Uncoordinate expressions of type I and III collagens, collagenase and tissue inhibitor of matrix metalloproteinase 1 along in vitro proliferative life span of human skin fibroblasts. Regulation by all-trans retinoic acid.

In vitro human skin fibroblasts aging was characterized by a continuous increase of collagenase mRNA levels. On the contrary, TIMP-1 mRNA level decreased only at late passages (> 65% of proliferative life span). Type I and III mRNA levels showed a high variability depending on cell strains studied. However, type I and III collagen expressions varied parallely. All-trans retinoic acid (RA) decreased collagenase expression and stimulated TIMP-1 expression. Under RA action, high variability in mRNAs levels encoding type I and III collagens was observed with HSF passages. However, RA tended to correct variations in collagens expressions observed along HSF life span.

[Tissue inhibitors of matrix metalloproteinases TIMP 1-2. Structures and functions]. / Inhibiteurs tissulaires des métalloprotéinases matricielles TIMP1-2 Structures et fonctions.

Tissue inhibitors of matrix metalloproteinases (TIMPs) represent a family of ubiquitous proteins which main biological function resides in their ability to control the activity of matrix metalloproteinases (MMPS). The structures and specificities of TIMP1 and TIMP2 are described. Moreover, TIMP1, but not TIMP2, is able to stimulate the growth of several cell types. TIMP1 interacts with keratinocytes via a receptor mediated pathway (Kd = 8.7 nM; 135,000 sites/cell). Thus, this inhibitor does contain several structural domains able to recognize i) MMP active site, ii) pro-MMP9 hemopexin-like domain, iii) cell membrane proteins.

Growth stimulation of human keratinocytes by tissue inhibitor of metalloproteinases.

Human recombinant tissue inhibitor of metalloproteinases (rTIMP) at 0.2-4.6 microM was found to stimulate the growth of normal human keratinocytes, in primary cultures on a plastic support, and to markedly increase their growth on a tridimensional culture system, the skin equivalent, as shown by histology, DNA measurements, and planimetry. In contrast, rTIMP had no effect on the growth of normal human fibroblasts. The growth of keratinocytes on extracellular matrix components produced by keratinocytes cultured in the presence or absence of rTIMP was similar, suggesting that rTIMP does not stimulate keratinocyte growth by modifying either the quantity or the composition of the extracellular matrix deposited. rTIMP was labeled with 125iodine in order to study its interaction with keratinocytes in culture. Binding of (125I) rTIMP to keratinocytes was found to be temperature and time dependent. Under steady-state conditions at 22 degrees C, one class of specific rTIMP binding sites was identified with KD of 8.7 nM and 135,000 sites/cell. Such findings are in keeping with the known potentiating effect of TIMP on erythroid precursors, and indicate that this protein has at least two distinct activities.

[Stimulation of epidermal growth in equivalent skin by tissue inhibitor of metalloproteases]. / Stimulation de la croissance épidermique dans la peau équivalente par l'inhibiteur tissulaire des métalloprotéinases.

Tissue inhibitor of metalloproteases (TIMP) is a major regulator of extracellular matrix synthesis and degradation. Moreover elevated tissue inhibitor of metalloproteases levels are found in the blister fluid of many diseases, suggesting that tissue inhibitor of metalloproteases plays a role in epidermal and dermal wound healing. The aim of this work was to study the effects of human recombinant tissue inhibitor of metalloproteases in a skin equivalent model used to evaluate the effects of drugs on epidermal and dermal components. Planimetry, DNA assays, and histologic studies showed that tissue inhibitor of metalloproteases enhanced the growth of normal human keratinocytes on dermal equivalent. Thus, the extracellular matrix regulator tissue inhibitor of metalloproteases also stimulates the growth of keratinocytes. Identity of the gene encoding tissue inhibitor of metalloproteases and Erythroid Potentiating Activity (EPA) has been reported. The effects of tissue inhibitor of metalloproteases on keratinocytes described herein are reminiscent of the stimulating effect of erythroid potentiating activity on the growth of erythroid precursors.

Growth of melanocytes in a skin equivalent model in vitro.

We have developed a pigmented human skin equivalent by inserting a punch biopsy of human infant foreskin as a source of epidermis into a collagen lattice (dermal equivalent). Using a conventional epidermal culture medium and stimulation with UVB irradiation or 8-MOP + UVA treatment, melanocytes were found to grow out from the biopsy with the epidermal sheet. In this newly formed epidermis, melanocytes and keratinocytes were maintained in an architectural relationship similar to that present in vivo and melanocyte outgrowth could be quantitatively evaluated. Consequently, this pigmented human skin equivalent is a useful model for investigating the biology and photobiology of human skin pigmentation.

Cantharide acantholysis: endogenous protease activation leading to desmosomal plaque dissolution.

Using a method which allowed us to study the morphological consequences of the expression and the inhibition of proteases in living tissues, we demonstrated that the primary detectable cellular event in cantharide acantholysis is the dissolution of the dense plaque, leading to the detachment of tonofilaments from desmosomes. This process is inhibited by neutral serine protease inhibitors. This suggests that the desmosome-tonofilament complex, more precisely the desmosomal dense plaque, is the primary target of activated proteases during cantharide acantholysis, and can be disrupted by a specific epidermal protease-anti protease system. Cantharide acantholysis may be useful model for studying desmosomal turnover.

Reconstructed human epidermis: absence of Langerhans cells and failure to stimulate allogeneic lymphocytes in vitro.

Whole human skin can be reconstructed in vitro, using dermal equivalents made of fibroblasts in a collagen matrix. We recently described a new method of epidermalization of dermal equivalents, based on the insertion of punch biopsies and the migration of epidermal cells (EC) on the reconstructed dermis. In the present study, we show that no MHC class II or T6 positive Langerhans cells (LC) can be detected in this new epidermis. Functional studies with EC of this reconstructed epidermis show that these EC completely fail to induce proliferation of allogeneic lymphocytes in mixed epidermal cell lymphocyte reactions and to raise an allogeneic T cell response. In contrast, fresh EC from the same donors induce a strong proliferative and cytotoxic response of the same effector cells. Moreover, the addition of fresh LC-containing EC autologous to effector lymphocytes does not restore an allogeneic proliferative and cytotoxic response directed against class I different EC of the new epidermis. Such a non-immunogenic whole skin model composed of two compartments, dermis and epidermis, completely devoid of class II-bearing antigen presenting cells, is thus a very promising technique for allogeneic skin grafting in the treatment of burns.

Autoradiographic study of the effects of psoralen and ultraviolet light on DNA, RNA and protein synthesis in cultured keratinocytes and psoriatic skin.

DNA, RNA and protein synthesis were studied by autoradiography in cultured keratinocytes, immediately, 24 and 48 h after 8-methoxypsoralen and ultraviolet light, (PUVA) treatment. Using the same technique, the immediate and long-term effects of PUVA therapy on DNA, RNA and protein synthesis were analysed in skin biopsies from psoriatic patients. In the cultures, immediately after irradiation, DNA and RNA syntheses were similarly inhibited in a dose-dependent manner, while protein synthesis was slightly affected only for the highest dose. After 24 h, RNA synthesis recovered whereas DNA synthesis was more severely inhibited suggesting that other cell components may be damaged by PUVA. In patients, DNA and RNA syntheses decreased immediately after PUVA sessions. During all the sessions until the psoriatic plaques had cleared, an impairment of DNA synthesis was observed in comparison with the synthesis in involved and uninvolved skin before treatment. These results suggest that the therapeutic efficiency of PUVA is based on the inhibition of DNA replication due to direct effects on nucleic acids but also to photoreactions with other cell components.

Psoriasis: a defect in the regulation of epidermal proteases, as shown by serial biopsies after cantharidin application.

The possible role of epidermal serine proteases in the genesis of psoriatic lesions was investigated by sequential biopsies of the epidermal damage induced by topical cantharidin. In the skin of normal subjects, epidermal damage was followed by the transient appearance of proteolytic activity in the upper epidermis accompanied by temporary hyperacanthosis and perivascular inflammatory cells in the superficial dermis. In the uninvolved skin of five patients with psoriasis this proteolysis persisted longer, for more than 7 days. Thereafter, in three of the patients, the proteolysis abated, and this was followed by disappearance of the hyperacanthosis and the dermal infiltrate; in the other two psoriatics the proteolysis and hyperacanthosis increased, and a typical Koebner phenomenon ensued. Migration of neutrophils into the epidermis occurred as a later event. Thus the abnormal persistence of proteolytic activity in the upper epidermis after cantharidin application distinguishes the normal from the psoriatic skin injury response and might initiate the psoriatic lesion.

Autoradiographic localization of 8-methoxypsoralen in psoriasis skin in vitro.

Slices of psoriatic (Pso) skin were incubated with tritiated 8-methoxypsoralen (8-MOP) and exposed to UVA irradiation. The photobinding of 8-MOP was studied by autoradiography at the cellular level and in the different layers of the epidermis and in the dermis. Silver grains were found in the nucleus and the cytoplasm of the various cell types. Keratin, collagen and lipoproteins were also labelled. The upper malpighian cells and the parakeratotic cells showed a greater degree of labelling than did the basal layers. In skin incubated with [3H]8-MOP and unirradiated, no measurable labelling was detected. These results suggest that the targets responsible for the therapeutic activity of 8-MOP might be not only nucleic acids, but also proteins.

Cellular events leading to blister formation in bullous pemphigoid.

Cellular events occurring in eight patients with bullous pemphigoid were studied by light and electron microscopy. Sections (0.5 micrometer) of large surface area, overlapping blisters and surrounding skin, were examined and correlated ultrastructural studies were performed on selected areas. The peroxidase contained in granules of neutrophils, eosinophils and young macrophages was visualized by incubation with diaminobenzidine and hydrogen peroxide. This cytochemical reaction was used as a marker to study the release of granule enzymes from these inflammatory cells. The release of such enzymes from eosinophils and occasionally from macrophages on the epidermal basement membrane (more precisely in the lamina lucida) was demonstrated in the skin surrounding the blisters in four patients. The release of these enzymes was also observed in the floor of the blisters in all eight patients. It is well known that these granules contain several proteolytic enzymes. These observations are therefore consistent with the proposal that proteolytic enzymes of eosinophils play a pathogenic role during the initial stages of blister formation in bullous pemphigoid.

Endogenous peroxidases: a morphological and functional marker to study inflammatory skin diseases.

The visualization of peroxidase positive lysosomes by diaminobenzidine plus H2O2 allowed easy identification of neutrophils, eosinophils, monocytes and young tissue macrophages in 0.2 micrometer to 0.5 micrometer skin plastic sections of large surface area. Evaluation of the lysosomal functions of these phagocytic cells was greatly improved by the possibility of correlating observations on the same cell at light and electron microscopic levels.

A simple method for correlating observations on skin at the light and electron microscopic levels.

A method is described which allows the embedding of 6 mm cutaneous punch biopsies for subsequent light and electron microscopic examination. Sections of 20 mm2 cut at 0.5-I micrometer are stained for light microscopy. The subtle tinctorial affinities, which approach those achieved on haematological smears, and the preservation of cellular detail frequently make subsequent ultrastructural examination unnecessary. However, when required, a special technique for trimming and resectioning the tissue blocks allows a good correlation between light and electron microscopic observations.