Immunopathological and ultrastructural findings in human allergic and irritant contact dermatitis

The histopathological features of allergic contact dermatitis were compared with those of irritant contact dermatitis in a group of 17 subjects. Each patient received simultaneous patch tests of a known allergen and a standardized irritant (benzalkonium chloride). The cellular changes occurring between 3 h and 7 days after patch test application were studied by light and electron microscopy and immunocytochemistry. No differences were observed between the induced allergic contact dermatitis (ACD) and the irritant contact dermatitis (ICD), either in the responding cell types or the sequence of cellular events. Both reactions showed a predominantly T lymphocyte infiltrate with no polymorphonuclear leukocyte involvement. Apposition of Langerhans cells to lymphocytes in the epidermis was seen in both types of response. Considerable variability in the intensity of reaction to irritant and allergen occurred within individuals. There was no statistically significant difference between the intensity of the reactions to the irritant and the allergen.     

Lymphocyte subsets and Langerhans cells in allergic and irritant patch test reactions: histometric studies

This study has attempted to distinguish between allergic and irritant reactions to patch tests by semiquantitative histological methods. The extent of perivascular chronic inflammatory infiltrate at 72 h in irritant patch test reactions to sodium lauryl sulphate was shown to be small and very consistent, whereas in allergic reactions to nickel sulphate it was generally larger and more variable in size (p less than 0.02). The two major lymphocyte subsets (T4 and T8) were randomly intermixed in both types of reaction and formed the major component of both the perivascular and diffuse dermal infiltrate, without any evidence of selective migration. The T4:T8 ratios were similar in focal and diffuse infiltrates. The number of T6 dendritic (putative Langerhans) cells in the epidermis (per mm inner epidermal length) was usually greatly reduced in irritant reactions (5-16 mm-1, mean 10 mm-1) but remained within normal limits in allergic reactions (6-33 mm-1, mean 21 mm-1) (p less than 0.001). Comparable results were seen with other irritants (mercuric chloride and benzalkonium chloride) and other allergens (neomycin sulphate, ethylene diamine and potassium dichromate). In additional experiments, pairs of biopsies were taken from the reaction and from adjacent unaffected skin. The T6 cell density in the epidermis did not significantly differ between allergic reactions and control skin. By contrast, the irritant reactions had fewer T6 cells than the control skin (p less than 0.001).     

The duration of Grenz ray-induced suppression of allergic contact dermatitis and its correlation with the density of Langerhans cells in human epidermis

Recent investigations have shown that Grenz rays can suppress the allergic contact dermatitis reaction completely and that Langerhans cells, identified by OKT6 antibodies and electron microscopy, disappear from the epidermis at the same time. It is not known for how long this suppression lasts. This has been investigated in 28 nickel-sensitive patients who were given Grenz rays (3 Gy) on the back, once a week for 3 weeks. The patients were then divided into four groups and tested with patch tests for nickel at 1, 7, 14 and 21 days after the last Grenz ray treatment. Biopsies were taken from positive patch test sites, and from the corresponding opposite control. They were labelled with OKT6 antibodies to detect Langerhans cells. The patch test reactions were suppressed and the Langerhans cell density was decreased initially. These changes were restored after 3 and 6 weeks, respectively. The results show that the effect of Grenz rays on eczematous reactions extends to a maximum of 3 weeks and imply that Langerhans cells are necessary for the elicitation of the efferent phase of allergic contact dermatitis.     

Dynamic changes in the epidermal OKT6 positive cells at mild irritant reactions in human skin

In the present study we induced mild irritant contact reactions by using 0.5% sodium lauryl sulphate (SLS) in distilled water or with distilled water in patch tests for 6 or 24 hours. The biopsies were taken at 6, 24, 48 and 96 hours. Light and electron microscopy were used to assess the irritant reactions produced and the monoclonal antibody OKT6 was used for the detection of the LCs. The number of the epidermal OKT6 positive dendritic cells was found to be increased at 48 and 96 hours after the exposure to SLS and at 96 hours in the water patch tests. It is concluded that mild irritant stimuli cause an increase in the LCs (OKT6 positive cells) and thus might influence and modulate the response to subsequent exposures to allergens.     

Changes in Lymphocyte and Langerhans Cell Populations in Allergic and Irritant Contact Dermatitis

We observed in situ changes in lymphocyte subpopulations and Langerhans cells during allergic and irritant contact dermatitis using immunohistochemical staining methods with monoclonal antibodies to cell surface antigens. In both types of contact dermatitis, there was a perivascular infiltrate of T lymphocytes, with helper/inducer T cells predominating. B cells were absent, and natural killer cells were absent or sparse. During the course of allergic contact dermatitis, Langerhans cells showed a striking sequential change in location, with the cells first in the epidermis, then perivascularly in the dermis (days 1-14), and returning to the epidermis (days 14-21). In irritant contact dermatitis, the Langerhans cells were initially identified in the epidermis and then appeared diffusely in the dermis (days 1-2). The numbers in the dermis then decreased abruptly (day 4). They were again identified in normal numbers in the epidermis (day 21). The response of Langerhans cells appears to be different between allergic and irritant contact dermatitis.     

Sequential immunophenotypic study of lymphoid infiltrate in allergic and irritant reactions

Sequential biopsies (4-72 h) of early allergic and irritant patch test reactions have been examined immunohistologically for reactivity with 19 monoclonal antibodies against antigens on lymphoid cells in order to investigate the nature/origin of the infiltrating lymphoid cells and assess their state of activation/proliferation. The composition of the infiltrates was similar in allergic and irritant reactions and consisted of T-lymphocytes of helper/inducer types in association with T-cell accessory cells, i.e., Langerhans cells and HLA-DR-positive macrophages. No differences in expression of T-cell or macrophage associated antigens were seen in early as opposed to late biopsies. In contrast, the proportion of cells positive for markers associated with activation (interleukin-2 receptor) or proliferation (transferrin receptor, the Ki-67 nuclear antigen) of lymphoid cells was found to increase with time in both types of reaction. These data substantiate the view that T-cell immune reactions are implicated in both allergic and toxic patch test lesions, and indicate that the lymphocytes in the infiltrates are activated and proliferate.     

Immunohistochemistry of lymphocytes and Langerhans' cells in long-lasting allergic patch tests

A long-lasting allergic patch test is a "normal" allergic patch test that remains positive for weeks or months. An immunohistochemical study of immunocompetent cells in the skin in this rare type of patch tests was performed. Most inflammatory cells were T11 positive T-lymphocytes. The majority of these cells were of the helper/inducer phenotype (T4+), but a relative increase of T8+ cells as compared to the initial (1-2d) stages of allergic patch tests was observed. T6+ Langerhans' cells (LCs) were normal or increased in number in the epidermis, while very few dendritic cells displayed Ial antigen in the epidermis, indicating loss of Ial-staining of LCs. High to very high numbers of T6+ cells were found in the dermis. An inflammatory reaction of hair follicles with moderate numbers of T6+ cells in the peribulbar infiltrate was observed indicating that hair follicles might act as shunt pathways for allergens. A defect in down regulation of the contact hypersensitivity reaction and/or a constant antigen stimulation could be responsible for the long-lasting allergic patch tests.     

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.     

The effect of grenz rays on irritant skin reactions in man

To investigate the effect of grenz rays on irritant contact reactions, eleven healthy volunteers were studied. They were given 3 Gy of grenz rays, once a week for 3 weeks, to a defined area of the back. Twenty-four hours after the last treatment, serial dilution sodium lauryl sulphate patch tests were applied both on the grenz ray treated area and on the untreated control skin. Biopsy specimens were taken from the irritant reactions both from the grenz ray treated area and from the control area and different cell populations in dermis and epidermis were identified by monoclonal antibodies (Leu 2, 3, 4, 7, Leu M1, B1, OKT6). In the grenz ray treated epidermis there was a pronounced reduction of OKT6-positive cells but the composition of the dermal cellular infiltrate did not differ between control and grenz ray treated skin. The assessment of the patch test reactions did reveal a tendency towards weaker reactions in the grenz ray pre-treated skin but this difference was not statistically significant. It is concluded that grenz rays do not have a marked effect on the elicitation of irritant reactions.     

Distribution of natural killer cells and lymphocyte subclasses in Jessner's lymphocytic infiltration of the skin and in cutaneous lesions of discoid and systemic lupus erythematosus

We studied the cell infiltrates in biopsies from lymphocytic infiltration of the skin (LIS), with six monoclonal T cell antigen-specific antibodies and compared the reactivity pattern with those in biopsies from discoid and systemic lupus erythematosus skin lesions and allergic contact skin reactions. A newly described antibody (NK₉) recognizing natural killer (NK) cells and activated cytotoxic T lymphocytes was included, and the numbers and activity of circulating NK cells was determined. Immunohistochemical staining revealed that the numbers of NK₉-positive cells were highest in LIS. The distribution of T lymphocytes (OKTii + ve), helper T cells (OKT₄+ ve), suppressor T celts (OKT8 + ve), Langerhans cells (OKT6 + ve) and activated T cells (anti-Tac + ve) in LIS differed from those in DLE, SLE and allergic contact reactions. However, the number of circulating NK cells (large granular lymphocytes) and the NK activity in peripheral blood were normal in LIS. We conclude that in LIS a distinct type of T cell activation occurs; the cause of this remains to be determined.     

Demonstration of Ia-like antigens on T lymphocytes in lesions of psoriasis, lichen planus and discoid lupus erythematosus

A combined indirect immunofluorescence technique with a murine monoclonal antibody against human Ia-like antigens (OKIal) and an IgG F(ab')2 preparation of a rabbit anti-T lymphocyte serum was used to study Ia-like antigens on T lymphocytes in skin lesions of psoriasis, lichen planus and discoid lupus erythematosus. The majority of the T lymphocytes in the various skin lesions expressed Ia-like antigens. T lymphocytes expressing Ia-like antigens were also demonstrated in the dermis and the epidermis in sections of unaffected skin, although of a markedly lower proportion than in affected skin. The results may indicate that the T lymphocytes in these skin lesions are activated cells involved in cell-mediated immune reactions.     

Antigen-presenting cells and keratinocytes express interleukin-12 in allergic contact dermatitis

Interleukin‐12 (IL‐12) has previously been suggested as playing a major rôle in the activation of cytotoxic lymphocytes. Recent reports indicate that cytotoxic CD8+ cells are critically involved in the elicitation phase of contact hypersensitivity reactions. In this study, the in situ expression of IL‐12 was investigated in normal human skin and in allergic contact dermatitis by immunohistochemistry. Skin biopsy specimens were obtained from allergic patch test reactions after 3 days, and from normal skin in 8 subjects. In contrast to normal skin, a strong enhancement of IL‐12 immunoreactivity was observed in the mononuclear cell infiltrate of allergic contact dermatitis. IL‐12 immunoreactivity was mainly located in the cytoplasm of dermal dendritic cells and macrophages as well as of some Langerhans cells. IL‐12‐positive cells were often found in close apposition to lymphocytes. Furthermore, positive immunostaining was also detected in keratinocytes at sites of marked exocytosis and spongiosis in the epidermis. In conclusion, the enhanced in situ expression of IL‐12 may contribute to the activation of cytotoxic lymphocytes and thereby represent an important factor in the pathogenesis of contact hypersensitivity reactions in humans.     

Immunohistologic studies of squamous cell carcinoma: possible participation of Leu-7+ (natural killer) cells as antitumor effector cells

Immunohistologic studies of 8 patients with squamous cell carcinoma (SCC) were undertaken using a series of monoclonal antibodies. In all of the patients, over 70% of the dermal infiltrates reacted with OKT3 and OKIal (HLA-DR), with a slight predominance of the OKT8+ suppressor/cytotoxic T subset (the mean OKT4/OKT8 ratio was 0.85). Both OKT4+ and OKT8+ subsets could be seen in contact with individual cancer cells. The percentage of OKB7+ (B) cells was less than 29% of the dermal infiltrates. Some Leu-7+ cells (less than 9% of the infiltrates) were seen in close association with individual cancer cells and none of these cells was present apart from the cancer cells. Few OKT6+ cells were observed in the papillary dermis and these had no relation to cancer cells. In the epidermis, OKT6+ dendritic cells remained within normal proportions. Staining with OKM1 revealed sporadic reactive cells. These results strongly suggest that besides T and B lymphocytes, Leu-7+ (natural killer) cells participate in a significant defense mechanism against SCC proliferation.     

Defined in situ enumeration of T6 and HLA-DR expressing epidermal Langerhans cells: morphologic and methodologic aspects

An essential prerequisite for the in situ enumeration of epidermal Langerhans cells (LCs) is the unequivocal identification of the desired cell type. We have examined over 250 cryostat sections of normal human skin to analyze morphologic and methodologic problems underlying the quantification of epidermal LCs, defined by anti-T6 (OKT6) and anti-HLA-DR (OKIal) immunoperoxidase staining. Our findings show that OKT6 reactivity of dendritic processes in cross-sectioned epidermis yields microscopic images which are not easy to analyze objectively. The morphology that we find leads us to categorize dendritic cells into 3 arbitrary types of T6+ LC profiles. In addition we describe criteria for the assessment of OKT6 staining patterns relating to the dendritic state of epidermal LCs. Preliminary quantitative data on this issue are discussed in relation to: epidermal thickness; the thickness of skin tissue sections; and the discrepancy between the number of T6+ and HLA-DR+ LCs. We hope that the principles outlined in this report may serve to overcome potential methodologic problems with quantitation of T6+ epidermal LCs in skin sections.     

In vivo cytokine profiles in allergic and irritant contact dermatitis

Local cytokine profiles in skin biopsies from allergic and irritant patch test reactions were determined by in vivo immunohistochemistry to differentiate between these 2 clinically identical afflictions especially at the time of final reading in diagnostic patch testing. Biopsies were taken from established allergic persons after specific allergic patch test.-, to epoxy resin (1%) and formaldehyde (1%) and from non-allergic individuals with irritant patch tests to sodium lauryl sulfate (10%) and formaldehyde (8%). At 72 h after application of the agents, significantly enhanced frequencies of dermal infiltrating cells, producing IL-1α, TNF-α. IL-2. and IFN-γ per 100 infiltrating cells in the dermis. were observed in allergic as well us irritant patch test reactions, as compared to normal skin. Significantly higher frequencies of IL- Iα-producing cells were observed in biopsies from epoxy resin (1%) allergen-affected and sodium lauryl sulfate (10%) irritant-affected skin as compared to formaldehyde (1%) allergen-affected skin. In addition, significantly higher frequencies of TNF -α reproducing cells were observed in epoxy resin allergen-affected skin us compared to Formaldehyde (1%) allergen-affected and formaldehyde (8%) irritant affected skin. The allergic and irritant patch test reactions showed similar levels of expression of the Thl cytokines IL-2 and IFN-γ in the dermis. confirmed by probe based detection of IL-2 mRNA and IFN-γ- mRNA, In conclusion, the described similarity shows that allergens and irritants can induce the same profile of IL-la. TNF-α. IL-2. and IFN-γ production, resulting in the near impossibility of discriminating between allergic and irritant contact dermal is at the lime of patch test reading.     

The cell population in the cutaneous infiltrate of mycosis fungoides: in situ studies using monoclonal antisera

T cell antigens were studied in cutaneous sections from five patients with mycosis fungoides (MF). The method allowed cell counting to be undertaken for each monoclonal antiserum. OKT3 (pan T cell) antiserum confirmed the predominantly T lymphocytic nature of the infiltrate, labelling the majority of infiltrating cells. OKT4 (helper/inducer) antiserum positively labelled 90% of the lymphocytes identified as OKT3⁺. OKT8 (suppressor) antiserum marked only single or small groups of dermal lymphocytes, which comprised 24% of the cells identified as T lymphocytes. OKT6 (anti-Langerhans) showed positive labelling of dendritic cells in the epidermis and dermis. Fewer positively labelled epidermal dendritic cells were observed in sections from patients receiving PUVA, but no difference was found in the number of OKT6 positive dermal cells. The ratio of helper to suppressor cells in the dermal infiltrate significantly exceeded the normal circulating ratio.     

LICHEN PLANUS: EVALUATION OF CELLS IN SKIN LESIONS AND OF T-LYMPHOCYTE SUBSETS IN BLOOD

The purpose of the present study was to examine the phenotype of cutaneous immunocompetent cells and to quantify Langerhans cells in Lichen planus by the use of monoclonal antibodies directed against T-cell populations. Helper cells (OKT4+) and Suppressor/cytotoxic cells (OKT8+) were observed in all cutaneous infiltrates, and numerous Langerhans cells were identified by OKT6, BL6, and BL2 (HLA-DR) in the epidermis and dermis. The quantification of Langerhans cells demonstrated that the number of dendritic cells in epidermis is greater in involved skin than in non-involved skin. In recent lesions, Langerhans cells are more abundant than in older lesions. The results in peripheral blood indicated a T Helper/Suppressor imbalance with a decreased T-Suppressor/cytotoxic subpopulation in patients with lichen planus diseases. In lichen planus, our results suggest an immunological reaction involving all the immunocompetent cell subpopulations with a first stage of information by Langerhans cells (OKT6+, BL6+, BL2+ (HLA-DR)) and Helper cells (OKT4+), and second stage mediated by Suppressor/cytotoxic cells (OKT8+).     

Fc epsilon R11/CD23 receptor distribution in patch test reactions to aeroallergens in atopic dermatitis.

There is increasing evidence that exposure to organic allergens may induce or exacerbate lesional skin in patients with atopic dermatitis. In this study, patients with atopic dermatitis were patch tested to 11 common organic allergens and to control chambers containing 0.4% phenol and 50% glycerin in 0.9% saline. In biopsies from positive patch test reactions, patch test control skin, lesional eczematous and non-lesional skin from atopic individuals, and normal skin from non-atopic volunteers, the presence and distribution of macrophages (RFD7+), dendritic cells (RFD1+), and Langerhans cells, and the expression of the low-affinity receptor for IgE (CD23) were investigated. In patch test reactions and lesional skin samples, inflammatory infiltrates of diffusely distributed macrophages (RFD7+), dendritic cells (RFD1+), T lymphocytes (RFTmix+), and Langerhans cells (CD1+) were seen, the latter being present in both the epidermis and the dermis. The numbers of Langerhans cells were reduced in the epidermis and increased in the dermis in patch test reactions and lesional skin compared to their controls. Double staining revealed a change in the distribution of CD23 antigen. In patch test control and non-lesional biopsies many macrophages and only a few Langerhans cells within the dermal infiltrates expressed this antigen. In patch test reaction and lesional skin samples, however, the proportion of CD23+ dermal Langerhans cells had increased compared to macrophages. Furthermore, in these latter samples an increased proportion of dermal CD1+ cells expressed the dendritic cell (RFD1+) marker. These results show that following antigen challenge there are marked similarities between the phenotype of the cellular infiltrate in patch test reaction and lesional skin biopsies, and also demonstrate a changing distribution of CD23 on antigen-presenting cells.     

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.