In situ identification of T6-positive cells in normal human dermis by immunoelectron microscopy*

Monoclonal anti-T6 antibody, which reacts with the majority of cortical thymocytes but not peripheral T cells, also reacts with human epidermal Langerhans cells, as shown by a four-step immunoperoxidase method and immunoelectron microscopy. To define whether T6-positive cells are also present in normal human dermis, we used these techniques to demonstrate two immunologically distinct populations of histiocyte-like cells in normal human dermis. The first population contains cells devoid of phagolysosomes or Birbeck granules. These cells react with anti-T6 antibody, but not with monoclonal anti-T3 antibody which defines peripheral T cells, and are found predominantly in and around dermal lymphatic vessels. The second is composed of phagolysosome-containing cells which do not react with anti-T6 antibody or anti-T3 antibody. Because to date, Langerhans cells are the only cells in normal human epidermis that react with anti-T6 antibody, these data provide immunological evidence for a specific link between Langerhans cells and a T6-positive dermal mononuclear cell, possibly the so-called indeterminate cell. In addition, application of these techniques should, for the first time, permit the immunological distinction of these T6-positive mononuclear cells from other cells bearing la antigens, such as dermal histiocytes and certain lymphocytes, in normal and diseased skin.     

T cell subsets and Langerhans cells in lichen planus: in situ characterization using monoclonal antibodies

Skin biopsies from four patients with lichen planus were studied using monoclonal antibodies directed against T lymphocytes. Anti-T1 and anti-T3 antibodies, which react with all peripheral T cells, stained most cells in the dermal infiltrates. The majority of infiltrating cells also stained with anti-T4 and anti-T4b antibodies, which react with helper/inducer cells, whereas a minority of cells stained with anti-T8 antibody, which reacts with cytotoxic/suppres-sor cells. Surface IgM was not identified on any infiltrating cells, providing evidence against B cell participation. Intraepidermal and dermal cells with long cytoplasmic extensions stained with anti-T6 antibody in all cases, defining them as Langerhans cells or their precursors. T6-positive cells were seen in greater number than in normal control epidermis and dermis. The results indicate that well-developed lesions of lichen planus are characterized by an influx of helper/inducer T lymphocytes and increased numbers of Langerhans cells. These observations support the contention that cellular immunity is important in the pathogenesis of this disorder.     

Immunophenotyping of the eczematous flare-up reaction in a nickel-sensitive subject

An eczematous flare-up reaction, occurring at a previously involved site, which followed oral challenge with 5.6 mg of nickel in a 29-year-old nickel-sensitive woman, was biopsied and studied by immunohistochemistry. The cellular infiltrate in the dermis and epidermis at 8 days was predominantly of Leu 3a phenotype (helper/inducer T lymphocytes), with smaller numbers of Leu-2a-reactive (suppressor/cytotoxic) T lymphocytes. Many infiltrating cells were DR-positive. No increase in epidermal Leu-6-positive Langerhans cells was seen but Leu-6-reactive cells were noted in the dermal infiltrate. Keratinocytes showed some expression of class II antigen (mainly DR). In comparison with the 48-hour allergic patch test reaction, the eczematous flare-up site showed no increase in epidermal Langerhans cell numbers nor infiltration with macrophages, but the responses were similar since both showed a superficial T cell reaction in the skin.     

Benign non-X histiocytosis: a unique case bridging several of the non-X histiocytic syndromes

We present a patient with a papular eruption of 4 years' duration that clinically resembled xanthoma disseminatum or the indeterminate cell disorder. On light microscopy his disorder resembled generalized eruptive histiocytoma or the indeterminate cell disorder. Special stains, cultures, and electron microscopy were noncontributory. Indirect immunofluorescence studies with monoclonal antibodies to cell surface markers demonstrated infiltrating cells of monocyte/macrophage lineage (OKM1, MAC-1, HLA-DR, and HLA-DQ positive) rather than Langerhans or indeterminate cell lineage (OKT6 negative). This case may overlap two or more of the previously reported non-X histiocytic syndromes, suggesting that perhaps these syndromes should be viewed as a spectrum of disease rather than as discrete entities. We recommend performing cell phenotyping on all new cases of non-X histiocytosis because clinical, microscopic, and ultramicroscopic findings often prove inadequate for classification.     

Clinical, immunohistochemical, and electron-microscopic findings in gold dermatitis

The clinical, immunohistochemical, and electronmicroscopic features of 13 consecutive patients with gold dermatitis were analyzed: 12 developed an eczematous dermatitis and one a lichenoid dermatosis. The patients had received intramuscular sodium aurothiomalate therapy from 1 month to 4 years before the dermatitis broke out. After cessation of gold therapy, the dermatitis persisted for 1-11 months. A relatively sparse perivascular mononuclear cell infiltrate was found in the affected skin in all cases. With immunoperoxidase staining, most of the infiltrating cells were shown to be OKT-4-positive T-helper lymphocytes. A majority of the infiltrating cells were Ia, i.e., HLA class II antigen, positive. Clearly increased numbers of dermal OKT-6-positive Langerhans' cells were also seen. In epidermis, on the contrary, the expression of both OKT-6 and Ia markers on dendritic cells was decreased. However, electron-microscopic examination revealed large numbers of macrophage-like cells and the Langerhans cells were activated, often in apposition to mononuclear cells within the epidermis. No correlation was observed between the immunohistological findings and the amount of gold received, the duration of gold therapy, and the interval between the last gold injection and biopsy, respectively, although peripheral blood eosinophilia was more common during 5-10 months of gold therapy. There were no specific findings in the patients in whom dermatitis lasted several months after discontinuation of the therapy. Our findings support the view that immunological mechanisms operate in the development of gold dermatitis, although the exact mechanisms remain unknown.     

T6 is superior to Ia (HLA-DR) as a marker for Langerhans cells and indeterminate cells in normal epidermis: a monoclonal antibody study

Previous studies in our laboratory using immunoelectron microscopy have shown that anti-T6 monoclonal antibody reacts with all epidermal Langerhans cells in normal skin. Comparison of the number of T6-positive (+) epidermal cells with Ia (HLA-DR) (+) cells, as defined by the monoclonal antibodies, anti-I1 and anti-I2, disclosed that these latter markers significantly underestimated Langerhans cell and indeterminate cell numbers (p less than 0.01 and p less than 0.001, respectively) when employed in a sensitive 4-step immunoperoxidase procedure. Thus, it appears that all epidermal Langerhans cells and indeterminate cells are not Ia-positive as defined in this system and that Ia(+)/T6(+) and Ia(-)/T6(+) subsets exist. These subsets may be analogous to the Ia(+) and IA(-) subsets of macrophages, in which the former are responsible for antigen interaction with T cells.     

Immunohistochemical Studies of Infiltrating Cells in Early and Chronic Lesions of Psoriasis

The expression of surface antigens on infiltrating cells, epidermal keratinocytes, and dendritic cells in biopsy specimens from 31 patients with psoriasis was examined immunohistochemically. The specimens were divided into early-phase and chronic-phase groups and then examined in a double blind manner. Among the infiltrating cells in the epidermis, CD4-positive cells were dominant in the early phase; CD8-positive cells were dominant in the chronic phase, resulting in a markedly decreased CD4/CD8 ratio in the latter. On the other hand, among the infiltrating cells in the dermal papillae, CD4-positive cells were dominant in both the early and chronic phases; both CD4-positive and CD8-positive cells were more dominant in the chronic phase than in the early one. However, the CD4/CD8 ratios were decreased in both the dermal papillae and the epidermis in the chronic phase. CD1-positive dendritic cells (probably Langerhans cells) were more numerous in the chronic phase than in the early phase. There were no significant differences between the early and chronic phases with regard to the expression of HLA-DR and HLA-DQ antigens on the infiltrating cells. However, the HLA-DR antigens and ICAM-1 (intercellular adhesion molecule-1) were more strongly expressed on epidermal keratinocytes in the chronic phase than in the early phase. LFA-1α (lymphocyte function-associated antigen-1α)-positive cells were also significantly more numerous in the chronic phase than in the early one, consistent with the expression of HLA-DR antigens and ICAM-1 on keratinocytes mentioned above. On the other hand, VLA-4 (integrin α₄β₁) positive cells were expressed more abundantly in the epidermis in the early phase than in the chronic phase. These results suggest, first, that the chronic phase of psoriasis is as immunologically active as or more active than the early phase. Second, CD4-positive T cells are more important than CD8-positive T cells in the early phase of psoriasis; CD8-positive rather than CD4-positive T cells are more important in the chronic phase. Third, the LFA-1/ICAM-1 pathway may play an important role with regard to cell adhesion of the infiltrating cells in the psoriatic lesions in disease exacerbation or prolongation, whereas the VLA-4/VCAM-1 (vascular cell adhesion molecule-1) pathway may be more important in disease onset.     

Immunohistologic identification of antigen-presenting cells in cutaneous sarcoidosis

Considerable evidence exists to show that activated T lymphocytes preferentially accumulate at sites of disease activity in sarcoidosis. Langerhans cells, which can be recognized by reactivity with an antibody to the T6 antigen are thought to play a primary role in T-lymphocyte activation by the skin, a tissue frequently involved in sarcoidosis. This immunohistologic study examined the distribution of OKT6-positive cells and surface expression of HLA-DR antigen in cutaneous sarcoid lesions. Skin specimens stained with an anti-HLA-DR antibody demonstrated diffuse staining of the granulomas. In addition, keratinocytes, which do not normally express HLA-DR antigens, were found to stain with monoclonal antibody to HLA-DR in an intercellular pattern. Examination of specimens for OKT6-reactive Langerhans cells revealed significantly greater concentrations in the epidermis overlying sarcoidal granulomas (33 +/- 7 cells/mm) than in the epidermis of age-, sex-, and race-matched controls (11 +/- 3 cells/mm, p less than 0.001). Of greater importance was the demonstration that significant numbers of OKT6-positive cells were present within the dermal sarcoid granulomas (19-208/mm2) in a distribution that paralleled that of Leu-3a-positive T lymphocytes. These data suggest that the epidermis may participate in activation of lymphocytes in cutaneous sarcoidosis, and implicate OKT6-positive cells in granuloma formation.     

Photohardening of polymorphic light eruption patients decreases baseline epidermal Langerhans cell density while increasing mast cell numbers in the papillary dermis

The pathogenesis of polymorphic light eruption (PLE) has been linked to a lack of UV‐induced immune suppression. To determine the role of Langerhans cells (LC), mast cells and regulatory T cells, biopsies from PLE patients were taken from exposed sites in spring before and after photohardening with 311 nm or PUVA as well as again in summer. Skin sections were assessed for the presence of Langerin/CD1a+ LC and CD3+, CD4+, CD25+ or FoxP3+ T cells and mast cells. Photohardening transiently decreased the density of epidermal LC and significantly increased a low baseline mast cell density in the papillary dermis of PLE patients. Baseline T cell numbers in the skin were low, and there was no difference in PLE patients among any time point. This suggests that LC suppression together with recruitment of mast cells into photohardened skin may be a key cellular event underlying the mechanism by which phototherapy protects from PLE.     

In situ characterization of cellular infiltrates in lupus vulgaris indicates lesional T-cell activation.

Skin biopsy specimens from nine patients with lupus vulgaris were examined in situ by means of monoclonal antibodies directed against phenotypes of lymphocyte subsets, Langerhans cells, HLA-DR antigens, and interleukin 2 receptor. The epidermis showed prominent changes, including intense expression of HLA-DR on keratinocytes, increase in epidermal cell layers, moderate to high Langerhans cell hyperplasia, and infiltration by CD3+ pan-T cells as well as CD8+ (cytotoxic/suppressor) and CD4+ (helper/inducer) T cells. The predominant lymphocyte in the dermal granulomas was the activated CD3+ T cell, expressing major histocompatibility complex class II antigens and interleukin 2 receptor. CD4+ and CD8+ cells were randomly distributed among the epithelioid cells, which showed intense staining for major histocompatibility complex class II antigens. In all except two patients, the CD4+ population was greater than that of the CD8+ cells. CD1+ Langerhans cells were scattered in moderate numbers in the dermal granulomas. Acid-fast bacilli were conspicuously absent in the biopsy specimens. These features suggest that T-cell activation and Langerhans cell hyperplasia are prominent features of dermal tuberculosis.     

Immunopathology of cutaneous human lupus erythematosus defined by murine monoclonal antibodies

Skin biopsy specimens obtained from involved skin from sixteen patients with systemic and discoid lupus erythematosus were studied. Murine monoclonal antibodies with a biotin-avidin-horseradish peroxidase staining system were used. The findings consisted of a marked reduction in the number of epidermal Langerhans cells defined by surface antigens, reduced HLA-DR (Ia-like) antigens on the surface of dermal capillary endothelium, and mononuclear cell infiltrates characterized by a predominance of helper T lymphocytes and an increase in the number of mononuclear phagocytic cells. B lymphocytes were rarely identified. The number of T lymphocytes within the dermis correlated inversely with both the number of HLA-DR-positive epidermal Langerhans cells (p less than 0.01) and the HLA-DR staining of dermal capillary endothelium (p less than 0.01). These findings suggest that a T lymphocyte-mediated immune response associated with a reduction in Langerhans cells and capillary endothelium HLA-DR antigens is involved in the inflammatory process of lupus erythematosus skin.     

Photosensitive dermatitis with actinic reticuloid syndrome: an immunohistological study of the cutaneous infiltrate

Cryostat sections of skin biopsies from five patients with chronic photosensitivity dermatitis with actinic reticuloid syndrome (PDAR) have been examined immunohistologically by the alkaline phosphatase:anti-alkaline phosphatase staining technique using a panel of 24 monoclonal antibodies against lymphoid cells and their subsets. The lymphoid infiltrates in all cases had an essentially identical cellular composition, containing a mixture of T-lymphocytes, T-cell accessory cells (Langerhans cells) and other types of HLA-DR positive dermal macrophages. In two patients there was an excess of T-helper/inducer cells relative to T-suppressor cells, while in the other three patients the numbers of T-cells in these two subsets were approximately equal. Many of the infiltrating T-cells expressed activation (HLA-DR, interleukin-2 receptor) or proliferation (the Ki67 nuclear antigen, transferrin receptor) associated markers. These data indicate that a T-cell immune response is operative in cutaneous PDAR lesions.     

In situ immunological characterization of the infiltrating cells in positive patch tests

Skin biopsies from positive allergic patch tests were analysed by immunoenzymatic labelling of frozen sections with monoclonal antibodies. In seventeen patients the cellular infiltrate consisted of T cells admixed with Langerhans cells/indeterminate cells, but in two patients there were also many B lymphocytes. The B cells were accompanied by dendritic reticulum cells forming B-cell follicles, indistinguishable from those of normal and hyperplastic lymph nodes. There was no correlation between these two immunohistological staining patterns and the sensitizing antigen, the extent of local reaction or the time from epicutaneous application of allergen to examination (2 to 16 days). The ratio between T-helper and T-suppressor cells varied considerably, and showed no correlation with these variables. In all patients the infiltrating T cells expressed HLA-DR antigen. Transferrin receptors were identified on the infiltrating T cells in biopsies from nine patients. These data indicate activation of T cells in the infiltrate from positive patch tests, and support the functional significance of Langerhans cells in the initiation and maintenance of cutaneous contact allergy. An involvement of B cells and B-cell accessory cells in the pathogenesis of contact allergic reactions is also suggested. The presence of dendritic reticulum cells in skin infiltrates from positive patch tests may reflect a functional implication of the skin in the development of B-cell memory.     

Macrophages and dendritic cells constitute a major subpopulation of cells in the mouse dermis

Macrophages and dendritic cells (DC) in tissues with close contact to the environment are of essential importance in host defense and are therefore present in sizeable numbers. Therefore, it is surprising that mononuclear phagocyte populations of the dermis have rarely been investigated in a quantitative manner. In this study, we examined mouse dermal skin immunophenotypically and related the observed numbers of observed cells to the total number of nucleated cells. These analyses show that about 70% of all dermal cells represent CD45+ leukocytes. The vast majority of these cells (approximately 60% of total) expresses the mononuclear phagocyte markers mMGL (ER-MP23), F4/80 and CD11b. In addition, these cells show avid phagocytic capacity and thus are identified as dermal macrophages. Different subpopulations can be defined using markers such as sialoadhesin, ER-HR3 and mSIGN-R1 (ER-TR9). Interestingly, MHC class II expression differs significantly between dermal cells from ear versus back skin. Moreover, we have identified small populations of dermal DC and migrating Langerhans cells (together approximately 10% of total). In summary, our findings show that mononuclear phagocyte populations form the majority of dermal cells and thus have been clearly underestimated so far.     

Characterization of cutaneous infiltrates in MRL/lpr mice monitored from onset to the full development of lupus erythematosus-like skin lesions.

The skin is a primary site injured in lupus erythematosus (LE), but it is still controversial whether the injury is due to cells of the mononuclear infiltrate and which immunocompetent cells play the major role in the development of cutaneous LE. To better characterize the role of immunocompetent cells, we performed an immunohistochemical examination of these cells in LE-like skin lesions in MRL/Mp-lpr/lpr (MRL/lpr) mice. Skin lesions in 60 female MRL/lpr mice were monitored from onset to full development. Skin specimens from each stage were stained for epidermal Ia+ Langerhans cells (Ia(+)-LC), for Thy-1+ dendritic epidermal cells (Thy-1+DEC), and for the phenotype of the mononuclear cell infiltrates. The numbers of Ia(+)-LC and Thy-1+DEC were decreased markedly in the skin lesions at the later stage. However, the numbers of Ia(+)-LC were increased significantly in the central portion of lesions at an early stage and in the peripheral portion of lesions later. L3T4+ cells were predominant, and the L3T4/Lyt-2 ratio was high in dermal infiltrates at an early stage. With advancing stage, the L3T4/Lyt-2 ratio gradually decreased in dermal infiltrates, whereas the Thy-1.2/Lyt-2 ratio in lymph nodes was reversed. L3T4+ cells were especially predominant in dermal infiltrates under the epidermis with increased numbers of Ia(+)-LC. This immunohistochemical analysis of a mouse model of cutaneous LE revealed changes in immunocompetent cell populations with the evolution of skin lesions, and we conclude that Ia(+)-LC and Thy-1+DEC, as well as L3T4+ and Lyt-2+ cells, may play pathogenic roles in the development of skin lesions.     

固定性药疹急性期免疫组织化学研究

本文用４种鼠抗人白细胞抗原的单克隆抗体，采用标记的链霉亲合素生物素技术对９例固定性药疹患者急性期皮损内浸润的Ｔ细胞及其亚群、Ｂ细胞进行标记。观察到损害内真皮中上部血管周转浸润的细胞中６０％－８０％为Ｔ细胞，其中ＣＤ４＾＋细胞和ＣＤ８＾＋细胞数量大致相等。     

Quantitative studies on nonlymphoid mononuclear cell subpopulations in cutaneous infiltrates. I. Stage-related changes of dendritic nonlymphoid mononuclear cells, Langerhans' cells, and macrophages in lichen planus lesions

In previous investigations on lichen planus, we suggested that in early lesions T4-positive cells might be antigen-specifically driven, whereas in late lesions T8-positive cells may be cytotoxic to keratinocytes. To verify this hypothesis, we investigated the following nonlymphoid mononuclear cell subpopulations in early versus late lichen planus lesions: interdigitating cells (phenotype: S100-positive, lysozyme-negative, T6-negative, M3-negative), Langerhans cells (phenotype: S100-positive, lysozyme-negative, T6-positive, M3-negative), macrophages (phenotype: S100-negative, lysozyme-positive, T6-negative, M3-positive). Interdigitating cells were moreover identified in semithin and ultrathin sections by distinctive morphological characteristics. The S100-positive/lysozyme-positive cell ratio was higher (p less than 0.01) in early lesions than late lesions. In dermis but not in epidermis (NS), of early lesions, T6-positive cells were less represented than S100 positive cells (p less than 0.025). Thus, Langerhans' cells largely predominated over interdigitating cells in epidermis, but the two populations were both represented in dermis. Lysozyme-positive and M3-positive cells, more abundant in late lesions than in early lesions (p less than 0.001), were often filled with pigment granules.     

Characterization of the mononuclear infiltrate involved in regression of halo nevi

Halo nevi are characterized by progressive degeneration of nevus cells surrounded by a mononuclear cell infiltrate. We studied the morphological features of the nevus cells and the composition of the mononuclear cell infiltrate in 15 cases of halo nevi using immunohistochemical techniques and a battery of antibodies to different subsets of lymphocytes and histiocytes. Regression could be divided into four more or less identifiable stages, associated with different subsets of lymphocytes and monocyte-macrophage lineage cells. Stage I (preregression): nests of unremarkable nevus cells were surrounded by a moderate number of T lymphocytes (relatively small percentage of helper/inducer T cells), occasional B cells and macrophages. Stage II (early regression): large number of T lymphocytes and FXIIIa-positive cells were in close contact with nevus cell clusters which showed ragged edges. Lysozymepositive cells and epidermal Langerhans cells were mildly increased. Stage III (late regression): single nevomelanocytes showing mild atypia were present. Numerous T lymphocytes and macrophages positive for lysozyme, KP1 and/or FXIIIa were interspersed between the nevus cells. Increased numbers of epidermal Langerhans cells were present. Stage IV (complete regression): no nevus cells were observed and moderate numbers of T lymphocytes only remained. These results suggest that T cells, especially T-suppressor cells, and different subsets of macrophages participate in the regression of the nevi.     

Immunohistopathology of light-induced skin lesions in lupus erythematosus

Recently we have been able to induce pathological skin reactions with UVB, UVA and visible light in patients with lupus erythematosus (LE). The pathological skin reactions had the appearance of spontaneously developed LE lesions. In the present study, using patients with polymorphic light eruption as controls, we subsequently investigated what types of immunohistochemical abnormalities were found in these lesions. It was shown that in the induced skin lesions, phenotypically similar inflammatory cells were found as in spontaneously evolved lesions. Granular deposits of immunoreactants, as found in most spontaneously evolved LE lesions, occurred in 12 out of 16 LE patients 7-10 days after onset of the artificial irradiation. The dermal infiltrates in light-induced LE lesions differed mainly from those in polymorphic light eruption, by the amounts of CD1+ cells (Langerhans' cells). In polymorphic light eruption, the relatively large amount of these cells suggests an active migration of antigen-presenting cells, a mechanism apparently not operative in LE. Our results underline the importance of the pathogenic action of light in LE.     

Atopic Dermatitis: Studies of Skin Permeability and Effectiveness of Topical PUVA Treatment

Ultraviolet light is effective treatment for patients with atopic dermatitis that is resistant to conservative therapy, or complicated by adverse effects of extended steroid use. We designed a protocol using topical psoralen chemotherapy with ultraviolet A (PUVA) to treat atopic dermatitis in 114 patients. Clinical results were excellent, with complete clearing in 50% of patients receiving daily treatment. Histologic and immunologic values correlated with the clinical response, including reduced epidermal thickness, and decreased numbers of epidermal Langerhans cells and dermal mast and mononuclear cell infiltrates. The pattern of keratin 14-positive keratinocytes returned toward normal. In addition, the water-holding capacity of the stratum corneum increased to near normal levels. We also studied stratum corneum permeability in lesional and nonlesional skin using the dimethyl sulfoxide whealing test and theophylline absorption studies. Compared with controls, permeability was markedly increased in lesional skin and mildly increased in nonlesional skin in patients with atopic dermatitis. These results suggest that immune abnormalities and barrier dysfunction participate in the pathogenesis of atopic dermatitis.