPF/VEGF and its receptors by tissue hypoxia in normal human skin explants and in cultured skin cells in vitro. VPF/VEGF mRNA expression was dramatically upregulated in epidermal keratinocytes, dermal fibroblasts, and dermal microvessels after 24 h of skin organ culture. Hypoxia also enhanced the expression of VPF/VEGF in cultured epidermal keratinocytes and dermal microvascular endothelial cells (predominantly VPF/VEGF121 and VPF/VEGF165) and in dermal fibroblasts (additional upregulation of VPF/VEGF189). The expression of the VPF/VEGF receptor Flt-1 was selectively induced on dermal microvessels in skin explant cultures and in dermal endothelial cell monolayer cultures under hypoxic conditions. In contrast, the KDR receptor was downregulated by hypoxia. These results suggest that hypoxia likely regulates cutaneous angiogenesis and microvascular permeability by two distinct mechanisms: (i) Induction of VPF/VEGF in epithelial and mesenchymal cells, including endothelial cells. (ii) Differential modulation of VPF/VEGF receptor expression by microvascular endothelial cells. These mechanisms may be of importance in the pathogenesis of healing wounds and some malignant tumors that are commonly characterized by hypoxia and overexpression of VPF/VEGF.

Changes in expression of two endogenous ?-galactoside-binding isolectins in the dermis of chick embryonic skin during development in ovo and in vitro

In order to elucidate the roles of metal-independent animal lectins, we systematically investigated changes in expression of 2 kinds of beta-galactoside-binding isolectins (MW 14 and 16 kDa) in the dermis of chick embryonic tarsometatarsal skin during the course of development. These lectins were immunohistochemically located at different stages of development both in ovo and in vitro by light and electron microscopy. Light-microscopic observation showed that while positive staining for the 14-kDa lectin was weak at days 8 and 10 it became intense after day 13. In contrast, staining for the 16-kDa lectin was intense at days 8, 10, and 13, but it became weak after day 17 when keratinization of the epidermis was completed. Immuno-electron-microscopic observation revealed that both the 14 and 16-kDa lectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts. Distribution of the 2 isolectins was also examined in cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by the addition of vitamin A, the distribution of the 14-kDa lectin in the epidermis was significantly affected. These results indicate that (1) the expression of the 2 isolectins is differently regulated in both the dermis and epidermis, (2) the 16-kDa lectin is involved in the early stage of the formation of the dermis and the basement membrane and is replaced by the 14-kDa lectin as keratinization of the epidermis occurs, and (3) the expression of the 2 isolectins in the dermis is not significantly affected by the induction of mucous metaplasia, in contrast to their drastic changes in the epidermis.

Pseudorabies virus infectivity for swine skin characterized in vitro.

The infectivity of pseudorabies virus (PrV) was demonstrated in a cell substrate derived from swine skin explant cultures designated primary porcine skin cells (c/cSLA PPSC). c/cSLA PPSC infected with either wild type or TK- PrV strain Kaplan (Ka) developed typical cytopathologic changes (CPE) as early as 4 h post inoculation (p.i.). The CPE caused by PrV on c/cSLA PPSC was specifically neutralized by covalescent swine sera. Synthesis of late viral proteins was demonstrated in PrV-infected c/cSLA PPSC by indirect fluorescent antibody staining using monoclonal antibodies (mAbs) specific for PrV gIII. PrV induced protein synthesis was further confirmed by specific immunoprecipitation of 35S-methionine labeled viral polypeptides from PrV-infected c/cSLA PPSC with PrV convalescent swine serum, PrV immune mouse serum or mAb to PrV gIII. Moreover, the virus progeny derived from c/cSLA PPSC was shown to be infectious for MDBK cells and this infection was specifically neutralized by PrV convalescent swine serum. The capacity c/cSLA PPSC to support a complete growth cycle of PrV and the relative ease of deriving these cells from pigs can be applied in an autologous fashion in studies of cellular immunity where the MHC needs to be matched.

Skin Explant Culture: A Reliable Method for Detecting Pemphigoid Antibodies in Pemphigoid Sera that Are Negative by Standard Immunofluorescence and Immunoblotting

We investigated the presence of bullous pemphigoid antibodies in bullous pemphigoid sera that are negative by standard indirect immunofluorescence. We incubated each of four indirect immunofluorescence-positive bullous pemphigoid sera, seven indirect immunofluorescence-negative bullous pemphigoid sera, one indirect immunofluorescence-negative herpes gestationis serum, three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera, five indirect immunofluorescence-negative epidermolysis bullosa acquisita sera, and two normal human sera with fresh human skin explants in medium 199 at 4 degrees C for 48 h. All bullous pemphigoid sera, herpes gestations serum, and the three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera had IgG that bound the basement membrane zone of skin explants with moderate to marked intensity as demonstrated by immunofluorescence. Normal sera and indirect immunofluorescence-negative epidermolysis bullosa acquisita sera failed to bind the explant basement membrane zone. Immunoblotting of bullous pemphigoid sera showed five of seven indirect immunofluorescence-negative bullous pemphigoid sera to bind high-molecular weight and/or low-molecular weight bullous pemphigoid antigens from epidermal extracts. We conclude that the skin explant culture system is a very sensitive method for the detection of bullous pemphigoid antibodies in all bullous pemphigoid sera.

Adhesion molecules and homeoproteins in the phenotypic determination of skin appendages.

We examined the roles of adhesion molecules and homeoproteins in the morphogenesis of skin appendages using feather as a model. The expression pattern of these molecules in different stages of feather development were very dynamic. For example, neural cell adhesion molecules are present first in the dermal condensations, then in distal bud epithelium, then in the dermal papilla, and finally in the marginal and axial plates. Tenascin is present first in the placode, then in the anterior bud epithelium and mesoderm, and then in the dermal papilla. The expression patterns suggest that the adhesion molecules are involved in forming the boundary of cell groups that interact to form skin appendages. Antibody perturbation of embryonic skin-explant cultures showed that liver cell adhesion molecules are involved in establishing the hexagonal pattern, neural cell adhesion molecules are involved in the formation of dermal condensations, tenascin appears to be involved in the growth of feather buds, and integrin is essential for epithelial-mesenchymal interactions. Using antibodies to XlHbox 1 (similar to Hox 3.3 or C6) and Hox 4.2 (or D4), we showed that there is a homeoprotein gradient within the feather buds, and that the expression pattern is position-specific. It is hypothesized that Hox codes, derived from the combined expression pattern of homeoproteins, determine the phenotypes and orientation of skin appendages. Experiments using retinoids in the media or retinoid-soaked beads to create a local retinoid gradient are consistent with this hypothesis. As demonstrated here, feather development provides an excellent opportunity to analyze the molecular cascade of skin-appendage morphogenesis.

Production of staphylococcal impetigo-like lesion on human skin explants in culture

We produced a highly reproducible experimental impetigo-like lesion in normal human skin explants in culture. The three Staphylococcus aureus strains we used were an isolate from a human impetigo (E strain), an isolate from a human furunculosis (N strain) and ATCC 29213 strain. E strain was a protein A positive, coagulase type V, producer of exfoliative toxin (ET) and β-toxin. N strain was a coagulase type IV, ET non-producer and α-toxin positive. ATCC 29213 was a coagulase type II, ET non-producer, and α-, β-, and δ-toxin positive. Normal human skin samples were obtained from 8 adult skin surgery patients. One specimen was obtained from human oral mucosa. Small pieces of the samples were slightly abraded on the epidermal surface and cultured on lens paper rafts floating in Eagle's Minimum Essential Medium in an atmosphere of 5% CO 2 and 95% air. Fifty microliters of the respective bacterial suspensions were applied to the epidermal surfaces of the explants. The inoculated surfaces were then occluded under sterile plastic plaster. Histologically, the formation of intraepidermal blisters at the granular layer level with acantholytic cells was observed in all 8 of the skin specimens at 10 h after inoculation with E strain. The specimen from an oral mucous membrane did not produce similar changes with any of the three S. aureus strains. Neither N or ATCC strains developed bullae in the epidermis at 6, 10 or 18 h after inoculation. Immunofluorescent examination revealed that the inner surfaces of blisters in the epidermis were lined with anti-ETA antibody. Under the electron microscope, the blisters of the specimens which had been inoculated with strain E contained only a few S. aureus cells. These results suggest that blister formation at the granular layer level with acantholytic cells is mediated by ET action at the granular layer level and occurs without invasion of lymphocytes or neutrophils, or the involvement of any serum components. Therefore, under appropriate conditions, impetigo could develop even in adults.

The Distribution of IgA Pemphigus Antigen in Human Skin and the Role of IgA Anti-Cell Surface Antibodies in the Induction of Intraepidermal Acantholysis

IgA pemphigus is an uncommon intraepidermal vesiculopustular disease that has clinical and histologic similarity to subcorneal pustular dermatosis and pemphigus foliaceus. All patients have IgA antibodies bound to the epidermal cell surface, and half of the patients have circulating IgA anti-cell surface antibodies detected by standard immunofluorescence testing. We studied the distribution of IgA pemphigus antigen in human skin and the pathogenetic role of circulating IgA antibodies in the induction of intraepidermal vesicle formation. We used skin specimens from numerous sites of two cadavers, as well as from neonatal foreskin, and serum specimens of two patients with IgA pemphigus. Organ culture and immunofluorescence studies revealed the following: (1) IgA pemphigus antibodies bound preferentially to the granular layer in the vast majority of skin sites that were tested. In one cadaver, binding was preferential to the spinous layer of plantar and buttock skin. No binding was observed in oral and esophageal mucosa. (2) Neither bound nor circulating IgA antibody was complement fixing. (3) One IgA pemphigus serum specimen that was negative by standard immunofluorescence had IgA antibodies that bound the epidermal cell surface after incubation under explant culture conditions. (4) Both IgA pemphigus serum specimens induced acantholysis in skin explant cultures. When antibodies from one IgA pemphigus serum specimen are used, the expression of IgA pemphigus antigen in human skin shows regional variability, interindividual variability, and variability in the microscopic distribution within the epidermal cell layers. IgA pemphigus antibodies play a role in the pathogenesis of IgA pemphigus. The skin explant culture is more sensitive than is standard immunofluorescence to detect circulating IgA antibodies.

Identification and partial purification of a large, variant form of type XII collagen.

A large, alternate form of type XII collagen has been identified in cultures of the human epidermoid cell line WISH. This form, designated XIIA, is comprised of alpha chains that are approximately 90 kDa larger than the 220-kDa alpha chain previously characterized in extracts of fetal chicken and bovine tissues. Results from both collagenase digestion and rotary shadow analysis of partially purified material show that the increase is due to a larger NC3 domain. While both the large (XIIA) and the small (XIIB) forms of type XII collagen are identified in pulse-chase radiolabeling of fetal bovine skin explant culture, they are not related in a precursor-product fashion. Inhibition studies with alpha, alpha'-dipyridyl indicate that proper folding of the collagen helix is required for complete assembly and secretion of type XIIA in WISH cell culture. The 310-kDa alpha 1A chain is likely to represent the bovine equivalent of a second translation product, estimated to be 340 kDa, predicted from analysis of one complete chick cDNA sequence. Additionally, the amino-terminal amino acid sequence of the 220-kDa bovine alpha 1B chain was determined. This sequence is very near a potential alternate splice site predicted from analysis of chicken type XII cDNA.

UVA- and UVB-induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice

The modifications induced in hairless mouse skin by chronic UV irradiation were investigated. Skin explant cultures were used to study UVA- and UVB-induced changes occurring in interstitial collagen (type I and type III) and fibronectin biosynthesis. To study the long-term effects, albino hairless mice were irradiated with UVA radiation alone from two sources with different spectral qualities or with UVB. UVA and UVB radiation produced a significant increase in the ratio of type III to type I collagen (more than 100% for UVA-irradiated skin and about 60% for UVB-irradiated skin) accompanied by a significantly increased fibronectin biosynthesis (50% or more in all irradiated groups). Irradiation with either UVA or UVB alone had no significant effect on the total collagen synthesis and resulted in only a slight decrease in the total collagen content of the skin determined as hydroxyproline. This decrease was significant only in the case of the group irradiated with UVA (xenon) (decrease of 25%, expressed as micrograms of hydroxyproline per milligram wet weight). A significant decrease in collagen hydroxylation (expressed as radioactive hydroxyproline/radioactive hydroxyproline plus proline in neosynthesized collagen) was observed of about 50% in skin irradiated with UVA (xenon) but not in UVB-treated skin. Several of the above modifications (increased fibronectin biosynthesis, increased collagen type III to type I ratio) correspond to the modifications observed during the aging of non-irradiated hairless mice. Therefore it appears that UV irradiation accelerates the modifications of extracellular matrix biosynthesis observed during aging.