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Background: It is widely accepted that CD40 plays a critical role in the regulation of immune response. However, the significance of CD40 expression on normal human keratinocytes is only partially known., Objectives: To perform a morphological re-examination of the role of CD40 on the differentiation of human keratinocytes and remodelling of the epidermis., Methods: Keratinocytes were grown on fibroblasts transfected with the CD40 ligand (CD40L) to investigate the formation of epidermal sheets in culture under the influence of the CD40L. Control experiments were carried out using the same cells but transfected with CD32. Further, three specific anti-CD40 monoclonal antibodies were used as soluble agonists to analyse the effect of CD40 ligation on keratinocyte differentiation., Results: Epidermal sheets developing from keratinocytes cocultured with fibroblasts transfected with CD40L but not with CD32 showed an up to 50% reduction in thickness compared with control sheets. This change depended mostly on cellular flattening and a decrease in the number of cell layers, and was coincident with a transient decrease in cell surface CD40 immunoreactivity. On the other hand, normal epidermis, and freshly isolated and cultured keratinocytes revealed a predominant CD40+/Ki-67- phenotype that was demonstrated by double immunocytochemistry. Consistent with these observations, keratinocytes primed with interferon-gamma responded to the three soluble agonists, but not to control IgG1, producing immunoreactive (pro)filaggrin and displaying morphological changes in shape and size equivalent to those seen in differentiated cells., Conclusions: As a whole, our findings provide evidence that CD40+ keratinocytes represent a poorly differentiated population, not actively engaged in the cell cycle, which under specific stimulation is committed towards terminal differentiation.

Background: Normal and dysregulated wound healing involves fibroblast activation and angiogenesis, in which polypeptide factors such as transforming growth factor (TGF)-beta, vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) play an important part. Ultraviolet (UV) A1 (365 nm) has recently received attention as a possible treatment for some dermal fibrotic disorders., Objectives: The aim of this study was to evaluate the effects of TGF-beta1 and UVA1 radiation, as well as that of cobalt chloride, reported to mimic hypoxia both in vivo and in vitro, on the expression of VEGF and ET-1 by cultured human dermal fibroblasts., Methods: Levels of VEGF and ET-1 were measured by enzyme-linked immunosorbent assay and expression of neutral endopeptidase (NEP, CD10), known to degrade ET-1, was quantified by flow cytometric analysis after cell trypsinization., Results: Our results showed that the cells released minor amounts of VEGF and ET-1. Both TGF-beta1 and UVA1 strongly increased VEGF secretion in a dose- and time-dependent manner, without significantly affecting ET-1 release. Irradiation of TGF-beta1-stimulated fibroblasts resulted in a synergistic effect on increasing levels of VEGF but not ET-1 after 48 h. Cobalt chloride stimulated the secretion of VEGF by fibroblasts; the effects of TGF-beta1 and cobalt were additive. However, no significant effect of cobalt chloride on ET-1 secretion was observed, suggesting that ET-1 production in fibroblasts is not oxygen-sensitive. The expression of NEP was not modified by TGF-beta1 or UVA1 radiation. Addition of a neutralizing anti-CD10 antibody to fibroblast cultures downregulated CD10 expression at the cell surface without changing ET-1 levels in cell supernatants after 24 or 48 h. This suggests that membrane-bound NEP has minimal or no activity against secreted ET-1., Conclusions: Taken together, these results underline the major role played by TGF-beta1 in increasing VEGF secretion by fibroblasts. This, as well as the documented effect of UVA1 on increasing VEGF production, may have implications for wound healing in vivo.

Hereditary skin disorders involving acantholysis, such as Hailey-Hailey disease and Darier's disease, have been genetically linked to distinct chromosomal parts which do not code for known structural proteins. Such evidence suggests that the genomic abnormalities underlying these dermatoses may concern functional/regulatory mechanisms of keratinocyte cohesion. Epidermal communication junctions (gap junctions) are responsible for direct coupling of cells and, thus, co-ordinate the behaviour of keratinocytes within the tissue. Consequently, they remain one of the potential, and poorly studied, elements in the pathogenesis of hereditary acantholytic diseases. We have investigated the distribution and fate of gap junctions during non-immune acantholysis, using fine immunolocalization methods at the light and electron microscopic levels. Our results demonstrate normal expression of epidermal gap junction proteins, connexins 26 and 43, in non-lesional skin of Hailey-Hailey and Darier's diseases. The gap junctions were not primarily dismantled during acantholysis, typical of both of the studied dermatoses, but underwent internalization and subsequent cytoplasmic dispersion in the portions of cells which were no longer attached to the rest of the tissue. In Darier's disease, perifollicular acantholysis did not specifically concern epithelium of appendages coexpressing connexin 26 in addition to connexin 43, further indicating that the observed changes in gap junction localization were secondary to the loss of cell-cell contact. We demonstrated that the sequence of changes was identical in both diseases and that the previously described putative differences were apparently related to the degree of acantholysis present in the studied biopsies. The fate of the junctional structures and proteins, documented in the present study, is most probably a form of recycling process also used by normal keratinocytes during organogenesis and tissue differentiation.

Erythema and the initiation of an inflammatory response are typical features of human skin after ultraviolet (UV) radiation (UVR) exposure. Among the soluble factors that account for the induction of an erythema, the most recently discovered is vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a potent inducer of microvascular permeability which is expressed by keratinocytes. As epidermal cells are the first target cells of UVR, we studied the effects of UVBR (312 nm) and UVA1R (365 nm) on the secretion of VEGF by normal human keratinocytes and evaluated the role of interleukin 1 alpha (IL-1 alpha) and tumour necrosis factor-alpha (TNF-alpha) in this process. UVBR (100 and 200 mJ/cm2) induced a dose-dependent increase in the release by normal human keratinocytes of VEGF, which is widely mediated through the release of TNF-alpha but not IL-1 alpha. Conversely, UVA1R (5 and 7 J/cm2) did not modify the basal level of VEGF and did not induce the secretion of TNF-alpha by keratinocytes. Moreover UVA1R, when associated with UVBR, inhibited the increase in VEGF induced by UVBR alone. Taken together, these findings indicate that UVBR and UVA1R have a contrasting effect on the release of VEGF, which is widely mediated by TNF-alpha. They may partly explain the minor erythematous effect of UVA1R and its beneficial role in cutaneous phototherapy.

The stratum corneum (SC) has long been considered as a sort of inert membrane destined to be shed at the surface of the epidermis. During the last two decades, however, several lines of evidence have been reported, suggesting that active physical and chemical changes take place in the horny layer despite the absence of intracytoplasmic organelles. In particular, processing of filaggrin, replacement of the plasma membrane by a ceramide envelope and constant, progressive modification of extracellular lipid multilayers have been put forward. Recently, attention has focused on the intercellular junctions, which may be involved in the regulation of SC desquamation. Corneodesmosin, a newly discovered protein of s.c. desmosomes (corneodesmosomes), is synthesized at the latest stages of keratinocyte differentiation and persists between the horny cells until desquamation occurs. In the present study, we performed immunohistochemical and immuno-ultrastructural investigations on corneodesmosin expression in various skin lesions characterized by abnormal production and/or retention of the horny layer. Our results suggest that corneodesmosin expression is independent from profilaggrin synthesis. We found corneodesmosin in almost all morphologically recognizable corneodesmosomal structures and specifically those which persisted up to the SC surface. Hyperkeratotic lesions which are characterized by an increased number of junctions showed intense immunoreactivity with anticorneodesmosin antibody. A complete absence of corneodesmosin was not observed in any disease. This finding, together with our previous biochemical studies, suggests that corneodesmosin may exert a protective function against proteolytic degradation of corneodesmosomes both in normal skin and in the pathological horny layer.

In allergic and irritant contact dermatitis, keratinocytes are major target cells that can be activated to take part in local reactions by secreting soluble mediators. Among the growth factors produced by keratinocytes, vascular endothelial growth factor (VEGF) is a powerful inducer of permeability of endothelial cells, and is involved in inflammation. We determined whether different contact allergens, dinitrosulphobenzene (DNSB), para-phenylenediamine (pPD) and the metals nickel and chromium, as distinct from cobalt, which has been shown to mimic the effects of hypoxia, can modify the basal level of VEGF in normal human keratinocytes when tested at various, non-toxic concentrations. The effects of an irritant, sodium lauryl sulphate (SLS), and of hydrocortisone were also tested. Our results showed an intense dose-dependent upregulation of VEGF release by keratinocytes after treatments by metals, pPD and SLS. DNSB induced only a moderate increase of VEGF. Hydrocortisone reduced the basal level as well as the nickel-induced upregulation of VEGF. These findings suggest that contact allergens and irritants probably upregulate VEGF in keratinocytes by different mechanisms and may contribute directly to the microvascular hyperpermeability which characterizes both contact and irritant dermatitis.

Somatostatin (SOM) is a ubiquitous peptide which is responsible for the inhibition of numerous biological functions. SOM is described as an antiproliferative molecule and an inhibitor of exocrine or endocrine secretion from a variety of tissues, including pancreas, gastrointestinal tract, central and peripheral nervous system. Mediation of SOM effects can be indirect or direct, respectively, through other molecules or receptors on target cells. We have searched for the presence of SOM in the epidermis using immunofluorescence, confocal laser scanning microscopy, radioimmunoassay, and chromatography. Immunofluorescence and confocal laser scanning microscopy studies were performed using rabbit antiserum anti-SOM and mouse monoclonal antibody directed to CD1a Langerhans cell (LC) marker disclosed with fluorescein or tetramethylrhodamine isothiocyanate conjugates. SOM was extracted from whole skin or epidermal cell suspension or LC-enriched suspensions and analysed by radioimmunoassay. We used an antiserum which was reactive for the 6-11 portion of native SOM. Chromatographic columns were performed on extracts from whole skin. The epidermis was SOM immunoreactive. LC were immunoreactive for SOM and the staining was membranous. SOM was extracted from the whole skin at about 0.13 +/- 0.02 fmol/mg of tissue (mean +/- SEM). The SOM concentration in epidermal cell suspensions was 1.5 +/- 0.9 fmol/10(6) cells. Data obtained with LC-enriched suspensions showed large variations between donors. Extracts from skin showed one peak with an elution profile like that of 14 amino acid SOM. This study demonstrates that 14 amino acid SOM is expressed in normal human epidermis.

The densities of feline epidermal dendritic cells expressing CD18, MHC class II and CD1a antigens were determined for four anatomical locations in 19 cats of European breed in blind conditions. The densities (+/- SD) of CD1a+ Langerhans cells in the skin of the abdominal wall (269 +/- 68 cells/mm2), the back (363 +/- 19), the internal side of the ear (572 +/- 30) and the external side of the ear (502 +/- 32) were significantly different, with young and old animals displaying less stained cells than adults. No significant differences in the mean densities were found with regard to sex, colour or antibody used.

Over the past 8 years, we have followed a child born as a harlequin baby, who survived due to treatment with retinoids. His condition evolved clinically towards the erythrodermic form of lamellar ichthyosis (non-bullous congenital ichthyosiform erythroderma, NBCIE). According to ultrastructural and biochemical criteria, our patient originally presented with type II harlequin ichthyosis. Investigations showed an abnormal keratinosome structure and extrusion, a keratin pattern characteristic for epidermal hyperproliferation, and an absence of conversion of profilaggrin to filaggrin. Persisting keratinocyte hyperproliferation, associated with the presence of a dermal infiltrate, is in agreement with the present clinical picture of severe NBCIE. However, abnormal lamellar body production and defective filaggrin processing, which is not one of the diagnostic criteria of NBCIE, persist in the patient's skin. Further studies of the epidermal lipid composition, and of possible mutations of the keratinocyte transglutaminase gene performed on epidermal cell cultures of harlequin ichthyosis, will be necessary before type II harlequin ichthyosis can be accepted as an extremely severe form of NBCIE.

ICAM-3 is a newly recognized adhesion molecule, which is a member of the immunoglobulin supergene family of ICAMs, and has been shown to be identical with the CD50 antigen. Recent functional studies have shown that ICAM-3 is a ligand for LFA-1, and plays an important part in immune reactions. To date, very few data exist in the literature concerning its expression in the skin. In the present study, we investigated the expression of ICAM-3 in normal skin and in 98 biopsy specimens of various inflammatory and neoplastic dermatoses. ICAM-3 was found to be expressed by epidermal CD1a+ Langerhans cells, by cells of Langerhans cell histiocytosis, by T and B lymphocytes infiltrating the dermis in cutaneous lymphomas and in a wide spectrum of inflammatory dermatoses. Epidermal keratinocytes were consistently negative; endothelial expression of ICAM-3 was observed in six of the 98 cases. These results show that ICAM-3 is constitutively and widely expressed by cells participating in inflammatory dermatoses (including Langerhans cells and T and B lymphocytes), and that it can be, albeit rarely, induced on endothelial cells and dermal dendrocytes. These results highlight the important part that ICAM-3 may play in cutaneous inflammatory and immune reactions.

In order to obtain greater insight into the nature of B-cell epitopes in bullous pemphigoid (BP), we generated a BP recombinant protein of 55 kDa M(r) (rBP 55) from a cDNA sequence encoding for the carboxyterminal region of the 230 kDa BP antigen. Serum IgG from guinea-pigs immunized with rBP 55 stained the basement membrane zone of normal human skin and immunoprecipitated the rBP 55 protein, and also the 230 kDa BP antigen recovered from extracts of cultured keratinocytes, thus confirming that the rBP 55 amino acid sequence is present in native BP antigen. The reactivity of sera from 60 patients with BP was analysed using an immunoblot assay on epidermal protein extracts and on the rBP 55 protein. Forty of the 60 BP sera (66%) contained autoantibodies to the 230 kDa polypeptide in an epidermal extract, and 37 of these 40 sera (92%) recognized the rBP 55 protein. In contrast, no reactivity against rBP 55 was detected with 20 BP sera devoid of autoantibodies against the 230 kDa antigen. Likewise, sera from patients with autoimmune blistering skin disorders other than BP (epidermolysis bullosa acquisita or pemphigus vulgaris), and control sera, were unreactive to rBP 55. These results clearly demonstrate the immunogenicity and antigenicity of the C-terminal end of the 230 kDa BP antigen. They confirm that this 555 amino acid segment, corresponding to rBP 55, contains major epitopes which can bind BP patients' autoantibodies, and suggest that the rBP 55 protein could be useful for further characterization of these B-cell epitopes.

Patch tests with nickel on sensitive subjects induce a characteristic allergic reaction involving epidermal and dermal cells, as well as modulation of cytokines and adhesion molecule production. In order to gain further insight into the role of keratinocytes in this phenomenon, we assessed their activation state induced by Ni2+ by studying interleukin 1 (IL-1) production and intercellular adhesion molecule 1 (ICAM-1) expression, using normal human keratinocytes cultured in defined medium. In comparison with controls, the addition of subtoxic NiSO4 concentrations (0.1-20 micrograms/ml) to keratinocyte cultures induced a significant, but low release of IL-1 alpha and IL-1 beta at 24 and 48 h, detectable by enzyme-linked immunosorbent assay of the supernatants of treated cells. Moreover, IL-1 receptor antagonist was significantly increased in the supernatants and the cell extracts. Using fluorescence-activated cell sorter analysis, ICAM-1 expression at 24 h was found to be induced in a dose-dependent manner, reaching a level comparable with that obtained upon interferon-gamma (10 IU/ml) stimulation. Overall, these data confirm the existence of direct interactions between Ni2+ and keratinocytes, which generate immunological signals of major importance in the pathophysiology of allergic contact dermatitis.

HB8 is a new monoclonal antibody, prepared against human lymphocytes, which has been found to react with elastic fibres, possibly the microfibrillar component. Samples of normal human and animal skin and human skin from various pathological conditions were subjected to immunolabelling with HB8, and alternate sections from each sample were stained by a standard orcein procedure. The individual fibres were clearly marked by the HB8 and this was confirmed by comparison with the standard elastic tissue stain. HB8 provides a new direct, practical and accurate method for the study of elastic tissue.

We report an ultrastructural study of the reactivity of an IgG monoclonal antibody, which reacts with human dermal elastic fibres. On electron microscopy, immunogold labelling demonstrated the reactivity of HB8 with the microfibrillar component of the fibres. In contrast, the electron-dense granular component which contains elastin was unlabelled with HB8. A fine network of microfibrils from the level of the oxytalan fibres to the large elastic fibres of the deep dermis was labelled with gold particles. No reactivity was detected on collagen fibres. These results demonstrate that HB8 reacts with the microfibrillar glycoproteins of the elastic fibres in normal skin. The precise biochemical nature of the antigens identified by HB8 is still unknown.

Two monoclonal antibodies, K20 and 4B4, assigned to the CDw29 cluster of differentiation antigens, were shown to react with basal keratinocytes (BK). The aim of this study was to identify the antigens recognized by K20 and 4B4 on epidermal cells, and to determine whether they were identical to those found on lymphocytes. Basal keratinocyte-enriched cell suspensions were labelled with 125I and then 1% NP40 cell lysates were used for immunoprecipitation. Under reducing conditions, K20 and 4B4 immunoprecipitated from basal keratinocytes a broad MW 105,000 band and proteins of MW 145,000, 90,000 and 80,000. Under non-reducing conditions, each band was shifted down by approximately 5000 MW. Metabolic labelling studies demonstrated that the MW 145,000 and 105,000 subunits were synthesized by basal keratinocytes. On lymphoid cells, K20 and 4B4 are known to precipitate glycoprotein complexes made of a broad MW 130,000 protein band (beta subunit) associated with a protein of MW 150,000 (alpha subunit) and proteins of MW 90,000 and 80,000 expressed in very low amounts. The MW 145,000 and 105,000 bands immunoprecipitated by K20 from basal keratinocytes correspond to the alpha and beta subunits present on lymphoid cells. It has recently been demonstrated that K20 recognizes the common beta subunit of the very late antigens family (VLA) and that 4B4 defines the helper-inducer subset of T lymphocytes. The present investigation provides evidence that basal keratinocytes share antigens of the VLA family with lymphoid cells and that they play an important role in the immune response in the skin-immune system.

Monoclonal antibodies to human alpha 1-microglobulin (alpha 1-m), a glycoprotein present in most biological fluids, reacted with the basement membrane of the epidermo-dermal junction and that of eccrine sweat glands in normal human skin. This immunofluorescence was specific in that control antibodies of known well-defined reactivity did not stain these structures. Therefore, alpha 1-m seems to be a new antigenic component of the junction and it may be an interesting antigen to study in diseases of this area.

Langerhans cells and indeterminate cells are the unique antigen-presenting epidermal cells participating in human lympho-epidermal interactions. They bear class II HLA-DR molecules, can substitute for macrophages in antigen presentation, induce a T-cell proliferative response to antigens and haptens in sensitized donors, and are necessary for alloantigen T-cell activation and generation of alloreactive cytotoxic T cells in vitro. Indirect immunofluorescence and immunogold electron microscopy on class II positive epidermal cell enriched suspensions (panning, FACS) indicated two populations of DR-positive epidermal cells: strongly DR-positive cells (25-30, 8% of positive epidermal cells) and faintly DR-positive cells, with a density of surface DR sites of respectively 5 X 10(5) and 1 X 10(5). Most Langerhans cells are among this second group while indeterminate cells are usually strongly DR-positive. OKT6-labelled cells were only typical Langerhans cells.

In the mixed skin cell lymphocyte culture reaction (MSLR), epidermal cells induce the proliferation in vitro of peripheral blood lymphocytes. Using monoclonal antibodies (MCAB) OKT6 specific for Langerhans cells among epidermal cells and an anti HLA-Dr, we analysed which epidermal cells are necessary in MSLR in man. Inhibition and reduction of the proliferative responses in MSLR conducted respectively with 'anti HLA-Dr and complement' and 'OKT6 and complement'-pretreated epidermal cells suggested that not only Langerhans cells but also other HLA-Dr bearing cells play a major role. Immuno-electron microscopic studies of either HLA-Dr (+) or OKT6 (+) enriched and depleted epidermal cells suspensions: (I) were well correlated with results in MSLR; (2) indicated that Langerhans cells express both T6 and HLA-Dr determinants while indeterminate cells express only the HLA-Dr. No other epidermal cells were found to be either OKT6 or HLA-Dr (+). The data strongly suggest that not only Langerhans cells but also OKT6 (-) HLA-Dr (+) indeterminate cells play a major role in lympho-epidermal interactions.

The presence of T lymphocytes in the situ in cutaneous infiltrates of contact dermatitis and lichen planus has been demonstrated by an immunoelectronmicroscope technique. A specific anti-human T cell antiserum labelled with peroxidase-conjugated anti-immunoglobulin serum was used to reveal the specific human T lymphocyte antigen (HTLA) which was visualized ultrastructurally. This technique shows that labelling is limited to the surface membrane and allows study of the in situ interrelationship of T cells.

Using electron microscopy, the immunological visualization of the membrane antigens of Langerhans cells (LC) can be performed by immunoperoxidase and immunogold techniques. The immunoperoxidase labelling permits the identification of only one antigen and the observation of qualitative variations of surface antigens. The immunogold method allows the identification of one antigen or simultaneously two or three surface antigens using gold particles of various sizes. This technique can be used to quantify the surface density of antigens on the cell membrane. The simultaneous identification of different surface antigens can be correlated with the ultrastructural characteristics of the cells. Using this technique we have recently demonstrated the existence of LC subsets in normal epidermis, and the presence of circulating T6-positive cells in normal subjects. In addition, a very low density of T4 antigenic sites on the LC membrane surface was observed. Several problems of a double-labelling immunogold technique related to steric hindrance and current artifacts are discussed.

Human keratinocytes from small skin specimens were grown on mouse 3T3 cell feeder layers into epidermal sheets free from Langerhans cells and MHC class II antigen. These were found to be suitable for the permanent coverage of wounds when used as autografts or allografts. We report here the ultrastructural differentiation of this cultured epidermis after grafting onto autologous or allogeneic recipients. The cultured epidermis was a thin but multilayered Malpighian epithelium composed of keratinocytes at different stages of differentiation. The dermo-epidermal basement membrane was newly synthesized during the first few days following transplantation onto de-epidermized wounds. The analysis of keratins and examination of various keratinocyte membrane antigens by immunofluorescence indicated that full terminal epithelial differentiation was only achieved after in vivo transplantation of the cultured epidermis. Langerhans cells, absent in cultures, progressively colonized the grafts, while melanocytes, not detectable in sections of the cultures, were identified among the keratinocytes 2 weeks after grafting.

We have used indirect immunogold electron microscopy to compare the respective density of cell membrane determinants revealed by OKT6 and OKT4 monoclonal antibodies on normal human Langerhans cells (LC): 12.9 +/- 3.5 gold granules were noted per cell section on OKT4-positive LC whereas 236.8 +/- 23.5 granules were counted per cell section on OKT6-reactive cells. These results confirm that human LC react with OKT4 antibody and they demonstrate a marked quantitative difference on LC surface between the antigenic determinants recognized by OKT6 and OKT4 antibodies.

Anti-D47 is a monoclonal antibody reacting with a surface antigen of human cortical thymocytes (different from the T6 antigen). It was prepared by immunization of mice with human thymocytes. The known cross-reactivity of monoclonal antibodies prepared against thymic cells with skin components prompted us to test anti-D47 on human skin as well as animal tissues. No labelling was observed on animal tissues. On human skin, anti-D47 was found to react with the secretory portion of eccrine sweat glands (ESG), i.e. with a cytoplasmic antigen of the secretory cells lining the deep portion of ESG glomeruli. No labelling was observed on apocrine sweat glands or on the excretory portion of ESG. Anti-D47 was also tested on histologically proven extramammary (vulvar) Paget's disease and basal cell epitheliomas: no staining of the neoplastic cells was observed. Anti-D47 appears to be an immunological marker of the secretory cells of human ESG and a new tool for the investigation of human sweat gland pathology.

We investigated the role of class II MHC (HLA-DR Ia-like) antigen-bearing Langerhans cells in the in vitro generation of human alloreactive cytotoxic T lymphocytes (CTL) directed against epidermal cells (EC). Normal EC in suspensions (from reconstructive surgery specimens) and anti-DR monoclonal antibody plus complement (C')-treated EC were used to stimulate peripheral blood (PB) T cells (T cells allogeneic or autologous to EC) in a classical 6-day human mixed skin cell-lymphocyte culture reaction (MSLR). CTL responses were then tested in 51Cr release assays against cultured EC (EC grown on collagen-coated plates in parallel to MSLR). CTL responses to EC were observed only after allogeneic MSLR and when targets and EC used in MSLR were from the same donor. They were comparable in magnitude to those seen in parallel studies using PBL from the same donor as the stimulating EC as target cells. They were abolished when EC used in MSLR were depleted in class II MHC Langerhans cells (preincubation of EC suspensions with anti-DR + C'). These data show: (a) alloreactive CTL to human normal EC may be generated in MSLR, as previously shown in animals; (b) the MSLR is an in vitro model of primary immunization against EC; (c) Langerhans cells are necessary for the generation of cell-mediated cytotoxicity reactions occurring against epidermal cells.

HLA-DR antigens are expressed only by Langerhans cells (LC) and indeterminate cells (IC) in normal human epidermis. Indirect immunofluorescence and ultrastructural immunogold labelling were used to study the HLA-DR expression by means of two anti-DR monoclonal antibodies (MCA). Freshly dispersed DR-positive epidermal cells expressed different densities of DR antigens on their membrane surface. Approximately 25% of DR-positive cells were strongly labelled by anti-DR-MCA and 75% were weakly stained. After 18 h in complete culture medium before labelling no significant difference in these percentages was observed. The lymphoid-like LC-enriched cells obtained by Ficoll-Hypaque centrifugation also had two populations of DR-positive cells: strongly labelled cells (30.8%) and weakly labelled cells (69.2%). DR-positive cells may be divided into two types according to the density of DR sites on their cell membrane: (I) type DR+ shows weak surface labelling by gold particles (8.8 +/- 3.0 gold particles/micron) and has cytoplasmic Birbeck granules, identifying such cells as typical Langerhans cells; (2) type DR shows strong membrane immunogold labelling (38.9 +/- 4.6 gold particles/micron) and may or may not contain Birbeck granules. The gold particles bound to the cell membrane were used to quantify the number of HLA-DR sites expressed on each cell type: 1 X 10(5) sites on DR+ cells and 5 X 10(5) on DR cells.

The lymphocytic infiltrate of lichen planus was studied by the identification of the human T lymphocyte antigen in frozen tissue sections using specific anti-human T lymphocytes antigen antiserum. The infiltrate was shown to be composed principally of T lymphocytes.

Ultrastructural study of a case of sclerosing lipogranuloma showed predominantly histiocytic-like cell infiltrate with numerous intracytoplasmic vacuoles of varied sizes without any limiting membrane and located in the deep dermis and subcutaneous fat. Fibroblasts appeared normal. Polymorphonuclear cells and macrophages were absent. This appearance was different from that described in 'classical' phagocytosis, mucopolysaccharidoses or polyvinylpyrrolidone storage syndrome.