Search

Langerhans cells (LC) act as potent antigen-presenting cells (APC) for primary and secondary T cell-dependent immune responses. LC express several costimulatory and/or adhesion molecules such as B7/BB1, which has been implicated as one of the important determinants for professional APC. Recent studies have shown that B7/BB1 antigens comprise three distinct molecules termed B7-1, B7-2, and B7-3. We have examined the regulatory properties of B7-1 expression in LC using various cytokines including interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, interferon (IFN)-gamma, granulocyte/macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha. We have demonstrated: 1) that the B7-1 expression of LC is reproducibly up-regulated by either GM-CSF, TNF-alpha, IL-1 alpha, IL-1 beta, or IL-4 in a dose- and time-dependent manner, 2) that GM-CSF exhibits the most active effect on B7-1 up-regulation in each experiment, 3) that IFN-gamma or IL-10 profoundly inhibits the B7-1 expression of LC in a dose- and time-dependent manner, and 4) that the down-regulatory ability of IFN-gamma or IL-10 neutralizes the activity of up-regulatory cytokines. The enhancing or inhibitory action of these cytokines on B7-1 expression occurs selectively because none of the cytokines consistently affects I-A expression of LC. These data suggest that the B7-1 expression of LC may be dynamically regulated by these up- and down-regulatory cytokines in normal and inflammatory epidermal microenvironment.

Human Langerhans cells (LC) express CD45, but clear data about the isoform(s) and their function(s) are lacking. In the present study, double labeling experiments reveal that freshly isolated LC from normal skin are CD45RO+/RA-/RB-. However, after isolation and short-time culture where LC undergo an in vitro maturation resembling that to lymphoid dendritic cells, CD45RB emerges whereas CD45RO expression decreases. This evolution results from dynamic alternative RNA splicing. Addition of granulocyte-macrophage colony-stimulating factor or tumor necrosis factor-alpha to short-time cultures has no significant effect on CD45RB, but both cytokines accelerate the loss of CD45RO. LC isolated from lesional skin of atopic eczema highly express CD45RO and CD45RB. Cross-linking of CD45 on LC isolated from atopic individuals inhibits the calcium mobilization in response to activation via Fc epsilon receptor type I (Fc epsilon RI). Hence, the protein tyrosine phosphatase CD45 from human LC is subjected to a splicing phenomenon related to the differentiation and activation stage of these cells and regulates their Fc epsilon RI-mediated activation.

Epidermal Langerhans cell heterogeneity is poorly understood with regard to phenotypic characteristics, such as the expression of human leukocyte antigen (HLA)-DR, integrin, and Fc receptor molecules, as well as functional characteristics, such as the ability to process and present antigens or produce cytokines during various phases of immigration and maturation. Technical limitations of Langerhans cell number have limited functional assays on putative Langerhans cell subsets in in vivo epidermis. Therefore, we used flow cytometry for simultaneous phenotypic and functional assessment at the single-cell level within the Langerhans cell population. Freshly isolated human epidermal cell suspensions were stained with a battery of monoclonal antibodies, including anti-HLA-DR, -CD1a, -CD1c, -CD11c, -Fc gamma RII, and -Fc epsilon RI. Two distinct Langerhans cell subsets were identified by their different levels of HLA-DR expression. The DRHi subset expressed higher amounts of CD11c and exhibited greater cytoplasmic complexity and higher baseline calcium than the DRLo subset (p < or = 0.03 for each). Some subjects also expressed high levels of Fc epsilon RI in the DRHi, CD11cHi subset. To determine whether these phenotypic subsets may exhibit differential signal-transduction functional properties, Langerhans cells were partially enriched over Ficoll-Hypaque and their cytosolic mobilization after the addition of ionomycin was analyzed using the calcium indicator, indo-1, in conjunction with quantitative analysis of HLA-DR expression. By this real-time flow cytometric analysis, a new subpopulation was revealed within the DRLo Langerhans cell subset. This subset increased its cytosolic calcium concentration much more than the other two subsets (change in indo-1 blue:violet emission ratio of 37.33 +/- 2.34 in the Hi Flux DRLo subset versus 13.23 +/- 0.29 in the Lo Flux DRLo subset, and versus 7.6 +/- 2.99 in the Lo Flux DRHi subset). These data indicate that functional, as well as phenotypic, subsets of Langerhans cells exist within normal human epidermis. Their responses to physiologic stimuli may relate to maturational stage or the level of in vivo activation.

Murine epidermal Langerhans cells (LC) synthesize and express E-cadherin, a homophilic adhesion molecule that mediates adhesion of LC to keratinocytes in vitro. To determine if E-cadherin expression is characteristic of LC or is a feature of all dendritic cells (DC), we studied DC from various lymphoid tissues and peripheral blood for reactivity with anti-E-cadherin monoclonal antibody. By flow cytometry, DC prepared from skin-associated lymph nodes (LN) expressed E-cadherin, whereas DC prepared from gut-associated LN and spleen did not. However, direct comparison revealed that levels of E-cadherin expressed by DC from skin-associated LN were approximately fivefold lower than those expressed by freshly-prepared LC. Immunohistochemical studies confirmed that E-cadherin was expressed by DC in skin-associated LN in situ, and demonstrated that the number of E-cadherin+ DC in LN draining skin previously treated with the contact allergen 2,4,6-trinitrochlorobenzene was increased relative to the number of E-cadherin+ DC present in LN draining normal skin. DC propagated from the blood of cyclophosphamide-treated mice in granulocyte/macrophage-colony stimulating factor-supplemented media also expressed E-cadherin. E-cadherin immunoprecipitated from DC co-migrated in SDS polyacrylamide gels with that from fibroblasts transfected with murine E-cadherin cDNA, and mRNA encoding extracellular and intracellular regions of E-cadherin was present in DC propagated from blood. These results indicate that E-cadherin expressed by murine dendritic cells is identical to E-cadherin expressed by epithelial cells, and suggest that E-cadherin represents a DC differentiation antigen characteristic of LC and lineage-related cells (skin-associated LN DC).

Epidermal Langerhans cells (LC) are major histocompatibility complex (MHC) class II (Ia)-positive dendritic cells that act as potent antigen-presenting or accessory cells for primary and secondary T cell-dependent immune responses. Recent studies have disclosed that the morphological, functional, and phenotypic characteristics of LC are variably and drastically modulated by external stimuli both in vivo and in vitro. However, little is known of the biological significance of diverse cytokines in regulating the surface molecules of LC. To determine the regulatory properties of ICAM-1, Ia, and MHC class I (H-2K) molecules in LC, we have examined the effects of interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the expression of these molecules. Among the cytokines examined, IFN-gamma markedly and reproducibly up-regulates the expression of H-2K, but not ICAM-1, in Ia+ LC in a time- and dose-dependent manner. TNF-alpha consistently up-regulates the expression of ICAM-1, but not H-2K, in a time- and dose-dependent manner. IL-10 slightly but reproducibly inhibits the expression of ICAM-1, but not H-2K, in a time- and dose-dependent manner. IL-10 potently inhibits the TNF-alpha-induced ICAM-1 up-regulation, but not the IFN-gamma-induced H-2K up-regulation. Moreover, no cytokine consistently affects the Ia expression of LC. In addition, slight enhancing effects have been observed on H-2K expression by IL-4, and on ICAM-1 expression by IL-1 alpha, IL-1 beta, or GM-CSF. The present data suggest that the selective regulation is operative in a certain cell surface moiety of LC by various cytokines. These results further facilitate our understanding of immunobiology of LC.

Selected aspects of the histopathology of Langerhans cell histiocytosis representing diagnostic difficulty and/or controversy are presented with emphasis on the composition of pathological lesions. Lesional cell phenotypes and the factors influencing variations are noted. Features of several skin-based histiocytic disorders, dermatopathic lymphadenopathy and Rosai-Dorfman disease are compared. Associations between Langerhans cell histiocytosis and juvenile xanthogranuloma and malignant disorders are considered. Observations of potential significance in the eventual elucidation of the pathogenesis of these enigmatic diseases are presented.

Langerhans cells (LC) belong to the dendritic cell lineage and are the principal antigen-presenting cells of squamous epithelia. Short-term cultured LC (cLC) exhibit a marked augmented capacity to stimulate allogeneic T cells and acquire the ability to activate naive T cells, probably in relation to enhanced expression of accessory signals. In this study, we evaluated the expression of B7 costimulatory molecule (CD80) in human freshly isolated (fLC) and cLC at both the protein and mRNA level. Staining of frozen skin sections did not reveal any epidermal dendritic cell reactive with either of two different anti-B7 monoclonal antibodies. fLC in suspension did not exhibit any B7 staining as evaluated by two-color flow-cytometry analysis and immunoelectron microscopy. In contrast, LC that were cultured for 24-72 h displayed strong surface B7 reactivity with a characteristic patchy pattern. Treatment with dispase and trypsin did not reduce B7 staining of cLC. Following warming to 37 degrees C, cLC tagged with anti-B7 monoclonal antibody and gold-conjugated secondary antibody could internalize surface B7 by using the organelles of receptor-mediated endocytosis. B7 mRNA, detected by the reverse-transcriptase polymerase chain reaction technique, was expressed at a low level in purified (> 90% HLA-DR+) fLC but not in LC-depleted epidermal cells, and was markedly upregulated in purified cLC. The results indicate that 1) fLC do not express B7 protein on their surface, but acquire B7 during culture, 2) surface B7 is not sensitive to trypsin, 3) B7 expression is regulated primarily at the mRNA level, and 4) membrane B7 can be internalized within cLC. B7 molecule on CLC may be relevant to their increased antigen-presenting cell potency and ability to stimulate naive T lymphocytes.

We investigated the ability of Aloe barbadensis gel extract to prevent suppression of contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) responses in mice by ultraviolet (UV) irradiation. Local immune suppression was induced in C3H mice by exposure to four daily doses of 400 J/m2 UV-B (280-320 nm) radiation from FS40 sunlamps, followed by sensitization with 0.5% fluorescein isothiocyanate (FITC) through the irradiated skin. Topical application of 0.167-1.67% Aloe gel after each irradiation significantly reduced this suppression. Aloe treatment partially preserved the number and morphology of Langerhans and Thy-1+ dendritic epidermal cells in skin, compared to those in the skin of mice given only UVR or UVR plus the vehicle. Experiments using a single (2 kJ/m2) dose of UVR followed by Aloe treatment showed that the effect of Aloe was not due to screening of the UVR. Systemic suppression of DTH to Candida albicans or CHS to FITC was induced in C3H mice exposed to 5 or 10 kJ/m2 UV-B radiation, respectively, on shaved dorsal skin and sensitized 3 d later with a subcutaneous injection of formalin-fixed Candida or FITC painted on unirradiated, ventral skin. Treatment of the UV-irradiated skin with Aloe immediately after irradiation prevented suppression of both DTH to Candida and CHS to FITC. Aloe treatment did not prevent the formation of cyclobutyl pyrimidine dimers in the DNA of UV-irradiated skin or accelerate the repair of these lesions. These studies demonstrate that topical application of Aloe barbadensis gel extract to the skin of UV-irradiated mice ameliorates UV-induced immune suppression by a mechanism that does not involve DNA damage or repair.

An immunoperoxidase technique was used to compare wax-embedded tissue with frozen tissue for quantitative immunohistochemistry of oral mucosal Langerhans cells. Initial experiments using anti-CD1a, -HLADR and -S100 antisera showed that phenotype, fixative, antibody dilution and trypsinisation of the tissue section significantly affected Langerhans cell counts. Only the anti-HLADR antibody detected Langerhans cells in both frozen and wax-embedded sections. Some 38% of S100-positive dendritic cells were situated in the stratum basale, and 41-84% of these contained melanin as determined by double-labelling. Sections from 39 volunteers were then reacted with the anti-CD1a and -HLADR antibodies. The morphology of Langerhans cells was more dendritic in frozen sections, and the mean HLADR-positive Langerhans cells count in frozen sections was significantly higher than that in wax-embedded sections from the same individual. The intra-individual ratio of counts between frozen and wax-embedded sections was variable; hence, the apparent loss of HLADR antigenicity as a result of tissue processing was unpredictable. Counts of CD1a-positive Langerhans cells were consistently higher. We conclude that the use of anti-CD1a antibody on frozen tissue is the optimum method for quantitative studies of oral mucosal Langerhans cells, and that such studies performed on wax-embedded tissue may be unreliable.

An immunocytochemical method was developed to study in vivo induction and removal of DNA damage in a specific cell population in the epidermis of hairless mice after ultraviolet B (UVB) exposure: the immunocompetent antigen-presenting Langerhans cells. To this aim, Ia+ cells, which are representative for epidermal Langerhans cells, were compared with the bulk of epidermal cells with respect to the nuclear level of cyclobutane thymine dimers. Mouse Langerhans cells were identified with a membrane-located immunoperoxidase stain, whereas DNA and DNA damage were revealed with fluorescent nuclear stains. After a low dose of UVB (approximately 1 minimal erythema dose), dimer levels were determined both in all murine epidermal cells and in Ia+ cells separately. At 24 h after irradiation, dimer removal was still incomplete, with a persistence of approximately 50% of the initially induced dimers in epidermal cells in general, and of approximately 75% in Langerhans cells. Possible applications of the method developed and the results presented here are discussed in relation to the immunosuppressive effect of UV.

Adhesion receptors and their ligands play a vital role in the immune system. We studied the expression of different adhesion receptors, using single- and double-staining immunohistochemical techniques, in both lesional and non-lesional skin specimens from seven psoriasis patients and in skin biopsy specimens from eight normal healthy controls. Our results showed an overall increased expression of several adhesion receptors in both lesional and non-lesional psoriatic skin. We consistently found an increased expression in particular of ICAM-1 and E-selectin on endothelial cells, and ICAM-1 on T cells and Langerhans cells. In contrast, a weak expression of VCAM-1 was found on endothelial cells and mononuclear cells in lesional psoriatic skin specimens alone. Interestingly, LFA-1 was also expressed on Langerhans cells, with a greater frequency in skin from lesional than from non-lesional sites, but was never expressed in skin from normal healthy individuals. Furthermore, significantly increased numbers of Langerhans cells and T cells with a positive reactivity for MAb HECA-452 were found in both lesional and non-lesional psoriatic skin. We hypothesize that the enhanced expression of adhesion receptors on migrating immunocompetent cells and endothelial cells of psoriatic skin in general facilitates the increased influx of activated T lymphocytes and other immunocomponent cells into the skin, and thus underscores the generalized character of the disease.

Adhesion receptors on endothelial cells are considered to be important for cellular influx in tissue. In this regard, skin constitutes a specialised environment for migration of leukocytes during inflammation. Using immuno-enzymatic staining techniques, we compared the in situ expression of ICAM-1, E-selectin, and VCAM-1 on endothelial cells and inflammatory infiltrates in both lesional and non-lesional biopsied skin from two immuno-inflammatory diseases, viz. psoriasis and contact dermatitis. The results were compared with those in skin specimens obtained from normal healthy individuals free from any history of skin disease. Our results show that ICAM-1 and ELAM-1 are upregulated in psoriatic non-lesional and lesional skin. On the other hand, in non-lesional biopsy from contact dermatitis patients, all three AR molecules are sparsely present, similar to the situation in normal skin although they are overtly expressed in the lesional sites. Moreover, VCAM-1 was found to be significantly increased on endothelial cells in the lesional sites of contact dermatitis as compared with biopsied psoriatic specimens. Interestingly VCAM-1 was also found to be present on some T-cells and Langerhans cells in contact dermatitis alone. The present data suggest that in different inflammatory dermatitis, varying adhesion receptor-ligand interactions involving endothelial cells and leukocytes are involved, which may be due to the differing cytokine profiles of perivascularly located T-cells.

In human epidermis, expression of CD1a is confined to Langerhans cells (LC), whereas CD1c expression has been observed in dendritic cells of the dermis, as well as the epidermis. In transfected fibroblasts, expression of CD1c at the cell surface appears to exclude expression of either CD1b or CD1a, despite continued transcription of the latter genes. In order to determine whether this mechanism might be operative in human skin, we have compared the expression of CD1a and CD1c on the surface of dermal and epidermal dendritic cells to their expression at the level of mRNA using a combination of dual-label immunofluorescence microscopy, northern blot hybridization, and reverse transcriptase-polymerase chain reaction (RT-PCR). By both immunofluorescence and Northern blotting, CD1c expression was observed in both dermal and epidermal cells, whereas expression of CD1a was confined largely to the epidermis. Moreover, as shown by immunomagnetic bead selection and RT-PCR, CD1a and CD1c were both expressed on epidermal LC, but were absent from other epidermal cell types. These results argue against cell surface exclusion as a mechanism for selective expression of CD1c in human dermis.

Quantitative and detailed 3-dimensional (3-D) morphological information can be obtained from the same tissue volume using a confocal laser scanning microscope (CLSM). In the present study, we used CLSM for evaluation of Langerhans' cells (LC) in human skin at 0 h, 24 h and 48 h following occlusion with patch tests for 48 h. The relative volume of epidermal CD1a reactivity was quantified with CLSM on 25 microns thick sections stained with indirect immunofluorescence. No statistically significant difference was found when comparing the values obtained on the biopsy specimens from occluded skin (n = 36) with those from non-occluded skin (n = 9). Nor were any statistically significant changes detected in the number of epidermal CD1a+ cells as determined with immunoperoxidase staining between occluded and non-occluded skin. The occlusion produced a transient mild inflammatory reaction with an induced expression of intercellular adhesion molecule-1 (ICAM-1) on keratinocytes and an increased number of CD3+ epidermal lymphocytes. In addition, 3-D reconstructions revealed spatial information on the distribution of LC dendrites towards the skin surface.

Granulocyte/macrophage-colony stimulating factor (GM-CSF) induces in vitro activation of Langerhans' cells. The association of GM-CSF and tumour necrosis factor alpha (TNF alpha) induces the differentiation of Langerhans' cells from CD34 positive haematopoietic progenitors. Intradermal administration of recombinant GM-CSF is associated with local accumulation of Langerhans' cells. We investigated the presence of GM-CSF in tissue samples of 10 patients with Langerhans' cell histiocytosis. Four patients had skin involvement, three had bone and three had diffuse disease. Eight normal skin samples were analysed as controls. Immunohistochemistry was performed on frozen tissue samples with two specific monoclonal antibodies directed against two different epitopes of GM-CSF. We detected GM-CSF in all the histiocytosis tissue samples. The GM-CSF was detected within the cytoplasm of all the tumoral Langerhans' cells. We did not find GM-CSF in any other cell type. These results suggest that GM-CSF may be implicated in the pathogenesis of Langerhans' cell histiocytosis.

Langerhans cells are characterized by specific markers, such as Birbeck granules and CD1a antigen. S100 protein and neuron-specific enolase are less specific but are expressed only on Langerhans cells among the cells of the phagocytic mononuclear system. In this study, the expression of these two antigens on many monocytic leukemic cells is shown. These cells could be precursors of Langerhans cells which have been transformed into malignant cells.

The LFA-1 integrin mediates its function in leukocyte intercellular interactions by recognition of at least one of its three identified counter-receptors, intercellular adhesion molecules 1 (ICAM-1), 2, and 3. The ICAM-1 molecule is expressed in an inducible-dependent manner by endothelial and epithelial cells, as well as by other cell types, including leukocytes. On the other hand, ICAM-2 is constitutively expressed mainly by endothelial cells. We have studied the tissue distribution of the ICAM-3 molecule by immunohistochemical staining of lymphoid and nonlymphoid organs. Only cells from the leukocyte lineage were found bearing the ICAM-3 antigen, showing a pattern of expression clearly distinct from those of ICAM-1 and ICAM-2. Interestingly, we have found that ICAM-3 is expressed by epidermal dendritic Langerhans cells as assessed by double immunostaining with antibodies specific for CD1. In contrast, staining of skin sections with anti-ICAM-1 and ICAM-2 antibodies showed an undetectable expression of these two molecules on Langerhans cells. However, CD1+ Langerhans cells localized in the paracortical area of dermatopathic lymph nodes expressed both ICAM-1 and ICAM-3 antigens. Our results indicate that ICAM-3 is the main LFA-1 counter-receptor in human resident epidermal Langerhans cells. ICAM-3 may have an important role in T-cell antigen stimulation driven by Langerhans cells during skin immune reactions.

Histochemical assessment of selected carbohydrate sequences on Langerhans cells of human oral mucosa was made by combined use of enzyme digestion and immunostaining with monoclonal antibodies against specific carbohydrate structures. In both frozen sections and epithelial sheets without the enzyme pretreatment, mucosal Langerhans cells, identified by positive staining with anti-CD1a and HLA-DR antibodies, did not express any carbohydrate antigens on their surface. In contrast, following neuraminidase pretreatment of both types of material, the fucosylated type 2 chain (Le(x)) became detectable on Langerhans cells, indicating that sialic acid is the terminal residue of this sequence. Other enzymes were ineffective in this apparent unmasking, and the staining patterns of the other related carbohydrate sequences (Le(y)+, Le(a), Le(b)) remained unaffected by pretreatment with any of the enzymes used. These findings suggest that the mucosal Langerhans cells possess a unique carbohydrate chain, the sialyl fucosylated type 2 sequence (sialyl Le(x) antigen).

The maintenance and regulation of continuously renewing tissues is ultimately controlled at the level of stem-cell proliferation. We have recently identified a reversible inhibitor of hemopoietic stem-cell proliferation (stem-cell inhibitor [SCI]), which is identical to the macrophage inflammatory protein, MIP-1 alpha, a 69-amino-acid heparin-binding cytokine. To test the cell/tissue specificity of the inhibition of proliferation by SCI/MIP-1 alpha, we have investigated its activity on epidermal keratinocytes, the principal cell type of another continuously renewing tissue. Here we show that SCI/MIP-1 alpha inhibits the proliferation of epidermal keratinocytes in vitro and that the MIP-1 alpha mRNA is present in epidermal Langerhans cells but not in keratinocytes. This suggests an important growth regulatory function for SCI/MIP-1 alpha in keratopoiesis, as well as hemopoiesis, and may also indicate a novel role for the epidermal Langerhans cell. As SCI/MIP-1 alpha can inhibit the proliferation of embryologically distinct precursor cells, this raises the possibility that it may also function in a number of other tissues.

The expression of the alpha 6 beta 4 and alpha 6 beta 1 integrins on epidermal Langerhans cells (LC) before and after mast cell degranulation was studied in cultured human neonatal foreskin by immunohistochemistry. Twenty-four hours after addition of mast cell secretagogues, morphine sulfate, or substance P, solitary mid-epidermal cells showed staining for the integrin subunits alpha 6, beta 4, and beta 1. This expression was not observed in cultured control explants, and immunostained cells were confirmed to be non-epithelial, dendritic cells by immuno-electron microscopy. The identity of these cells as LC was further established by coincident staining for alpha 6 and CD1a using double immunofluorescence labeling. Addition of tumor necrosis factor-alpha (TNF alpha), the predominant cytokine in mast cell granules, also induced LC to express alpha 6 integrins. Furthermore, preincubation of skin organ cultures with anti-TNF alpha antibodies or the mast cell inhibitor cromolyn sodium abrogated the ability to induce alpha 6 integrins on LC consequent to experimental mast cell degranulation by substance P. These data implicate a role for mast cell-derived TNF alpha in the regulation of the integrins alpha 6 beta 4 and alpha 6 beta 1 on LC. These findings may have important implications relevant to mechanisms for spatial localization of LC within the cutaneous compartments during immune responses.

Expression of the adhesion molecules ICAM-1 and LFA-1 (CD11a/CD18) on mouse epidermal Langerhans cells (LC) and on spleen dendritic cells (DC) from BALB/c mice was examined by staining with specific mAb and was evaluated by flow cytometry. LC were shown to express both ICAM-1 and LFA-1, whereas spleen DC expressed only LFA-1. The contribution of these adhesion molecules to LC- or DC-induced activation of keyhole limpet hemocyanin (KLH)-specific, Iad-restricted, Th1 or Th2 clones was investigated in mAb blocking studies. At optimal doses, anti-CD11a or anti-CD18 mAb completely inhibited Th1 proliferation induced by either LC or DC. Anti-ICAM-1 also abrogated Th1 proliferation induced by LC, but only moderately reduced Th1 proliferation induced by DC. Inhibition in these experiments was specific, since isotype-matched control Ab against other Ag constitutively expressed on LC (NLDC 145) or DC (33D1) had no effect on Th1 proliferation. In marked contradistinction, the capacity of LC to present KLH to our Th2 clones was resistant to treatment with the same mAb against ICAM-1, CD11a or CD18. We conclude that interactions between ICAM-1 and LFA-1 on epidermal LC and LFA-1 on spleen DC with their respective ligands on our Th1 clones are required for optimal presentation of protein Ag to Th1. Our results also indicate that neither ICAM-1 nor LFA-1 is required for the analogous activation of our Th2 clones by LC.

Eosinophilic ulcer of the oral mucosa (traumatic eosinophilic granuloma) mimics oral cancer clinically and is occasionally misdiagnosed as lymphoma on microscopic examination. Trauma is believed to play a role in its development, but its exact pathogenesis is not known. The demographic, clinical, and histologic features of 38 previously unreported cases of eosinophilic ulcer of the oral mucosa are reviewed. Nine representative cases were studied immunohistochemically. Microscopically, the lesions contained a polymorphic inflammatory infiltrate extending deep into the submucosa, underlying muscle, and salivary glands. Numerous eosinophils and large mononuclear cells with pale nuclei and frequent mitoses were seen in all lesions. Lymphocytes, histiocytes, plasma cells, granulocytes, and mast cells were also present. Immunohistochemical stains showed that the lymphocytic infiltrate was composed predominantly of T cells. T-cell-specific antigen-presenting cells were more numerous than the non-antigen-presenting cell type. The large cells with pale nuclei stained positively only for vimentin; the possible myofibroblastic nature of these cells is discussed. Although trauma might have an etiologic role, the pathogenesis of eosinophilic ulcer of the oral mucosa is probably T cell mediated.

In the present study we investigated whether there is a correlation between Langerhans cell density, peritumoral infiltrate or intraepithelial T-lymphocyte density within 17 epithelial skin tumors using immunohistologic and morphometric methods. We found a significant difference between seborrheic keratosis, basal cell carcinoma, and squamous cell carcinoma. No correlation was found between the Langerhans cell density and the number of intraepithelial T lymphocytes or between Langerhans cell density and the peritumoral infiltrate. A significant (inverse) correlation was found between mean nuclear area of the epithelial tissue and the number of Langerhans cells. These data might indicate that the number of Langerhans cells does not influence the extent of the anti-tumoral immune response, but that there is an association between the differentiation state of the epithelium and Langerhans cell density.

Lichen planus-like keratosis (LPLK) (benign lichenoid keratosis) is a common skin lesion that shows some morphologic features of lichen planus (LP) and lichenoid actinic keratosis (LAK). To try to detect differences among these three entities, immunohistochemical staining for S100 protein and HLA-DR (with the antibody LN3) was performed in 31 cases of LPLK, 26 of LAK, and 25 of LP. Langerhans cells (LC) were counted per linear mm of epidermis in the S100 and LN3 slides. With anti-S100 staining, LP cases showed higher numbers of LC (mean = 25.3, SE = 2.84, median = 21.2) than did LPLK (mean = 17.3, SE = 2.26, median = 14.5) and LAK (mean = 9.7, SE = 1.5, median = 5.4). With LN3 stains, LP cases also showed higher numbers of LC than did LPLK and LAK. These results suggest that the involvement of LC in the production of lesions may be different in these three entities. However, due to the overlap in distribution of values observed, the use of these stains does not allow a definite diagnosis to be made exclusively based on the number of LC.

After hapten application, epidermal Langerhans cells migrate into the regional lymph nodes through dermal lymphatics. Recently, we have demonstrated that some of them take the phenotypic and functional characteristics similar to those of in vitro cultured Langerhans cells, before disappearing from the epidermis. To analyze the mechanisms underlying the migration of Langerhans cells, we studied the expression of several adhesion molecules on freshly isolated LC and cultured LC. Pgp-1 (CD44), intercellular adhesion molecule 1, and alpha 4 integrin were strongly expressed on cultured Langerhans cells. Among them, only alpha 4 integrin was strongly up-regulated by cultured Langerhans cells, because its expression by freshly isolated Langerhans cells was very weak. This up-regulation of alpha 4 integrin was also observed on in vivo activated Langerhans cells in the epidermis and draining lymph nodes after hapten application. These data suggest a possible role played by VLA-4 in the migration of Langerhans cells from the epidermis into the regional lymph nodes after hapten application.

Double immunostaining for OKT-6 and S-100 protein antibodies was carried out. However, it was impossible to stain using two antibodies at the same time, or using one antibody prior to another. We first stained using FITC labeled OKT-6 antibody, and secondary stained for S-100 protein by the ABC method, after taking photographs and removing OKT-6 reaction products. The exact same cells could be stained by both OKT-6 and S-100 protein antibodies. It has been elucidated that both OKT-6 and S-100 protein positive cells are LCs, and only S-100 protein positive, but OKT-6 negative cells are IDCs. LCs only exist in the superficial and hilar lymph nodes. However IDCs exist not only in these lymph nodes, but also in the mesenteric lymph nodes and spleens.

The mechanism of transmission of HIV from the male to the female genital tract or in the reverse order is not clear. CD4 glycoprotein is the receptor for HIV and Langerhans cells and the related dendritic cells could play a role in the initial transmission of HIV. Fc receptors (FcR) for IgG might be involved in antibody-mediated binding of HIV. We carried out an immunohistological study of normal human cervical and vaginal epithelia for the presence of CD4 glycoprotein, Langerhans cells and FcR to IgG. CD4+ glycoprotein was not found in the vaginal or cervical epithelium, with the exception of a few endocervical epithelial cells. A small number of CD4+ mononuclear cells were found in the endocervical epithelium of a third of the specimens but a large number of CD4+ cells was found in the submucosa of most of the cervical and vaginal specimens. Langerhans cells expressing CD4, HLA class II, Fc gamma R2 and Fc gamma R3 were detected in most vaginal, ectocervical and transformation zone epithelia and in 9/14 endocervical tissues. Fc gamma R3 was detected in about two-thirds of the columnar endocervical epithelium and the transformation zone. A smaller number of specimens expressed Fc gamma R2 in these epithelia, but Fc gamma R1 was not detected. We then demonstrated mRNA for Fc gamma R3 in the columnar endocervical epithelial cells and transformation zone by in situ hybridization, using a CD16-RNA probe. Fc gamma R3 and Fc gamma R2 gene transcripts were also found in fetal cervical tissue by applying the polymerase chain reaction to amplify portions of the Fc gamma R3 and Fc gamma R2 coding sequences in cDNA prepared from fetal RNA. HLA-DR was found in the endocervical cells, transformation zone and in Langerhans cells of all specimens. The presence of Langerhans cells, Fc gamma receptors and HLA class II antigen offers three potential mechanisms for cervico-vaginal HIV transmission: (i) direct HIV infection of Langerhans cells, (ii) binding of HIV antibody complexes to cervical epithelial Fc gamma receptors and (iii) binding of HIV infected CD4+ cells to cervical HLA class II antigen which may infect these or the adjacent CD4+ cells.

Interaction of T-cell antigen receptors with antigen/major histocompatibility complex (MHC) molecule complexes on antigen-presenting cells (APC) leads to T-cell activation. Additional interaction between adhesion molecules on the APC and their ligands on T cells is required for optimal T-cell activation. In vitro cultured human epidermal Langerhans cells (cLC) are powerful APC that express adhesion molecules LFA-3 and ICAM-1. Both molecules on cLC serve a functional role in the generation of a T-cell response.

Immunocompetent cells of the epidermis can interact by the elaboration and recognition of cytokines. Although much new information has been reported concerning the cytokines secreted by keratinocytes, little is known about cytokines derived from Langerhans cells (LC). To address this deficiency, we examined cytokine mRNA profiles in different epidermal preparations from BALB/c mice, taking advantage of the sensitive technique of polymerase chain reaction (PCR), after reverse transcription of mRNA. In assays of epidermal sheets separated from dermis by ammonium thiocyanate, mRNA for IL-1 alpha, IL-1 beta, IL-6, IL-7, tumor necrosis factor alpha (TNF alpha), TNF beta, granulocyte macrophage/colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) were unequivocally present. By contrast, faint bands were detected for IL-4, IL-5, and interferon gamma (IFN gamma), and no PCR signal was detected for IL-2. Importantly, assays of epidermal cells (EC) dissociated with trypsin revealed similar mRNA profiles. To determine the effects of cell isolation, fluorescence-activated cell sorter (FACS)-purified Ia- EC were first analyzed; all of the previously cited cytokine mRNA were present except for IL-1 beta and MIP-1 alpha. EC depleted of LC by a second technique, lysis using anti-Ia monoclonal antibody and complement, revealed similar profiles, with substantially reduced PCR signals for IL-1 beta and MIP-1 alpha. Finally, FACS-purified LC (Ia+ EC) clearly expressed IL-1 beta and MIP-1 alpha mRNA, a finding that was verified by Southern blotting using internal oligo probes. We conclude that these cell-isolation procedures did not produce substantial alterations in basal mRNA profiles and that LC are the principal source of mRNA for IL-1 beta and MIP-1 alpha among unstimulated EC in mice.

The hepatic alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP) binds and endocytoses alpha 2-macroglobulin-proteinase complexes in plasma. In addition, it binds lipoproteins, a novel 40 kDa protein, and complexes between plasminogen activators and plasminogen activator inhibitor type-1. This study shows, for the first time, the tissue distribution of alpha 2MR/LRP as determined by immunohistochemistry with specific monoclonal antibodies. The analysis revealed alpha 2MR/LRP-expression in a restricted spectrum of cell types, including neurons and astrocytes in the central nervous system, epithelial cells of the gastrointestinal tract, smooth muscle cells, fibroblasts, Leydig cells in testis, granulosa cells in ovary, and dendritic interstitial cells of kidney. Monocyte-derived cells displayed marked alpha 2MR/LRP expression in the phagocytes of liver, lung and lymphoid tissues, but no or low expression in antigen-presenting cells including Langerhans' cells of the skin. The high abundance of alpha 2MR/LRP in certain cell types of most organs suggests two main routes for alpha 2MR/LRP-mediated ligand clearance: (1) systemic removal in liver of circulating ligands, and (2) non-hepatic interstitial removal in different organs, including the brain.

The arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) is the main eicosanoid formed by epidermal cells and is assumed to play an important role in skin physiology and pathophysiology. Our aim was to find out whether epidermal Langerhans cells possess specific receptors for 12-HETE which would mediate the effects of this eicosanoid in their skin microenvironment. By radioligand binding studies on isolated human Langerhans cells, we could identify specific binding sites for 12(S)-HETE. The analysis of binding data revealed a single class of binding sites with a Kd of 3.32 +/- 0.45 nM and a Bmax of 691,000 +/- 58,000 receptors per cell. The binding was saturable, readily reversible, and specific for 12(S)-HETE. The receptor is likely to mediate the potent chemotactic response of human Langerhans cells towards 12(S)-HETE, which we previously described. Our results strongly suggest that extremely low concentrations of 12-HETE which is formed by epidermal keratinocytes may dramatically influence the biology of Langerhans cells by receptor-mediated effects.

cDNA isolated from a human lymphocyte library were analyzed and shown to encode a novel cell-surface glycoprotein, termed HB15, expressed by dendritic cell subsets and activated lymphocytes. The predicted mature 186 amino acid protein was composed of a single extracellular V-type Ig-like domain, a transmembrane region, and a 39-amino acid cytoplasmic domain. In contrast to most Ig-like domains, analysis of a partial genomic DNA clone revealed that the extracellular Ig-like domain of HB15 was encoded by at least two exons. Northern blot analysis revealed that HB15 derived from three mRNA transcripts of approximately 1.7, 2.0, and 2.5 kb expressed by lymphoblastoid cell lines. Two mAb reactive with HB15 were produced and used to show that HB15 is expressed as a single chain cell-surface glycoprotein of M(r) 45,000. HB15 expression was specific for lymphoblastoid cell lines and mitogen-activated lymphocytes, and HB15 was not expressed at detectable levels by circulating leukocytes. Immunohistologic analysis revealed that HB15 had a unique pattern of expression, being found predominantly in hemopoietic tissues with strong expression by scattered interfollicular interdigitating reticulum cells and weak expression by germinal center cells. HB15 was also expressed by Langerhans cells within the skin. HB15 therefore serves as a unique marker for the subset of dendritic cells represented by Langerhans cells and interdigitating reticulum cells. Thus, the HB15 glycoprotein represents a newly identified member of the Ig superfamily that may play a significant role in Ag presentation or the cellular interactions that follow lymphocyte activation.

Langerhans' cells (LC) were investigated immunohistochemically and electron microscopically in normal equine epidermis and 133 equine cutaneous papillomas experimentally induced in five 2-year-old Thoroughbred horses. Class II major histocompatibility complex antigen-positive dendritic LC were found in the normal epidermis and ultrastructurally had the characteristic Birbeck's granules. In the developing phase of the papillomas, LC were significantly decreased in number and size, indicative of a hypofunctional state. In the regressing phase of the papillomas, LC were markedly increased in number, especially at the epidermis-dermis junction. LC with long dendrites were rich in cytoplasm with well-developed cytoplasmic organelles, including Golgi apparatus, lysosomes, Birbeck's granules, and multivesicular bodies. These LC were hyperfunctional. An infiltration of many T lymphocytes was also observed at the epidermis-dermis junction.

Langerhans cells (LC) serve as antigen presenting cells and provide immune surveillance within epithelia. Since depression of LC number and/or function may allow tolerance to antigens, we evaluated LC in median rhomboid glossitis (MRG), a condition linked to persistent candidal infection of lingual mucosa. Material included a total of 36 cases of MRG (7 of which did not show PAS + fungi) and 6 controls. LC were identified by their expression of S-100 and HLA-DR antigens and quantified using image analysis. Equal numbers of LC were identified using S-100 + and HLA-DR + markers. The density of LC (cells/mm of basement membrane, mean +/- SD) in both PAS + MRG (2.6 +/- 1.3) and PAS-MRG (3.0 +/- 1.7) was markedly depressed compared with controls (17.2 +/- 6.4), (P less than 0.001). These findings indicate that the LC network is perturbed in MRG, and are consistent with the view that of localized defect in immune surveillance may contribute to persistent fungal infection of the oral mucosa.

It is now becoming clear that a collection of adhesion molecules is required on the surface of epidermal cells (EC) to establish the cell interactions that are necessary for skin immunologic reactions. In previous studies, we showed that human resting Langerhans cells (LC) express at least two members of the "integrins" family of adhesive molecules, as well as the intercellular adhesion molecule-1, which is a member of the immunoglobulin-related superfamily of molecules. This latter family includes another adhesive moiety, namely, the lymphocyte function-associated antigen-3 (LFA-3), which is the ligand for the T-lymphocyte-associated CD2 molecule, and has a broad tissue and organ distribution. In the present investigation the colloidal gold-immunoelectronmicroscopy immunostaining system and a quantitative analysis of the labeling provided decisive evidence for the weak but clear LFA-3 expression on virtually all keratinocytes (KC) and LC freshly isolated from normal human skin. Such constitutive expression of LFA-3 molecule on EC may be relevant for a number of functional interactions between LFA-3-positive EC and CD2-positive T lymphocytes within the cutaneous environment.

S-100 positive epidermal dendritic cells were counted in skin biopsies from 48 Kveim tests and four known foreign-body reactions. Counts in histologically positive Kveim biopsies (mean 11.3 per 200 basal cells) were significantly higher than in either negative biopsies (5.1; P less than 0.001) or foreign-body reactions (4.7; P less than 0.05). A similar difference was found, irrespective of the histological appearances, between biopsies from patients diagnosed clinically as having sarcoidosis (10.5) and those in which another diagnosis had been made (4.1; P less than 0.001). In biopsies from patients with sarcoidosis 70% had a positive Kveim test, 70% had a raised epidermal dendritic cell count and one or the other was positive in 90%. All cases in which both the Kveim test was positive and the dendritic cell count was raised had a final clinical diagnosis of sarcoidosis. Counts of S-100 positive epidermal dendritic cells are useful in differentiating positive reactions to Kveim suspension from non-specific reactions to foreign material and increase the diagnostic confidence of the Kveim test.

The cellular and subcellular distribution of 2,4-dinitrophenyl (DNP) groups in the epidermis and regional lymph nodes of the mouse was investigated after epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) to sensitized and non-sensitized mice. The peroxidase-antiperoxidase method and the immunogold technique were used to visualize the DNP groups at both light and electron microscopic levels. The highest intensity of immunolabelling was found on tonofilaments of keratinocytes present in the upper layers of the epidermis. On the other hand, in vitro experiments showed that DNFB has the capacity to bind keratin which, together with immunocytochemistry, suggests that this molecule may be one of the skin protein carriers for DNFB. In addition, intense immunostaining for DNP was observed in the Golgi area of some epidermal Langerhans cells. Cells immunoreactive to DNP were also observed in the marginal sinus of cervical lymph nodes 6, 12 and 24 h after challenge. Immunoelectron microscopy revealed immunoreactive DNP groups in phagosomes of Langerhans cells at this site. The present findings support the hypothesis that the hapten DNFB penetrates passively into the cytoplasm of Langerhans cells, concentrates in the Golgi area and, during the migration of Langerhans cells to the lymph nodes, it is probably processed in the lysosomes before its presentation to T lymphocytes.

We report a case of T cell lymphoma which was exclusively located within the epidermis. The T cells of this lymphoma were all of suppressor-cytotoxic phenotype. They did not express the CD7 antigen and a molecular biology study showed T cell type genotypic rearrangements. Both immunohistochemical and ultrastructural studies showed close contacts between lymphoma and Langerhans cells.

The presence of melanin macroglobules, and sometimes that of melanosome complexes also, in epidermal melanocytes has been considered a feature of various skin diseases. Opinions differ as to whether these structures can occur in normal skin. We have studied these melanin inclusions in normal Caucasian skin in the entire soma of 116 melanocytes and the occurrence of melanosomes in phagosomes of 77 Langerhans' cells obtained in different seasons. During winter the melanocytes contained few melanosomes but many melanosome complexes and melanin macroglobules. These melanosome inclusions were in 86%, localized in the most basal part of the melanocytes, particularly in the dermal protrusions. It is suggested that these structures can be transferred from epidermal melanocytes to dermal cells and that melanin macroglobules derive from melanosome complexes. Irrespective of the season, most of the Langerhans' cells contained melanosomes in their phagosomes, which suggests a phagocytic capacity of these cells and a role in the elimination of the melanin.

The presence of human cytokines was examined in parallel skin biopsies and epidermal single cell preparations obtained from normal individuals. Using biotin-avidin-peroxidase and immunofluorescence techniques and antibodies against recombinant cytokines, a granular intercellular/membrane-associated staining for interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF alpha), but not IL-1 alpha or beta, was observed. An epidermal cytoplasmic staining pattern was also detected, which was most pronounced using the anti-rIL-6 antiserum. In the epidermal single cell preparations, membrane-associated staining was detected for both IL-6 and TNF alpha. Double staining revealed that CD1-positive Langerhans cells (LC) failed to express any of the examined cytokines. In vitro binding of rIL-6 or rTNF alpha to skin sections and epidermal single cell preparations indicated that the cell surface-associated IL-6 and TNF alpha originally demonstrated on keratinocytes were truly membrane-bound. Finally, co-cultivation of epidermal cells with an IL-6 responsive cell line, B9, and testing of epidermal cell supernatants in this assay, indicated that the in vivo membrane-bound IL-6 had biological activity.

The expression pattern of well-defined cell surface adhesion receptors called VLA-family, LFA-1 and ICAM-1 was determined semiquantitatively in biopsies of oral (n = 12) and dermal lichen planus (n = 5) and compared to normal uninvolved human oral mucosa (n = 12) and skin (n = 12) using an indirect immunoperoxidase technique. In both oral and dermal lichen planus, an induction of the beta 1-integrins VLA-1 and VLA-3 and an upregulation of VLA-6 was found in T cells infiltrating the basement membrane zone. These cell surface molecules function as receptors for collagen, fibronectin and laminin. A focal induction of ICAM-1 on basal keratinocytes could be detected at sites of intramucosal T cells. These results suggest that investigated adhesion receptors are crucially involved in the aggregation of T cells in both conditions. Further investigations have to be done to determine the functional role of these adhesion receptors in lichen planus.

Cyclosporin A (CSA)-induced gingival overgrowth was immunohistochemically compared with that phenytoin-induced and nonspecific inflammatory gingiva, and CSA concentration was determined for dental plaque. Leu-6+ epithelial dendric cells (EDC) were found to significantly decrease in number in CSA-induced gingival overgrowth, while the ratio of HLA-DR+ EDC to Leu-6+ EDC did not change significantly. The expression of class II major histocompatibility complex antigens, such as HLA-DR, -DP and -DQ on keratinocytes did not change by CSA-treatment. Leu-4+ mononuclear cells in CSA-induced gingival overgrowth were located primarily in the connective tissue far outside the epithelium. CSA concentration was much higher in dental plaque than in blood and other tissues. Immune response thus appears to be suppressed in the epithelial layer of CSA-induced gingival overgrowth through decrease in Leu-6+ HLA-DR+ EDC and T cell infiltration, both due to CSA in dental plaque. DNA polymerase alpha was detected in much fewer basal keratinocytes of CSA- and phenytoin-induced gingival overgrowth. Epithelial hyperplasia may thus be not due to increased keratinocyte proliferation, but rather to enhanced keratinocyte life span.

A variant of calcifying epithelial odontogenic tumor (CEOT) with Langerhans cells is reported. Compared to a typical CEOT, the tumor islands of this case were thin and composed of a small number of polyhedral epithelial cells. Almost no calcification of homogeneous eosinophilic materials was observed. In addition, clear cells which structurally corresponded to Langerhans cell were intermingled in the epithelial islands. These cells stain positively for S-100 protein, lysozome, MT 1, LN-3 and OKT 6 antibodies, but not for keratin antibody. Electronmicroscopic examination revealed the rod-shaped and racket-shaped structures called Birbeck's granules in the cytoplasm of these clear cells. Our observations indicate a variant case of CEOT with Langerhans cells in tumor nests.

In the skin of healthy subjects linear deposits were found of fibronectin at the dermoepidermal junction and in the walls of blood vessels, and reticular deposits in the papillary and reticular layers of the skin. In the epidermis of 12 out of 51 patients with allergic contact dermatitis fibronectin deposits were found in relation to Langerhans cells and keratinocytes. This was not observed in the epidermis in healthy subjects. The authors suggest that fibronectin on the surface of Langerhans cells and keratinocytes may play a role in antigen presentation.

A family of dendritic cells has been identified in situ and in vitro by microscopy and immunolabeling. The members of this family include the dendritic cells isolated from lymphoid organs, Langerhans cells [LC] of the epidermis, veiled cells in afferent lymph, and interdigitating cells [IDC] in the T-cell areas. Some common features to all members of the family are high levels of MHC class II antigens, a lack of most B and T cell markers, and an absence or low levels of macrophage/granulocyte antigens. This review summarizes the markers of mouse dendritic cells as assessed by a panel of monoclonal antibodies, and stresses a few recent findings. 1) In spleen, there are two populations of dendritic cells. More than 75% of isolated cells are 33D1+, NLDC145-, and J11d-, while the remainder have the reciprocal phenotype and thus share the NLDC145 antigen of IDC. Thymic dendritic cells, released by collagenase digestion, and epidermal LC also are 33D1-, NLDC145+, J11d+. 2) When epidermal LC are placed in culture, there are changes in cell function and phenotype. There is a decrease in Fc gamma receptors and the F4/80 macrophage antigen, an increase in class I and II MHC products and p55 IL-2 receptors, and persistence of the NLDC145 IDC antigen. The cultured LC thereby resembles the IDC. 3) A new antibody N418 shows that dendritic cells express the p150/90 member of the leukocyte beta 2 integrin family. Immunolabeling of tissue sections of spleen indicates that N418+ dendritic cells not only are present in the periarterial sheaths, the location of IDC, but also in "nests" at the periphery of the T area where 33D1 has been found. The peripheral collections interrupt the marginal zone of macrophages that separates white and red pulp, and places the dendritic cells in the path of T cells as they move through the white pulp. Therefore the members of the dendritic cell family have important markers in common, as well as differences that are associated with state of immunologic function and location.

In order to determine the antigenic phenotype of the proliferating cells in Langerhans cell histiocytosis (LCH), we studied 15 such examples by using formalin- and B5-fixed, paraffin-embedded tissues. We used a panel of antibodies that are known to react with lymphocyte- and histiocyte-associated antigens. These included LN-1, LN-2, and LN-3 monoclonal antibodies (MoAbs), MoAbs to leukocyte common antigen (LCA), Leu-M1 antigen, vimentin, and epithelial membrane antigen (EMA), as well as polyclonal antibodies to lysozyme and S100 protein. The antigens encountered most frequently in LCH cells were S100 protein (93% of cases), vimentin (86%), and those detected by LN-2 (80%) and LN-3 (82%). Lysozyme was detected focally in two cases and diffusely in one case. The LCH cells were negative for LN-1, LCA, Leu-M1, and EMA. There was only one specimen in which S100 protein was not demonstrated; in this case, LN-3, vimentin, and T6 on frozen section were positive. The phenotype of LCH cells is similar to that of Langerhans' cells and interdigitating histiocytes. Our results demonstrate the value of using a panel of antibodies, including anti-vimentin MoAb, LN-2, and LN-3 for the immunophenotypic diagnosis of LCH in addition to an antibody to S100 protein.