HLA class II expression on human epidermal Langerhans cells in situ: upregulation during the elicitation of allergic contact dermatitis.

An immunoelectron-microscopic technique was applied to investigate the localization of molecules that are involved in the elicitation of allergic contact dermatitis in human epidermal cells in situ. Langerhans cells in the epidermis of lesions showed a strongly increased cell surface expression of HLA class II molecules as compared with normal skin. In addition, a high number of intracellularly located HLA class II molecules were present in Langerhans cells of lesional epidermis, suggesting increased biosynthesis of these molecules during the elicitation process. In contrast, no differences in the expression of CD1a by Langerhans cells was observed between normal and lesional skin. Frequently, the Langerhans cells were found in close apposition to mononuclear cells, which also exhibited a strong cell surface HLA class II expression. The number of Birbeck granules that are characteristic intracellular Langerhans cells organelles was increased in lesional Langerhans cells as compared with normal-skin Langerhans cells, which may correlate with the activated state of lesional Langerhans cells. These Birbeck granules were always HLA class II or CD1a negative. The increased synthesis and expression of HLA class II molecules on the cell surface of Langerhans cells suggests a direct role for these HLA class II molecules in the elicitation process of allergic contact dermatitis.

Internalization of MHC class I molecules is a prerequisite for endocytosis of endorphin by lymphocytes.

The nature of the interaction between gamma-type endorphins and the HLA class I molecules was studied by immunoelectronmicroscopy. The HLA molecules were not involved in the actual binding of endorphin to the cell. In contrast, for the endocytosis of gamma-endorphin, co-internalization of the HLA class I molecules is essential. The internalization process starts with clustering of gamma-endorphin and HLA class I molecules in coated pits. Cells that do not carry HLA class I molecules (Daudi) or do not internalize HLA class I molecules (EBV-transformed B cells) bind but do not internalize gamma-endorphin. On the basis of these observations, we suggest that the MHC class I molecules may function as transport molecules. Whether it is a general phenomenon that non-immunological ligands use the HLA class I molecules to get into the cell and immunological ligands (viral proteins) to reach the cell surface, remains to be established.

Differences in low density lipoprotein receptor expression in the suprabasal layer of normal and psoriatic epidermis.

Previous morphological experiments on the distribution of binding sites for low density lipoprotein (LDL) on normal and psoriatic epidermis in situ, done with the LDL-gold technique [Mommaas-Kienhuis AM, et al. J Invest Dermatol 89: 513-517, 1987.] showed an unequivocal correlation between the ability to bind LDL-gold complexes and the state of keratinocyte differentiation. To determine the involvement of the LDL receptor in this phenomenon, we applied immunoelectronmicroscopical methods in conjunction with a monoclonal anti-LDL receptor antibody. Biopsy specimens of normal and psoriasis skin were fixed before being embedded in Lowicryl K4M. Ultrathin sections were incubated first with the anti-LDL receptor antibody, and then with a second antibody conjugated to colloidal gold. On basal cells of both normal and psoriatic epidermis the LDL receptor was distributed evenly between the cell surface and the cytoplasm. No obvious differences in the density of LDL receptors were observed. However, cells from the suprabasal layer showed two striking differences in the localization of the LDL receptor: 1) normal epidermis showed fewer LDL receptor molecules, whereas in psoriasis epidermis the number increased relative to those on basal cells; and 2) in normal suprabasal cells most of the LDL receptors were located inside the cell, but in psoriasis the majority was found on the cell surface. Both phenomena are discussed and we postulate that the higher expression of LDL receptors in psoriasis suprabasal cells and the high expression of the receptor on the cell surface is connected with the hyperproliferative state of the disorder.

Ultrastructural localization of HLA-DR and HLA-DQ molecules in Langerhans cells and B cells: an immunoelectronmicroscopic study.

Using the immunoelectronmicroscopic techniques of Lowicryl embedding and ultracryomicrotomy, the intracellular distribution of HLA class II molecules was investigated on a human B cell line and on human Langerhans cells. These techniques enabled us to localize the HLA class II molecules on fixed specimens in which mobilization or clustering induced by cross linkage with antibodies is ruled out. Compared with Lowicryl embedding, the ultracryomicrotomy clearly showed more labeling. Both in B cells and Langerhans cells, the HLA-DR molecules were predominantly found in intracellular vesicular structures and on the plasma membrane. The HLA-DQ molecules were less abundant than HLA-DR molecules in the B cells and Langerhans cells. In the B cells, HLA-DQ molecules were found on the plasma membrane and in the cytoplasm. In the Langerhans cells, some HLA-DQ molecules were also found in intracellular vesicular structures. These experiments show a different localization of HLA-DR and -DQ antigens both in Langerhans cells and B cells, suggesting a different function for these molecules. The localization of HLA-DR molecules may indicate that these molecules take part in the recycling process of the plasma membrane in B cells and Langerhans cells. The HLA-DQ molecules are not internalized or recycled by the B cells, and a limited internalization of these molecules takes place in Langerhans cells. Finally, no HLA class II or clathrin molecules were found on the Birbeck granules, suggesting that, when no cross-linking with antibodies takes place, Birbeck granules, suggesting that, when no cross-linking with antibodies takes place, Birbeck granules do not play a role in recycling of HLA class II molecules in Langerhans cells.

Low density lipoprotein receptor expression on keratinocytes in normal and psoriatic epidermis.

Biochemical and morphologic studies on the interaction of low density lipoprotein (LDL) with cultured normal keratinocytes and squamous carcinoma cells have shown a negative correlation between LDL receptor activity and terminal differentiation of the epidermal cells [Ponec M et al, J Invest Dermatol 83:436-440, 1984 and Vermeer, BJ et al, J Invest Dermatol 86:195-200, 1986]. Whether such in vitro studies pertain to the epidermis in vivo is not known. To obtain information on the distribution of LDL receptors in the epidermis in situ, morphologic studies were performed using LDL-gold as an ultrastructural marker. When freshly isolated mouse and human epidermal cells were incubated with LDL-gold complexes, only keratinocytes with the morphologic characteristics of basal cells showed binding and uptake of LDL-gold. No LDL receptor activity was found on Langerhans cells, melanocytes or highly differentiated keratinocytes. Since cell separation techniques can destroy receptors, the staphylococcal epidermolytic toxin was utilized to produce intercellular and intra-epithelial splitting of the epidermis. In preparations of both normal mouse and human epidermis, LDL-gold binding was restricted to basal cells and a few suprabasal keratinocytes. In contrast, in psoriatic epidermis, and to a lesser extent, essential fatty acid-deficient mouse epidermis, cells in the stratum spinosum showed abundant LDL-gold binding. Thus LDL-gold may be a useful marker for epidermal differentiation.

Culture and characterization of rat middle-ear epithelium.

This study was performed to design a method for the culture of rat middle-ear epithelium and to apply the method to investigate the characteristics of this epithelium. Culture of explants of middle-ear epithelium in the presence of the epidermal growth factor was successful, whereas serial cultivation required 3T3 feeder cells in addition to the epidermal growth factor. Cultured middle-ear epithelium was studied by phase-contrast microscopy, transmission and scanning electron microscopy, and combined light and scanning electron microscopy (LM/SEM). These techniques showed similarity between the cultured and the natural middle-ear epithelium. Explants and outgrowths showed both flat polygonal and ciliated epithelial cells. In serial cultivation, however, only the first of these cell types was observed. Frequently, a single primary cilium was found on the cell surface. Transmission electron microscopy showed cross-linked envelopes whose formation was promoted by ionophore X537A. Cytokeratin was demonstrated by immunoblotting, immunofluorescence, and immunoperoxidase methods, using an anti-cytokeratin monoclonal antibody. The model described here permits study of the differentiation of middle-ear epithelium in vitro and may be of future value for the study of chronic middle-ear diseases.

Binding and internalization of low-density lipoproteins in SCC25 cells and SV40 transformed keratinocytes. A morphologic study.

Binding of low-density lipoproteins (LDL) to the plasma membrane and internalization of low-density lipoprotein receptor complexes were investigated in an epithelial tumor cell derived from the tongue (SCC25) and in SV40-transformed keratinocytes (SVK14 cells). For light microscopic studies an immunofluorescence technique with antiapoprotein B as well as conjugation procedure by which a fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanide (DIL) was conjugated with LDL (LDL-DIL) was used. Binding of LDL to the plasma membrane at 4 degrees C was observed in most SCC25 cells but not in SVK14 cells. The internalization of LDL-DIL was absent in SVK14 cells and was excessive in SCC25 cells. In SCC25 cells, internalization of the LDL-DIL particles was heterogeneously distributed over various cells. When a pulse-chase experiment was performed with LDL-DIL, less LDL was internalized into the SCC25 cells in comparison with a continuous label experiment. For the ultrastructural studies LDL conjugated with colloidal gold was used. In the binding experiments at 4 degrees C most LDL-gold particles were attached to the plasma membrane outside coated pits. During internalization experiments with LDL-gold particles it was observed that within 5-15 min at 37 degrees C several LDL-gold particles were seen in electron-dense structures near the plasma membrane. The electron-dense structures containing LDL-gold, as observed after an internalization period of 5-15 min, may represent the first endosomal compartment as described for transferrin receptors in A431 cells. After a period of 30 min at 37 degrees C the LDL-gold particles were observed in electron-lucent vesicles (multivesicular bodies) and dense bodies. However coated vesicles containing LDL-gold particles were seen sporadically. It is concluded that the route of internalization of LDL into the SCC25 cells differs from that of other cell types. No internalization of LDL gold was found in SVK14 cells, thus, in this respect, the SVK14 cells resemble normal keratinocytes. The morphologic data are in good agreement with biochemical studies published earlier (Ponec M et al, J Invest Dermatol 83:436-440, 1984). Both investigations suggest that LDL receptor activity is modulated during the process of terminal differentiation.

Morphological studies on cellular detachment induced by antibody reactions directed against membrane associated antigens. An ultrastructural study.

The skin explant model was used to determine the effect of antibody reactions against membrane associated antigens on normal human keratinocytes. Addition of specific allo-antibodies against HLA class I antigens induced characteristic changes in the cells on the outermost region of the explant-outgrowth. A disorganization of the filopodia of these cells occurred and the edges of the cellular border were lifted from the substratum. These signs of detachment were also found when pemphigus serum was added. In both experimental conditions the detachment of the cells was complement independent. After removing the antiserum a recovery took place, but the cells once lifted from the substratum remained recognizable as a ridge of cells. No changes were observed when the explants were incubated with antibodies against HLA class II antigens. Incubation with specific antibodies against HLA class I antigens not present on the explant had also no effect. We propose that antibody reactions against various membrane associated antigens can induce within a few hours characteristic changes of the cellular margins.

Modulation of low density lipoprotein receptor activity in squamous carcinoma cells by variation in cell density.

Morphological and biochemical studies on low density lipoprotein (LDL) receptor metabolism were performed in squamous carcinoma cells (SCC-15 and SCC-12F2). Modulation of terminal differentiation was achieved by culturing these cells at different cell densities. Studies on these cells cultured at low density (hardly any terminal differentiation) showed the following results: High affinity binding of LDL was excessive; LDL binding to SCC-15 cells was twice as high as that in SCC-12F2 cells and in fibroblasts. The distribution of the LDL binding visualized by LDL receptor antibodies was non-linear. There was no contact inhibition of LDL binding. LDL-gold particles were mainly bound to the plasma membrane outside coated pits. LDL-gold particles were internalized and delivered to dense bodies (= lysosomes). Degradation of LDL took place after a lag period of 10 min. Dissociation of LDL from the plasma membrane was substantial (more than 40% after a 120 min chase period). The same experiments on the cells cultured at high density (terminal differentiation present) showed several differences: A sharp decrease in high affinity LDL binding in both cell types. The internalization of surface bound LDL was defective in most of the squamous carcinoma cells. Dissociation of LDL from the plasma membrane was substantial, and after a chase period of 120 min at 37 degrees C still more than 20% of LDL remained intracellular and was not degraded. We postulate that LDL receptor-mediated endocytosis and degradation take place in squamous carcinoma cells but that during the process of terminal differentiation modulation of LDL-receptor metabolism occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages.

The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4 degrees C some LDL- or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization. However, after incubation of the cells at 8 degrees C with either LDL-gold or AcLDL-gold, lipoprotein-gold complexes were present in clusters on the plasma membrane, often in coated pits. Cells incubated at 37 degrees C for various time periods showed internalization of both LDL- and AcLDL-gold complexes via small coated and non-coated vesicles and processing of the complexes in smooth-walled endosomes. When the cells were pulse-chased with LDL- or AcLDL-gold for 30 min at 37 degrees C, the gold conjugates occurred in dense bodies, probably lysosomes. The results suggest that although native and modified LDL are reported to be metabolized differently by macrophages, the morphological aspects of the endocytosis of LDL and AcLDL by cultured human monocyte-derived macrophages are similar.