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Immune sera against total keratin, as well as three keratin polypeptide subunits, were raised in guinea pig with different bands of stratum corneum fibrous proteins derived from normal human epidermis and isolated by SDS polyacrylamide gel electrophoresis. The staining pattern of the different polypeptide antisera using the indirect immunofluorescence and immunoperoxidase methods in biopsy specimens of normal human epidermis confirmed recently published results. Biopsy specimens from 10 lesions of lichen planus were studied by indirect immunofluorescence and immunoperoxidase methods utilizing the same four different specific antikeratin immune sera. Most often the staining pattern with the different immune sera was similar to that seen in normal keratinized epidermis. In all specimens, intense staining of the colloid bodies was detected with the different immune sera. This finding favors the concept that colloid bodies originate from keratin filaments. Control staining with pre-immune serum was always negative. In one case of bullous lichen planus, colloid bodies were not labeled by 67 K and 62 K immune sera, but were intensely stained with 55 K and total keratin antisera. Moreover, with 55 K immune serum there was more intense staining of certain basal cells. The correlation between these findings and previously published studies by electron microscopy is discussed.

Experimental anti-keratin polypeptide sera (KPS) which were prepared by immunizing guinea pigs with the P1 polypeptide (molecular weight: 67 0000 dalton) of alpha-keratin of normal human stratum corneum, were shown to react in immunofluorescence only to cell cytoplasmic antigen of the upper layers of the epidermis. No staining was detected in the basal layer. An immunolabeling performed on free epidermal cells obtained after trypsinization demonstrated by electron microscopy that receptors for KPS were tonofilaments. Minor proportions of negative cells containing tonofilaments and numerous melanosomes detected might correspond to the basal cells. An IF pattern similar to that seen with KPS was observed with some human epidermal cytoplasmic sera (ECS). However, reciprocal blocking tests performed on both rabbit lip and normal human skin with the different sera showed no inhibition. No cross-reaction was detected by immunodiffusion test between whole purified alpha-keratin and human ECS. In electron microscopy, the receptors for ECS appeared not to be keratin-like, but were located on a granular part of the peripheral keratinocyte cytoplasm. These findings confirmed two steps (basal and malphighian layers) in epidermal differentiation defined by antigenic markers and especially by the alpha-keratin component of MW 67 000 d present in normal stratum corneum.

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.

We report on application of the highly sensitive and specific immunogold labeling method for ultrastructural investigation of keratin intermediate filament antigens in human epidermal cell suspensions. Triton X-100 pretreated cells proved accessible to the colloidal gold conjugate, thus enabling keratin filament bundles to be labeled. Anti-keratin KL1 and KL2 monoclonal antibodies were raised in mice after immunization with either human stratum corneum-isolated keratins or keratins extracted from human epidermal cells suspensions, respectively. Immunoelectron microscopy confirmed immunofluorescence and immunoperoxidase results of epidermal keratinocyte staining, and revealed two different antibody reactivity patterns: KL2 reacted with keratin filaments in keratinocytes of all epidermal layers, whereas antigen to KL1 was detected only on keratin of the suprabasal layers, not on the basal keratinocyte tonofilaments. The monoclonal antibody-recognized epitopes were specific for the keratin filaments. Vimentin-rich cells (melanocytes) were not stained in the same epidermal cell suspensions. Additionally, two distinct ultrastructural patterns of keratin filament epitope labeling were observed. KL1 and KL2 monoclonal antibodies react with two different antigenic determinants, depending on the stage of keratinocyte differentiation, and may therefore be used for immunohistochemical studies of various keratin-containing cells in normal and pathologic conditions.

We have used tritiated thymidine (3HT) labelling and immunoperoxidase staining of 67 K polypeptide in human epidermis to study normal skin before and after irritation induced by application of croton oil (20% and 50% in olive oil). By this double labelling method, it was possible to identify, on the same skin section, DNA-synthesizing nuclei and epidermal cells which contained 67 K polypeptide. Our results clearly indicate that the germinative compartment of normal epidermis is a mixed population comprising basal and adjacent suprabasal cells; as a rule, absence of expression of 67 K polypeptide, which characterizes all basal cells, could not be regarded as a good marker to distinguish the germinative from the differentiating compartments. In mild primary irritation, the ratio of 3HT-labelled undifferentiated cells to keratinizing ones was similar to that observed in normal epidermis. In severe irritation, this homeostatic process was altered, as 3HT-labelled cells were mainly 67 K negative.

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.

Anti-keratin polypeptide sera were obtained against the different bands of polyacrylamide gel electrophoresis of fibrous proteins of stratum corneum derived from normal human epidermis. The sera were tested by indirect immunofluorescence of immunoperoxidase techniques. It was demonstrated that antibodies against P1 and P2 polypeptides, of MW 67,000 and 62,000 daltons respectively, were directed towards cytoplasmic antigens of keratinocytes of the upper Malpighian layers, while no labelling could be detected in the basal cell layer. Anti-P3 polypeptide sera and the anti-whole keratin serum labelled the whole epidermis, including the basal cell layer. Ultrastructural immuno-labelling performed on free epidermal cells obtained after trypsinization demonstrated that receptors for anti-P1 polypeptide sera were tonofilaments. These results showed that some keratin components (P1 and P2 polypeptides) might be absent in basal cell tonofilaments. This in in favour of various differentiation stages of the keratinizing cells. Keratin polypeptide 1 could be a useful marker of keratinization. According to preliminary studies, the expression of this keratin antigens was markedly disturbed in tumors such as basal and squamous cell carcinoma, in warts and in ichthyosis.

Comparative studies on the specificity of the so-called antiepidermal antibodies (Abs) found in human sera were performed by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM). After a screening test by indirect immunofluorescence (IF), sera obtained from patients with various diseases and controls could be classified in 5 different groups according to the IF patterns on the epidermis: sera reactive with: (1) the stratum corneum (SC); (2) the upper layer (U-Cyt); (3) the whole epidermis (G-Cyt); (4) basal cells (B-Cyt); and (5) negative ones. By immunoblotting, all the 23 IF-positive sera were found to bind to one or more keratin bands, and did not show any reactivity with epidermal Nonidet P-40 soluble proteins. SC-Abs were mainly directed against a 67 kD Keratin band, whereas U-Cyt- and G-Cyt-Abs bound to both 58-56 kD and 67-63 kD keratins. B-Cyt-Abs reacted strongly with 63 kD Keratins and slightly with a 50 kD band. Antikeratin Abs were detected by immunoblotting even in the IF-negative sera. The ELISA study showed that sera with high IF titers contained high levels of antikeratin Abs. In the IEM study using sera containing U-Cyt- or B-Cyt-Abs, 2 distinct reaction patterns were demonstrated: U-Cyt-Abs stained tonofilaments of suprabasal keratinocytes, while B-Cyt-Abs characteristically reacted with those of basal cells. Moreover, SC-, U-Cyt-, and G-Cyt-Abs were absorbed out by insoluble epidermal proteins, and B-Cyt-Abs were decreased in titer after the absorption test. The present study provides strong evidence that most, though not all, human antiepidermal Abs are directed against different keratin polypeptides, and that antikeratin Abs commonly occur in almost all human sera.

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.

Psoriatic epidermis is characterized by increased DNA synthesis and disturbed differentiation. Even though these processes are closely associated, most investigations do not give insight into temporal/spatial relationships between both events. We previously developed a double labeling method for the simultaneous demonstration of the germinative and differentiated epidermal compartments in normal human skin by using tritium-labeled thymidine ([3H] Thd) incorporation and immunoperoxidase staining of 67 kD keratin polypeptides. In this paper we report the results of combined evaluation of these compartments in stable plaques of psoriasis. Scanning of skin sections with an automatic image analyzer allows objective quantification of areas of total epidermis, 67 kD+ differentiated epidermis and numbers of [3H] Thdr+ nuclei. Our data indicate that the 67 kD- undifferentiated psoriatic epidermis is expanded. Increased numbers of [3H] Thd+ basal and suprabasal psoriatic keratinocytes are present and most of them (97.9%) pertain to the 67 kD- compartment. Keratin identification in scales taken from the same sites showed a variable but distinct decrease of 67 kD keratin polypeptides. Hence, the hyperplastic epidermis of stable plaques of psoriasis is characterized by the presence of increased numbers of [3H] Thd+ cells, which primarily belong to the undifferentiated (67 kD-) basal and suprabasal compartments, especially in the lowermost parts of the elongated interpapillary rete ridges. These changes are associated with a relative decrease of synthesis of 67 kD polypeptides and the presence in the scales of keratins that confer a characteristic hyperproliferative epidermal keratin pattern to the psoriatic plaque.

The expression of an epidermal keratin subunit and a specific antigen of the keratinocyte membrane, two differentiation antigens in normal human epidermis, was studied in benign and malignant epidermal lesions by use of monoclonal antibodies KL1 (anti 55-57 Kd keratins) and KL3 (anti keratinocyte membrane antigen). In normal human epidermis, KL1 labelled all keratinocytes from the suprabasal layers, KL3 stained the intercellular spaces in all epidermal layers with a fluorescence intensity increasing from the basal to the more upper layers and recognized a keratinocyte membrane antigen as demonstrated in electron microscopy. Frozen or deparaffinized sections of basal cell carcinomas (BCC), squamous cell carcinomas (SCC) malignant melanomas, warts, and skin biopsies from benign lesions (psoriasis, lichen planus, bullous pemphigoid, lupus erythematodes, pemphigus, vasculitis) were tested with either KL1 or KL3 by indirect immunofluorescence and/or immunoperoxidase. Benign and malignant lesions in which modifications of the keratinization process and cell differentiation are known to occur (BCC, SCC, warts, psoriasis) showed the most severe alterations as compared to normal epidermis. With KL1 we observed an irregular staining of basal cells; a reorganization of keratin filaments and variable staining intensities within tumoral cells which did not express high MW keratins. With KL3 drastic alterations in the epidermal intercellular patterns and loss of reactivity of tumoral cells were noted. Conversely, the positivity of epidermal basal cells with KL1, in some cases, was the only modification noted in other skin lesions.

The aim of this study was to analyze the filament distribution in the parotid gland and their tumors. A correlation to the histogenetic implications and histological properties was attempted. Normal rat and human parotid glands as well as pleomorphic adenomas and squamous cell carcinomas of this gland were examined by the indirect immunoperoxidase technique using antibodies to the keratin polypeptide of 67,000 dalton, and 55,000 dalton and anti-actin auto-antibodies. Both keratin and actin antigens were demonstrated in the duct system and in the myoepithelial cells of the normal salivary glands. The acinar cells remained negative. In pleomorphic adenomas, there were numerous keratin-positive spindle-shaped cells which represented the so-called myoepithelial cells. These cells were demonstrated to contain actin, too. The tubular duct-like structures were labeled by keratin antiserum and by anti-actin auto-antibodies. In squamous cell carcinomas, the majority of the tumor cells were strongly labeled by keratin antibodies. Actin was detected in these malignant cells, too. Our results show important differences in the cellular elements of the normal salivary glands with regard to their filament distribution. In normal and tumoral conditions, our findings support the hypothesis of the epithelial nature of the myoepithelial cells. Our preliminary results encourage the research of filamentous structures for scientific and diagnostic purposes.

A murine hybridoma secreting an IgM monoclonal antibody (KL3) was produced by cell fusion of mouse myeloma cells with spleen cells from mice immunized with human epidermal keratins. On normal human epidermis KL3 stained the intercellular spaces from the stratum germinatum to the stratum granulosum with a fluorescence intensity increasing from the basal layer to the upper layers. Basal cells were not stained on the side facing the basement membrane. About 90% of free keratinocytes isolated after trypsinization were labelled by KL3 in a punctate staining. Immunoelectron microscopy allowed us to show that the antigen recognized by KL3 was exclusively localized on the keratinocyte membrane especially in the desmosomal plaques. KL3 reactivity was not modified by preincubation of skin sections with lectins showing a selective intercellular labelling of upper layers of epidermis or pemphigus antisera, nor by adsorption of the antibody on NP40 soluble proteins of the epidermis. Though KL3 reactivity was completely abolished after adsorption of purified keratins, no immunological reactivity of KL3 was detected with epidermal keratin polypeptides blotted on nitrocellulose paper. In psoriatic epidemis and epidermal tumors KL3 reactivity was drastically modified. These results suggest that KL3 recognized a keratinocyte membrane antigen implied in the epidermal differentiation process.

Anti-keratin polypeptide sera (K.P.S) were obtained by immunizing guinea pigs with fibrous proteins from stratum corneum, which were acquired from normal human epidermis by m eans of S.D.S. polyacrylamide gel electrophoresis. After absorption with red blood cells and liver powder the sera were tested by indirect immunofluorescence technique on different substrates. Antibodies against polypeptides P1 and P2 of M.W. 67,000 and 62,000 dalton, respectively, were directed toward cytoplasmic Ag of keratinocytes of spinous and graunular layer of normal human and rabbit epidermis. No labeling could be detected in the basal cell layer. This finding is in favor of various differentiation stages of the keratinizing cells. P3 of M.W. 53,000 dalton induced low titre anibodies which labelled the whole epidermis, including the basal cell layer. The fourth polypeptide of M.W. 49,000 dalton seemed not to be immunogenic in such experiences. In tumors, such as basal cell carcinom,a squamous cell carcinoma, and warts, the expression of keratin antigens is markedly diminished. No analogy could be drawn between experimental keratin polypeptide antibodies and the human epidermal cytoplasmic antibodies which were detected in some patient sera.

We have investigated on human skin the reactivity of a panel of 42 anti-thymus monoclonal antibodies (MCA) supplied by the Third International Workshop and Conference on Human Leucocyte Differentiation Antigens, Oxford, 1986. MCA of the first cluster of differentiation (CD1) define a group of surface molecules expressed by cortical thymocytes. Some of them (OKT6, M241 and Na1/34) have been shown to react in normal human skin with the epidermal Langerhans cells (LC). Twenty-two CD1 MCA were investigated in the present study. On normal human skin, 13 MCA reacted with LC in situ. This result suggests and confirms the heterogeneity of CD1 MCA. Recently, some of them were shown to recognize biochemically different molecules and/or epitopes of thymocytes. In addition, 20 anti-thymic epithelium MCA were tested on human skin. The MCA which only reacted with the thymic epithelial cell network (except Hassall's corpuscles) decorated only the epidermal basal cell layer. The MCA which reacted with all the thymic epithelial cells (including Hassall's corpuscles) decorated all the epidermal cell layers. These results confirm the heterogeneity of the thymic epithelial microenvironment and underline the antigenic similarities between the thymic epithelial structures and the different epidermal cell layers. The existence of bone-marrow-derived CD1-positive cells (thymocytes or LC) in an epithelial cell network (the thymus and the epidermis) focus the speculation around the immunological role of the epidermal basal cell layer in the T cell education and the exact lineage of the epidermal LC.

The keratin polypeptide composition of different types of warts was studied by means of SDS polyacrylamide gel electrophoresis. It was composed of five main polypeptides, similar to those found in normal epidermis (mol. wt. 67K to 55K) and an additional polypeptide (72K) was often detected. The distribution of each polypeptide was expressed as percentage of total keratins in the different samples. The most characteristic feature observed in warts was the marked decrease of the 67K polypeptide. There were increased amounts of the 67K-63K proteins and some difference was observed in the distribution of these proteins in the different types of warts. Thus, the viral infection seems mainly to modify the proteins which are only involved in the latest stages of the differentiation of keratinocytes.

The variations in the expression of epidermal keratins occurring during retinoid therapy were studied in patients with psoriasis before and during oral administration of etretinate (aromatic retinoid Ro 10-9359) and compared with normal epidermis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis densitometric readings were performed on keratins extracted from epidermal cells obtained through trypsinization of skin specimens. Epidermal cells were also tested by immunofluorescence (IF) for the presence of BMZ antigens as markers of basal cells and for the presence of TK and KP (67 K, 63 K, and 55 K) with sera with antibodies against BMZ antigens and specific antisera for TK and KP. In psoriasis-involved epidermis, SDS-PAGE anaLysis showed lower amounts of 67 K and increased amounts of 63 K and 55 K, as compared with normal epidermis. Low proportions of cells expressing by IF the 67 K and the 63 K were also noted, with a defective expression of these two KP by suprabasal keratinocytes in psoriasis-involved epidermis. During etretinate administration, a return toward the normal electrophoretic pattern and a correction of the defective cellular expression of these two KP were obtained parallel with clinical improvement. These findings indicate the presence in involved psoriatic epidermis of a population of suprabasal keratinocytes that do not express the high-molecular-weight KP and show a normalization of the relative proportions of the KP with etretinate.