A method for the isolation and serial propagation of keratinocytes,endothelial cells,and fibroblasts from a single punch biopsy of human skin

Summary When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment toUlex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.     

Development of a bilayered living skin construct for clinical applications

An in vitro construct of human skin (living skin equivalent, LSE) has been engineered using serially passaged human epidermal keratinocytes and human dermal fibroblasts with a matrix of type I collagen. Cells are obtained from neonatal foreskin. LSE is cast, cultured, and shipped in a single culture insert. The size and shape of theinsert determines thesize and shape of the LSE. The dermal matrix consists of dermal fibroblasts within a condensed collagen lattice. The overlying epidermis is developed at the air-liquid interface to generate a protective cornified layer. Serum was not necessaryfor development of the epidermis. LSE for graft (Graftskin) has handling characteristics similar to split-thickness skin allowing it to be meshed, stapled, and sutured. LSE was cryopreserved using 65% glycerol an rapid freezing. Viability and in vivo performance on athymic mice were similar to fresh LSE. Cells derived from human eccrine gland were able to invade and form tubules rudimentary appendages may be possible.     

Regional specificity of anuran larval skin during metamorphosis: dermal specificity in development and histolysis of recombined skin grafts

Summary Skins from back and tail were dissected from tadpoles of Rana japonica prior to resorption of the tail and separated into epidermis and dermis by treatment with neutral protease. Homotypically and heterotypically recombined skins were constructed from the separated epidermis and dermis and transplanted into the tail of the original tadpole. Skin grafts using dermis from tail region degenerated simultaneously with resorption of the tail. However, skin grafts containing dermis from back region survived on the posterior part of the juvenile frog beyond metamorphosis. Furthermore, all epidermis underlaid with dermis from back region formed secretory glands and became flattened epithelia characteristic of adult back skin, regardless of region from which the epidermis came. Even when epidermis isolated from tail skin was cultured inside a back skin graft, the tail epidermis survived forming an epithelial cyst and developed secretory glands. These results suggest that regional specificities of anuran larval skin, i.e., development of back skin and even histolysis of tail skin, are determined by regionally specific dermis. The results also suggest that some of epidermal cells of tail skin are able to differentiate into epithelial cells similar to back skin of the adult under the influence of back dermis.     

In vitro and post-transplantation differentiation of human keratinocytes grown on the human type IV collagen film of a bilayered dermal substitute

Using human type IV and type I + III collagens and a new, nontoxic cross-linking procedure, we have developed a cell-free bilayered human dermal substitute for organotypic culture and transplantation of human skin keratinocytes. We have studied the formation of the basement membrane, and the differentiation of keratinocytes grown on the type IV collagen layer of this dermal substitute, in vitro and after grafting onto nude mice. These studies demonstrated the formation of essential constituents of the basement membrane in culture: hemidesmosomes and deposition of extracellular matrix on the top of the type IV collagen were observed as early as 6 days after plating of human keratinocytes. Although the keratinocytes formed a well-organized multilayered epithelium, they exhibited limited differentiation when grown submerged in liquid medium. However, the multilayered sheet obtained after 14 days in submerged culture was composed of a regular basal cell layer, several nucleated suprabasal cell layers containing granular cells, and several dense, anucleated cell layers. The grafting experiments have shown a good biocompatibility of the dermal substitute. It is repopulated by fibroblasts, newly synthesized collagen, vessels, and a few mononuclear cells. At Day 14 after grafting, the type IV collagen layer was still present and very dense, and the basement membrane appeared as in culture, with numerous well-structured hemidesmosomes and deposition of extracellular matrix resembling lamina densa. At Day 55 after transplantation, even if the epidermal graft did not exhibit all the characteristics of the normal epidermis in vivo, it was very close to it. At this stage, the basement membrane was complete, with structures clearly indicative of anchoring fibrils. This new dermal substitute offers many advantages. It is stable and easy to handle. Its production is standardized. The oxidation induced by periodic acid led to a nontoxic cross-linked matrix. This dermal substitute is the first one entirely composed of human collagens. The type I + III collagen underlayer is reorganized when grafted. It supports a type IV collagen top layer which offers an excellent substrate for keratinocytes, favors their anchorage, and favors the formation of the basement membrane in vitro. This dermal substitute could be useful for wound coverage or as an in vitro model for toxicological and pharmacological studies.     

Expression of the WE3 antigen in newt epithelium originating from subcutaneous grafts of skin

mAb WE3 recognizes an antigen that is developmentally regulated in the wound epithelium of regenerating newt limbs. The antigen is precociously expressed when pieces of WE3-negative wound epithelium are grafted subcutaneously (Tassava et al.: Recent Trends in Regeneration Research. New York: Plenum Publishing Co., pp. 37-49, 1989). In the present study, we investigated whether the WE3 antigen is expressed in epidermis of subcutaneous grafts of skin. Small pieces of limb skin were grafted into small tunnels in the lower jaw, limb, and tail, oriented either the same as (epidermis facing out) or opposite to (epidermis facing in) the orientation of the host skin. In most cases, the epithelium migrated from the graft along the wounded surface of the tunnel, closed onto itself, and formed a multilayered "emigrant" epithelium. Infrequently, the migrating epithelium combined with the wound epithelium of the insertion wound. In no case did the epithelium migrate over the cut edge of the grafted dermis. Reactivity to mAb WE3 was first seen at 4 days after grafting, when the migrating epithelium had almost closed over onto itself. By 6 days and thereafter, the entire emigrant epithelium was reactive to mAb WE3. While initially restricted to the emigrant epithelium, at 10 days after grafting and thereafter, reactivity was also seen in the epidermis that remained in contact with the dermis. Expression of the WE3 antigen was not influenced by the orientation of the graft nor by the graft site. The results show that, compared to amputated limbs, the epithelium originating from these grafts precociously expresses the WE3 antigen. Also, epidermis of grafted skin is capable of expressing the WE3 antigen.     

Pigmented Human Skin Equivalent: New Method of Reconstitution by Grafting an Epithelial Sheet Onto a Non-Contractile Dermal Equivalent

We have established a new protocol for reconstituting a pigmented human skin equivalent (PSE) and have evaluated its functional responses to environmental stimulus, UVB. The PSE is reconstituted by grafting an epithelial sheet consisting of keratinocytes and melanocytes onto a porous non-contractile dermal equivalent populated with mitotically and metabolically active fibroblasts. i) The PSE has a multilayered, well-differentiated epidermis with cuboidal basal cells and highly organised dermis with newly synthesised extracellular matrix components. ii) Ki67-positive proliferating keratinocytes (18.1 ± 7.4%) were detected on the basal layer of the epidermis. iii) Melanocytes located exclusively within the basal layer were detected by monoclonal antibody against tyrosinase-related protein (TRP-1). iv) After exposure to UVB (100 mJ/cm² per day) for 7 consecutive days, the intensity of TRP-1 staining was increased in the PSE, showing their functional state, whereas the number of melanocytes was not changed. This non-contractile and functioning new PSE is potentially useful as a model for studying the role of melanocyte-keratinocyte-fibroblast interactions in photoprotection of the skin in more complex cutaneous microenvironment than monolayer culture, and for developing in vitro disease models and therapeutic protocols with genetically altered cells both in epidermis and dermis.     

FORMATION AND ORIGIN OF BASAL LAMINA AND ANCHORING FIBRILS IN ADULT HUMAN SKIN

The purpose of this investigation was to study the formation and origin of basal lamina and anchoring fibrils in adult human skin. Epidermis and dermis were separated by "cold trypsinization." Viable epidermis and viable, inverted dermis were recombined and grafted to the chorioallantoic membrane of embryonated chicken eggs for varying periods up to 10 days. Basal lamina and anchoring fibrils were absent from the freshly trypsinized epidermis before grafting although hemidesmosomes and tonofilaments of the basal cells remained intact. Basal lamina and anchoring fibrils were absent from freshly cut, inverted surface of the dermis. Beginning 3 days after grafting, basal lamina was noted to form immediately subjacent to hemidesmosomes of epidermal basal cells at the epidermal-dermal interface. From the fifth to the seventh day after grafting, basal lamina became progressively more dense and extended to become continuous in many areas at the epidermal-dermal interface. Anchoring fibrils appeared first in grafts consisting of epidermis and viable dermis at five day cultivation and became progressively more numerous thereafter. In order to determine the epidermal versus dermal origin of basal lamina and anchoring fibrils, dermis was rendered nonviable by repeated freezing and thawing 10 times followed by recombination with viable epidermis. Formation of basal lamina occurred as readily in these recombinants of epidermis with freeze-thawed, nonviable dermis as with viable dermis, indicating that dermal viability was not essential for synthesis of basal lamina. This observation supports the concept of epidermal origin for basal lamina. Anchoring fibrils did not form in recombinants containing freeze-thawed dermis, indicating that dermal viability was required for anchoring fibrils formation. This observation supports the concept of dermal origin of anchoring fibrils.     

Migration and differentiation of Langerhans cell precursors

Epidermal Langerhans cells (LC) are the first sentinels of the skin immune system. To study immigration of human LC precursor cells into the skin, we established a two-compartmental skin model consisting of a dermal matrix and an epidermal sheet of keratinocytes. We tested the individual components of the skin model for their influence on phenotype and function of LC precursors. A time window at day 5/6 of differentiation was determined, during which in vitro generated LC precursors expressed adhesion molecules and chemokine receptors required for transmigration across endothelial cell layers and the dermis towards the epidermis. They expressed L-selectin, integrins, platelet endothelial cell adhesion molecule-1, E-cadherin and CC-chemokine receptor 6 and were thus fitted out for transendothelial migration and immigration into the dermis. In a transwell system, these LC precursors migrated towards the chemokine MIP3alpha, demonstrating functional integrity of chemokine receptor 6. For the in vitro reconstituted skin, keratinocytes were grown on a de-epidermized dermis for one to three weeks and formed an epidermal sheet. We allowed LC precursor cells to migrate into this two-compartmental model from the dermal side and examined the presence of CD1alpha--positive cells. LC precursors migrated through the dermal matrix towards the layer of keratinocytes representing the epidermis and could be identified by immunohistology. Experiments designed to investigate the influence of signals provided by both the skin components and by the LC precursors on LC immigration into the skin are in progress.     

The keratin polypeptide patterns in heterotypically recombined epithelia of skin and mucosa of adult mouse

Epithelial-mesenchymal interactions were investigated considering both morphologic criteria and keratin polypeptide expression in homotypic and heterotypic recombinants of adult mouse skin and oral mucosa. Two series of cross-recombinants of epithelia with different morphology and keratin patterns were chosen: (a) footpad epidermis/ear dermis and ear epidermis/footpad dermis; (b) palate epithelium/cheek connective tissue and cheek epithelium/palate connective tissue. Homotypic and heterotypic recombinants were prepared after EDTA-separation of the original tissues and then grown on syngeneic mice in subcutaneously prepared protected graft chambers. EDTA-separation is especially suited to completely separate the epidermal-dermal union, and the transplantation procedure used strictly prevents contamination with host epithelium. Five weeks after implantation keratins were analyzed by one and two-dimensional gel electrophoresis and peptide mapping. In both series, homotypic recombination of the tissues did not alter the original morphology and keratin polypeptide composition of the individual epithelial components. Ear epidermis displayed no significant changes in structure or keratin pattern in heterotypic recombinants. Recombined with ear dermis, footpad epidermis showed acquisition of some morphologic features typical for ear epidermis and slight changes in keratin composition which were, however, difficult to interpret due to the normal similarities of footpad keratin with that of ear. In contrast, the heterorecombinants of the palate/cheek series exhibited considerable alterations in their keratin patterns. Either epithelium showed suppression of distinct keratin subunits and de novo expression of subunits characteristic of the epithelium normally associated with the connective tissue component. The keratin patterns of both matches closely resembled each other and represented patterns intermediate between the normal patterns. This partial, however, significant modulation in the expression of differentiation markers was paralleled by similarly directed changes in the architecture of the heterotransplanted tissues, thus indicating that both morphogenesis and cytodifferentiation of certain adult epithelia can be influenced by extrinsic mesenchymal factors.     

Monoclonal antibody analysis of skin in chronic murine graft vs host disease produced across minor histocompatibility barriers

Chronic graft vs host disease (GVHD) across minor histocompatibility barriers was produced in BALB/c mice by the injection of spleen cells from B10.D2 mice. Changes in the skin were analyzed in frozen sections using a panel of monoclonal antibodies detected by immunoperoxidase methods. Compared to control animals, a number of changes occurred in the skin of animals with chronic GVHD. In the epidermis, there were increased numbers of Thy-1-positive dendritic cells; keratinocytes expressed Thy-1 and Ia antigens. T lymphocytes appeared in both dermis and epidermis. In the early stages, cells with "helper" and "suppressor" phenotypes were present, while at later times "helper" cells remained in the epidermis and "suppressor" cells remained in the dermis. Cells bearing markers of macrophages were prominent in both dermis and epidermis after the second week. Of great interest was the appearance of spindle-shaped cells in the dermis which expressed Thy-1 and Ia. These cells resembled fibroblasts which may be activated to produce the excess collagen seen in the skin of chronic GVHD.     

Composite autologous-allogeneic skin replacement: development and clinical application

A major unsolved problem in skin restoration in severe burns is replacement of lost dermis. We report the development and clinical application of a composite grafting technique in which allogeneic skin is the source of dermis, and cultured autologous keratinocytes generate epidermis. Excised burn wounds are resurfaced with unmatched allograft. Immunosuppression from the burn and reduced immunoreactivity of the allograft permit successful allograft engraftment. Keratinocyte cultures are initiated from the patient. Allogeneic epidermis is removed, and the dermal bed is resurfaced with keratinocyte cultures. The allogeneic dermis promotes rapid (less than 7 days) stratification, maturation, and integration of the cultures and the synthesis of anchoring fibrils. One case followed 11 months has shown no evidence of rejection. We reason that removal of the epidermis from allograft eliminates the majority of cells constitutively expressing alloclass II antigens, leaving behind a viable allogeneic dermal bed that serves as an ideal substrate for engraftment and integration of keratinocyte cultures but does not initiate rejection.     

Expression of acyl-CoA synthetase 5 in human epidermis

The human epidermis is characterized by a constant renewal of keratinocytes embedded in a matrix enriched with lipids. Numerous proteins involved in lipid metabolism are found in human epidermis, especially in keratinocytes. Long-chain acyl-CoA derivatives, which are catalyzed by human ACSL5, are important metabolites in several biochemical pathways, including ceramide de novo synthesis. The aim of the present study was to investigate expression of acyl-CoA synthetase isoform 5 (ACSL5) in human epidermis by an in situ, as well as a molecular approach. We show that ACSL5 mRNA and protein are found in human epidermis, as well as in non-differentiated and differentiated HaCaT cells. Keratinocytes of stratum spinosum are the main source for ACSL5 expression in both meshed facial or abdominal skin and ridged skin of upper or lower extremities including TUNEL-positive cells in upper cellular layers. Single keratinocytes of chronic solar-exposed meshed facial epidermis occasionally display a stronger ACSL5 immunostaining. In conclusion, our study indicates that epidermal ACSL5 expression might be involved in differentiation and the stress response of keratinocytes.     

Adult corneal epithelium basal cells possess the capacity to activate epidermal, pilosebaceous and sweat gland genetic programs in response to embryonic dermal stimuli

Recent work has shown remarkable plasticity between neural and hematopoeitic, as well as between hematopoeitic and muscle stem cells, depending on environmental stimuli (Fuchs, E. and Segre, J. A. (2000) Cell 100, 143-155). Stem cells give rise to a proliferative transient amplifying population (TA), which is generally considered to be irreversibly committed. Corneal epithelium provides a particularly useful system for studying the ability of TA cells to activate different genetic programs in response to a change in their fibroblast environment. Indeed, corneal stem and TA cells occupy different localities - stem cells at the periphery, and TA cells more central (Lehrer, M. S., Sun, T. T. and Lavker, R. M. (1998) J. Cell Sci. 111, 2867-2875) - and thus can be discretely dissected from each other. It is well known that pluristratified epithelia of cornea and skin display distinct programs of differentiation: corneal keratinocytes express keratin pair K3/K12 and epidermal keratinocytes keratin pair K1-2/K10; moreover, the epidermis forms cutaneous appendages, which express their own set of keratins. In our experiments, central adult rabbit corneal epithelium was thus associated either with a mouse embryonic dorsal, upper-lip or plantar dermis before grafting onto nude mice. Complementary experiments were performed using adult mouse corneal epithelium from the Rosa 26 strain. The origin of the differentiated structures were identified in the first case by Hoechst staining and in the second by the detection of beta-galactosidase activity. The results show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. They give rise first to a new basal stratum, which does not express anymore corneal-type keratins, then to pilosebaceous units, or sweat glands, depending of the dermis, and finally to upper layers expressing epidermal-type keratins. Our results provide the first evidence that a distinct TA cell population can be reprogrammed.     

Fibroblast subpopulations: a developmental approach of skin physiology and ageing

Skin is an organ whose function is far beyond a physical barrier between the inside and the outside of the body. Skin as the whole organism is subjected to ageing which concerns skin mostly in its dermal and deepest component which is also its matricial component. The dermis is a tissue rich in matricial elements and poor in cellular content and it is generally admitted that modifications occurring in the matrix are those which mostly contribute to skin ageing, by altering its biomechanical properties. Therefore it is common to address questions related to skin ageing by considering alterations in matrix molecules like collagen. Actually the dermis is a complex tissue both matricial and cellular and is divided between a superficial dermis close to epidermis and a deep dermis much thicker and histologically different. Several years ago we have undertaken investigations related to fibroblasts which are the cells responsible for the formation and maintenance of the dermis, aiming at isolation, culture and characterization of the fibroblasts from the superficial dermis also called papillary dermis and fibroblasts from the deep dermis also called reticular dermis. We were able to show that these fibroblasts in classical culture on plastic exhibit very different morphologies associated with different secretion properties and we have confirmed and expanded such observations revealing different phenotypes by incorporating these cells in reconstructed skin which allows the reproduction of a three-dimensional architecture recalling skin in vivo especially after grafting onto the nude mouse. We also raise the question of how these two dermal regions appear during the formation of the dermis and the question of their fate during ageing. Progress in solving these questions would certainly appear to be very useful for a better understanding of skin physiology and ageing and would hopefully provide new strategies in anti-ageing research.     

Taurine levels and localisation in the stratified squamous epithelia

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.     

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.     

Development of interfollicular epidermis on the surface of collagen framework of the dermis in experimental animals]

The fate of interfollicular epidermis keratinocytes which filled the hair follicles bursas of collagen dermis framework was investigated in the autotransplantation experiment. Collagen dermis framework was prepared from the skin flap. Interfollicular epidermis was detached with the help of suction method and transferred to the collagen dermis framework surface which was placed previously on the full thickness skin defect surface. It was established that in this environment keratinocytes not only developed, but some of them migrated into the hair follicle bursas cavities. It resulted in the follicular-like structures formation, cell elements of which differentiated in the manner characteristic of interfollicular epidermis. However, in spite of the fact that the epidermal cells partially retained their proliferating ability, ingrowing would completely disappear by the 15th to 20th day after the transplantation.     

Take percentage and conditions of cultured epithelium were improved when basement membrane of the graft was maintained and anchoring mesh was added

To achieve a higher take rate for epithelial grafts, this study investigated grafting techniques. Seventy-seven nude mice received flap grafting in which cultured human epithelium was grafted inside the flap, and 55 nude rats received transplantation of epithelium to a full-thickness skin defect. In each group, four models were studied, including model 1, in which epithelium was cultured with the conventional method; model 2, in which epithelium was cultured with fibrin gel to avoid sheet damage, then absorptive mesh was incorporated into the epithelium for anchoring to the graft bed; model 3, in which epithelium was cultured with fibrin gel and combined with absorptive mesh and artificial dermis containing fibroblasts; and model 4, in which the model 2 epithelium was grafted after artificial dermis was transplanted. The take for these models was evaluated grossly and histologically. The results show that the take percentage of models 2 and 3 was significantly higher than that of model 1 (conventional epithelium) and that there was no significant difference between model 3 (simultaneous grafting) and model 4 (two-step grafting). The difference in the take percentages of the grafts to the flap and to the full-thickness skin defect was also insignificant. In immunohistochemistry, human keratin appeared in all epidermis layers and diversification of the layer was observed in models 2, 3, and 4. In these three models, type IV collagen appeared in the basal layer and the formation of basal membrane was confirmed. These findings suggest that epithelia cultured on fibrin gel and combined with absorptive mesh could be used in a new technique for better, more stable take.     

Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder

Transforming growth factor beta1 (TGFbeta1), a potent keratinocyte growth inhibitor, has been shown to be overexpressed in keratinocytes in certain inflammatory skin diseases and has been thought to counteract the effects of other growth factors at the site of inflammation. Surprisingly, our transgenic mice expressing wild-type TGFbeta1 in the epidermis using a keratin 5 promoter (K5.TGFbeta1(wt)) developed inflammatory skin lesions, with gross appearance of psoriasis-like plaques, generalized scaly erythema, and Koebner's phenomenon. These lesions were characterized by epidermal hyperproliferation, massive infiltration of neutrophils, T lymphocytes, and macrophages to the epidermis and superficial dermis, subcorneal microabscesses, basement membrane degradation, and angiogenesis. K5.TGFbeta1(wt) skin exhibited multiple molecular changes that typically occur in human Th1 inflammatory skin disorders, such as psoriasis. Further analyses revealed enhanced Smad signaling in transgenic epidermis and dermis. Our study suggests that certain pathological condition-induced TGFbeta1 overexpression in the skin may synergize with or induce molecules required for the development of Th1 inflammatory skin disorders.     

Expression of Ia antigen on epidermal keratinocytes after the grafting of normal skin to nude mice

During the course of experimentation designed to evaluate the migration of host Langerhans cells (LC) into normal skin grafted onto nude mice, we observed that the epidermal cells of these grafts were induced to express la determinants solely of graft origin. The data presented herein indicate that the expression of la by normal epidermal cells correlates with the infiltration of host LC into the graft. This la expression is restricted to the keratinocytes within the epidermis of the grafted skin, is first observed 7 to 9 days post-grafting, and persists within the grafted skin for greater than 12 wk. The induction of la expression by keratinocytes appears to be the result of the environment that is provided by the nude mouse host and is independent of both passenger lymphocytes within the skin graft and allogeneic differences between graft and host. We strongly believe that these studies provide the basis for the development of an experimental animal model system for investigating the potential role that la expression by epidermal cells may play in enhancing the immune response to antigens encountered in the skin.