Culture of putative Langerhans cell bone marrow precursors: characterization of their phenotype.

We searched for the presence of human CD1-positive cells in bone marrow populations in order to characterize putative Langerhans cell precursors. Bone marrow progenitors were cultured in 0.8% methylcellulose supplemented with 10% granulocyte-macrophage (GM) colony-stimulating factor(s) GCT and HTB9. We compared the kinetics of these two factors and found that GCT was the more appropriate for our study. After 8 days of culture, colony-forming units of granulocyte-macrophages (CFU-GM) were tested for the presence of CD1-positive cells using the immunofluorescence technique. Positive cells were counted by cytofluorometric analysis: 9.4% CD1a (BL6), 13.4% CD1c (L161), 4.3% CD1b (NuT2), 4.6% CD2 (T11), and 25.5% CD33 (My9). Ultrastructural features and phenotype were then specified by the immunogold labeling technique using electron microscopy. A subpopulation of CD1-positive cells showed the ultrastructural morphology of bone marrow pro-monocyte/monocyte cells. By using well-characterized monoclonal antibodies, it was demonstrated that these cells expressed the following phenotype: CD14+, CD33+, CD4+, HLA-DR+, HLA-DP+, HLA-DQ-, OKT10-, CD2-. These data indicate that these bone marrow promonocyte/monocyte progenitors express a phenotype similar to that of epidermal Langerhans cells but the density of each antigen is much lower than that observed on mature skin dendritic cells.

An immunoelectron microscopic localization of wart associated antigens present in human papilloma virus (HPV) infected cells.

Wart associated antigens were observed by the indirect immunoperoxidase technique in light and electron microscopy. Positive reaction products could be found in the nuclei of wart cells, in which virus particles were labeled with a specific immune rabbit serum and with human sera from patients with warts. Tissue antigens, differing from the viral labeled inclusions, were detected within the cytoplasm of some infected cells, by means of IgM and IgG antibodies from patients with warts. In these particular cells the positive reaction occurred in irregular patterns giving microgranular precipitates, which suggests that the antigen was not uniform. No staining was observed on the cell surface. This study has shown direct immunomorphological evidence in vivo of a specific immune reaction in man directed against whole virus and wart specific cellular antigens.

GP37 is different from filaggrin.

The identity of two differentiation markers of human epidermis, filaggrin and a Concanavalin A (Con-A) reactive glycoprotein of 37 kD, has been studied. Human epidermis was extracted in Nonidet P-40 buffer, and the soluble proteins were separated by two-dimensional electrophoresis. Con-A reactive glycoproteins were identified by incubating gels with iodinated lectin followed by autoradiography. Identical, parallel gels were electrophoretically transferred to nitrocellulose paper and filaggrin-related molecules labeled by the specific monoclonal antibody AKH1. We found that the 37-kD Con-A reactive component was resolved by two-dimensional gel electrophoresis into several glycoproteins and that the lectin Con-A does not bind to filaggrin. Under these conditions, the anti-GP37 serum failed to identify any component. However, when applied to human keratinocyte culture extract, AKH1 and the anti-GP37 serum reacted in a similar way. These data show 1) that the 37-kD band is not homogeneous but contains distinct markers of differentiation (filaggrin and Con-A reactive glycoproteins) and 2) that the GP37 antibody's specificity is for the filaggrin precursor.

Flow cytometry for separation of keratinocyte subpopulations from the viable epidermis.

Human epidermal cell suspensions were analyzed and sorted with flow cytometry. The desmosome and differentiation-related KM48 monoclonal antibody was used for indirect immunofluorescence and permitted staining of keratinocytes at various stages of the cell maturation. Intensity of the staining correlated with the degree of differentiation. Three sorting gates were chosen to obtain subpopulations which varied distinctly in KM48 expression. The flow cytometry-sorted cells were characterized by their ultrastructural appearance and by the bullous pemphigoid antigen expression. According to the ultrastructure criteria, about 50% of the cells obtained from the "IF negative" gate were basal layer keratinocytes (45.5% expressed bullous pemphigoid antigen); 90% of the "intermediate" gate cells were spinal layer keratinocytes, and over 80% of the cells sorted through the "strongly IF positive" gate were of the granular layer type. The method of keratinocyte separation proposed allows samples to be obtained for further biochemical and functional studies on keratinocyte subpopulations in normal and pathologic skin.

Cyclosporin A inhibits directly in vivo keratinocyte proliferation of living human skin.

A direct in vivo antiproliferative effect of cyclosporin A (CsA) on human epidermal keratinocytes (EK) grafted onto nude mice was evaluated. Using pulse-labeling of 5-bromo-2'-deoxyuridine (BrdU), a thymidine analogue incorporated into the nuclei of DNA-synthesizing (S-phase) cells, the antiproliferative effect of CsA was revealed as a decrease in the number of BrdU-positive human EK grafted onto nude mice receiving a daily subcutaneous injection of 50 mg/kg of CsA. The blood level of CsA in the treated mice, evaluated by a radioimmunologic assay, was 679 +/- 501 ng/ml (n = 3). Using an antibody to leukocyte common antigen, it was shown that no human lymphocytes were present in the grafted skin. Therefore, this antiproliferative effect of CsA on human EK seems to be due to a direct effect on EK rather than to lymphocyte regulation.

Langerhans cells in skin from patients with psoriasis: quantitative and qualitative study of T6 and HLA-DR antigen-expressing cells and changes with aromatic retinoid administration.

Using a monoclonal antibody against human HLA-DR antigens and OKT6, we investigated by indirect immunofluorescence the distribution of Langerhans cells in normal human skin and involved and uninvolved skin from patients with psoriasis before, during, and after systemic aromatic retinoid administration. In parallel, enumeration of HLA-DR and of OKT6+ cells was also performed. In involved psoriatic epidermis the distribution of positive cells was disturbed; OKT6+ cells were reduced in number, as were HLA-DR+ cells which were seen in clusters. In control skin sections, a regular pattern of fluorescent dendritic epidermal cells was noted. In normal-appearing human skin, in nonlesional psoriatic skin, but not in diseased psoriatic skin, the number of OKT6+ cells per epidermal section surface unit was higher than that of HLA-DR expressing cells. Changes in the number and distribution of OKT6 and HLA-DR+ cells in psoriatic involved epidermis were corrected by oral retinoid treatment.

PUVA-induced repigmentation of vitiligo: scanning electron microscopy of hair follicles.

PUVA-i-duced repigmentation of vitiligo was studied using both the split-dopa reaction and scanning electron microscopy. Proliferation of hypertrophic, Dopa-positive melanocytes were observed in the lower portion of some hair follicles, whereas other giant melanocytes were observed along the middle portion. The existence of a melanocyte reservoir in human hair follicles is postulated.

Detection of human immunodeficiency virus-DNA and RNA in the skin of HIV-infected patients using the polymerase chain reaction.

The presence of HIV genomic-associated nucleic acids (DNA and RNA) within biopsies of normal-appearing skin and various skin lesions obtained from a group of 33 HIV-infected patients was investigated by using the polymerase chain reaction (PCR). In order to define the localization (dermal vs. epidermal) of HIV, the PCR was carried out separately on the dermis and the epidermis in 21 of the specimens. Altogether, HIV-DNA and HIV-RNA were detected, respectively, in 89% and 47% of the specimens included in this study; both DNA and RNA were detected more frequently in the dermis (90% and 43%, respectively) than in the epidermis (62% and 5%, respectively). No correlation could be established between the presence of HIV genomic material, the nature (normal-appearing vs. diseased) of the skin specimen studied, and the clinical or biologic severity of HIV infection, as evidenced by the CDC stage classification and the number of peripheral CD4+ cells. It seems, therefore, that the HIV is very frequently present within the skin during the course of HIV infection; however, its precise cellular localization and pathologic significance await further investigation.

Immunoglobulin-producing cells in the inflammatory infiltrates of cutaneous tumors. Immunocytologic identification in situ.

An immunocytochemical technique has been developed for the identification in situ of immunoglobulin-producing cells in tissues fixed in Bouin's solution and embedded in paraffin. Technical details are discussed as well as the application of the technique to the study of plasma cells in the inflammatory infiltrate around cutaneous tumors. Preliminary results have been obtained with basal cell epitheliomas, squamous cell carcinomas, and malignant melanomas. IgA-producing cells were present in all tumors. IgG-producing cells were present in variable frequency, depending on the type of tumor, and IgM-producing cells were found only in basal cell epitheliomas and squamous cell carcinomas.

Flow cytometric sorting of human epidermal pure basal cell suspensions using a specific antikeratin monoclonal antibody.

A method is described for flow cytometric sorting of epidermal basal cells, based on the cellular keratin content. KL1, a monoclonal antikeratin antibody specific for the 56.5 kD polypeptide present in suprabasal cells was used to distinguish suprabasal from basal cells. After the action of Triton X-100, epidermal cells were stained in suspensions with KL1 antibody, and KL1-positive cells were separated by flow cytometry from KL1-negative cells (basal cells). This makes it possible to sort and analyze pure fractions of human epidermal basal cells since no less than 99% of the KL1 sorted cells were bearing bullous pemphigoid antigen. Therefore, the use of specific antibodies to cytoplasmic antigens can be of help in sorting pure fractions of cells of a particular tissue for further studies.

Epidermal cell-derived lymphocyte differentiating factor (ELDIF) inhibits in vitro lymphoproliferative responses and interleukin 2 production.

We have examined the biologic characteristics and immunologic properties of epidermal cell-derived lymphocyte differentiating factor (ELDIF), a lymphocyte differentiating factor produced by cultured human keratinocytes. The ELDIF was semipurified by a gel filtration procedure. This factor, which is distinct from prostaglandins, epidermal cell-derived thymocyte activating factor (ETAF), and the well-known thymic hormones (thymulin, thymopoietin, and thymosin alpha 1) did not exhibit any interleukin (IL)-1, IL-2, or IL-3 activity. It strongly inhibited in vitro lymphoproliferative responses of normal mouse spleen cells to phytohemagglutinin, concanavalin A, and lipopolysaccharide. This dose-dependent phenomenon was associated with a suppression of IL-2 production rather than any toxic effect. It can be concluded that ELDIF, a product of human epidermal cells, which displays in vitro T-cell differentiation and regulatory activities, could be of major importance in vivo in the control of cutaneous inflammatory reactions.

ConA- and PNA-binding glycoproteins of human epidermis.

Concanavalia ensiformis agglutinin (ConA) and Arachis hypogaea agglutinin (PNA) binding glycoproteins in NP-40 extracts of human epidermis and epidermal cell preparations have been investigated by incubation of SDS-polyacrylamide gels with 125I-labeled lectins. In whole epidermis, 125I-ConA labels numerous glycoproteins, of which some appear to be restricted to the more differentiated upper layers of the epidermis and thus may represent markers of epidermal differentiation. These glycoproteins are not expressed by cells cultured on collagen. 125I-PNA labels a limited number of components, of which the major one is intercellular and/or extremely trypsin-sensitive. This material is expressed in low amounts after 13 days in culture.

Reactivity pattern of a monoclonal antikeratin antibody (KL1).

A monoclonal antibody against keratins (KL1) from normal human stratum corneum was obtained using hybridoma techniques. Spleen cells from immunized BALB/c mice were fused with NS1, a mouse myeloma cell line, to produce hybrids. Antibody activity to epidermal keratins was tested using an indirect immunofluorescence test on cryostat sections of human epidermis and rabbit lip. A stable clone producing antikeratin antibody was isolated and an ascitic fluid was produced and used as a source of antibody (IgG1 kappa). KL1 was characterized by its immunohistochemical staining of various epithelia and by its recognition of 55-57 kilodalton (kd) keratin polypeptide from normal epidermis using the immunoblot technique. Frozen and deparaffinized sections of normal human epidermis, mucosa, and esophagus were stained by this antibody only in the upper cell layers as demonstrated by both indirect immunofluorescence and immunoperoxidase techniques. Approximatively 80% of normal keratinocytes isolated after trypsinization were labeled by KL1 whereas most negative cells showed basement membrane zone antigens. This confirmed differences in the expression of medium-sized polypeptides between basal and supra-basal cells during the course of human epidermal differentiation. All epithelial cells from other human epithelia (thymus, thyroid, bronchial mucosa, stomach, intestines) were positive with KL1 whereas nonepithelial cells and tissues did not show any staining. In view of these results KL1 promises to be a useful tool in the exploration of human epithelial differentiation.

Clearance mediated by splenic macrophage membrane receptors for immune complexes in cutaneous vasculitis.

This study concerns 11 patients with immune complex associated cutaneous vasculitis (5 leukocytoclastic vasculitis or Gougerot Ruiter's disease, 3 essential mixed cryoglobulinaemia, 2 Henoch-Schönlein purpura and 1 Waldenström's hypergammaglobulinaemic purpura). By determining the clearance of 51Cr-labeled IgG sensitized erythrocytes we showed a slight modification in the splenic mononuclear phagocyte system. In patients with Gougerot-Ruiter's disease the clearance of the autologous IgG-coated erythrocytes was delayed in 1 patient, and normal in 2 patients. In contact, the 8 other patients showed accelerated rates of IgG-mediated clearance. There was no statistically significant correlation between clearance rate, serum complement component levels and composition and/or levels of circulating immune complexes. Thus, the accelerated clearance rate suggests an enhanced activity of the mononuclear phagocyte system IgG-Fc receptors.

Immunocytochemical and ultrastructural localization of keratin polypeptides in normal epidermal and mucosal cells and tissues.

Previous studies revealed that the filaments of cytokeratin express different antigenic sites in the basal and suprabasal cell compartment of the skin and oral mucosa. It has been demonstrated that the keratin polypeptide subunit of molecular weight 67,000 dalton (67K) is a valuable marker of the suprabasal cells, since the normal basal cells remain unlabeled by antibodies against this large polypeptide. In the present study, the distribution of the 67K polypeptide has been investigated on human and guinea pig keratinocyte suspensions, purified basal cells of guinea pigs, normal human abdominal skin and normal human buccal mucosa, using the indirect immunoperoxidase method. The results of this investigation confirmed ultrastructurally that the 67K-antigen expression is lacking in the epidermal basal cell compartment. However, it could be shown that 67K polypeptide is a component of the tonofilaments in the upper Malpighian cell layers of the skin and oral mucosa. These observations suggest that keratinocyte differentiation is accompanied by modifications of antigenic determinants of the keratin filaments.

Response of discoid and subacute cutaneous lupus erythematosus to recombinant interferon alpha 2a.

Ten patients suffering from discoid lupus erythematosus (DLE) or subacute cutaneous lupus erythematosus (SCLE) were treated with interferon alpha 2a. A marked improvement or clearing of cutaneous lupus erythematosus lesions was observed in eight of them. However, the response to interferon was of short duration and within a few weeks after interferon withdrawal all patients who were improved or cleared relapsed. This study suggests that interferon alpha 2a represents a new interesting approach in the treatment of DLE and SCLE. Ongoing trials will define the optimal treatment schedule for the maintenance of interferon-induced improvement of cutaneous lupus erythematosus.

Immunogold technique applied to simultaneous identification of T6 and HLA-DR antigens on Langerhans cells by electron microscopy.

A double-labeling immunogold technique in electron microscopy and specific monoclonal antibodies to surface antigens of Langerhans cells (OKT6 and BL2) were applied to assess directly the coexpression of two cell surface antigens (T6 and HLA-DR antigens) in a heterogeneous epidermal cell suspension. Electron microscopic examination of double-labeled cells revealed that all Birbeck granule-containing Langerhans cells bound OKT6 and BL2. The preparation of markers with colloidal gold particles and the procedure for double labeling are described. Several problems related to the steric hindrance and current artifacts are illustrated by micrographs and also discussed.

Epithelial differentiation of human skin equivalents after grafting onto nude mice.

Human skin equivalents were developed in vitro with a unidimensionally retracted dermal equivalent made of human type I + III collagen and human MRC5 fibroblasts, and a multilayered epithelium grown in liquid medium from normal human keratinocytes in suspension. We investigated the degree of epidermal differentiation that could be achieved after in vivo grafting onto nude mice by means of light and electronmicroscopy as well as by immunohistochemistry. All transplanted grafts showed a primary take. The grafts formed an epidermis with a stratum corneum and from day 7 to 14 after transplantation a distribution of a 56.5 Kd keratin protein, involucrin, profilaggrin/filaggrin, and MHC-I antigens that was similar to what is noted in normal human epidermis. These data indicate that a full terminal differentiation was only achieved after in vivo transplantation of the cultured epithelium. Pigmentation was present, but no marker of Langerhans cells was seen at 4 weeks. Although there was no evidence of the dermal equivalent after 2 weeks, we noted a strong adherence of the graft to the wound bed, with the presence of type-IV collagen, laminin, and bullous pemphigoid antigen at the dermo-epidermal junction (day 7) and hemidesmosomes, a lamina lucida and a lamina densa (day 30). No epithelial damage was noted in spite of an inflammatory infiltrate in the underlying tissue. This represents a preliminary step in the use of such a skin-equivalent in the treatment of human patients with wounds.

A surface glycoprotein complex related to the adhesive receptors of the VLA family, shared by epidermal Langerhans cells and basal keratinocytes.

A murine monoclonal antibody, designated K20, was raised by immunization with a human malignant T-cell line. It reacted specifically with membrane glycoprotein complexes on early haematopoietic cells, T cells, and monocytes. In epidermis, K20 specifically reacted with Langerhans cells and basal keratinocytes, as demonstrated by double labeling experiments. Membrane immunoprecipitation analysis demonstrated that the antigen identified by K20 on lymphoid cells and epidermal cells was different. While on lymphoid cells, K20 recognized glycoprotein complexes made of a constant 130-kD subunit associated with subunits of higher molecular weight ranging from 150 to 200 kD, a complex of 105-145 kD was precipitated from Langerhans and basal cells. Metabolic labeling studies demonstrated that these proteins were synthesized by the basal cells. The antigen identified by K20 was thought to belong to the integrins, a family of cell surface receptors that play a role in cell adhesion, cell interactions, wound healing, and immune defense mechanisms. K20 is the first monoclonal antibody that specifically recognizes a membrane antigen common to Langerhans and basal cells. Additionally, K20 is the first of five reported monoclonal antibodies to have been characterized on the epidermal cells that detect antigens shared by lymphoid subpopulations and normal basal keratinocytes.

Langerhans cells in S-phase in normal skin detected by simultaneous analysis of cell surface antigen and BrdU incorporation.

We report on a double immunofluorescence staining for the detection of the Langerhans Cell (LC) population in S-phase. After trypsinization, the epidermal cell suspensions were enriched for LC and exposed to 10 microM BrdU for 2 h. Studies of BrdU labeled cells included the determination of their cell surface phenotype. Both membrane labeling and incorporated BrdU as revealed using anti-BrdU demonstrated the BrdU as revealed using anti-BrdU demonstrated the presence of LC in S-phase. We observed 6% of the epidermal LC in S-phase. This is a proof that LC are able to proliferate in the epidermal microenvironment. This technique, besides being rapid and free of radioactivity, allows the cytokinetic study of phenotypically defined LC in heterogeneous epidermal cell populations.