Characterization of the dermal infiltrates in Jessner''s lymphocytic infiltrate of the skin, polymorphous light eruption and cutaneous lupus erythematosus: differential diagnostic and pathogenetic aspects

In the present study a comparative immunohistochemical study was performed on skin biopsies from of patients with Jessner's lymphocytic infiltration of the skin (LIS), polymorphous light eruption (PLE), discoid lupus erythematosus (DLE) and subacute cutaneous lupus erythematosus (SCLE) using a large panel of monoclonal antibodies against T cell differentiation antigens (CD3, CD4, CD8), immunoregulatory T cell subsets (CD7, 4B4, 2H4, Leu 8), B cells (CD22), activated cells CD25, OKT9, HLA-DR), Langerhans cells (CD1) and macrophages (Leu-M5). The results showed many similarities between LIS and PLE. The most important differences between these conditions and CDLE/SCLE were the high proportions of cells reactive with monoclonal antibody Leu-8 and the absence of T cells expressing HLA-DR antigens in LIS and PLE, suggesting absence of local T cell activation in these conditions. The differential diagnostic and pathogenetic aspects of these findings will be discussed.     

Lymphocytes and Langerhans cells in patch tests

The distribution of immunocompetent cells was analysed in allergic (nickel) and irritant (dithranol) patch tests using conventional transmission electron microscopy and labelling with monoclonal antibodies in an avidin-biotin immunoperoxidase study. The biopsies were taken 24 or 48 h after the allergen/irritant application. In allergic and irritant reactions, most inflammatory cells were OKT11 positive (pan T lymphocytes). The majority of these cells were also OKT4 positive (helper/inducer T lymphocytes), while the minority were OKT8 positive (suppressor/cytotoxic T lymphocytes). NK9 positive cells (natural killer cells) were observed in small numbers. The number of dendritic OKT6 and OKIal positive cells (Langerhans cells) in the epidermis was unaffected in allergic reactions. In irritant reactions, a normal number of OKT6 positive Langerhans cells was observed, while the number of OKIal positive cells had increased in the epidermis. Dithranol caused prominent fine structural changes in the mitochondria of the Langerhans cells, while the keratinocytes appeared largely unaffected. The present study indicates that allergic and irritant patch tests cannot be differentiated reliably using current immunohistopathological or electron microscopic techniques, in spite of the small differences observed.     

In situ characterization of cells in the dermal infiltrates of lepromin reaction using monoclonal antibodies

A study was made on the in situ characteristics of dermal infiltrates in the early and late lepromin reaction with monoclonal antibodies defining T cell subsets, Langerhan cells and Ia like antigens. The early reaction (24 hrs) was elicited either with standard Dharmendra lepromin or leprosin-A and the late reaction (3-4 weeks) was elicited with standard Dharmendra lepromin. In all, 15 biopsies were studied. Most lymphocytes in the infiltrates of both the lepromin and leprosin reactions were positive for OKT 11, Leu 3a, OKT 8 and Ia like antigens indicating thereby the presence of activated T cells. A high proportion of OKT6 + cells were also noticed in the infiltrates of these reactions. In the late reaction, the lymphocytes in the granulomas were predominantly activated T lymphocytes expressing OKT 11, Leu 3a, OKT 8 and Ia like antigens. Leu 3a + cells were scattered diffusely amidst the epithelioid cells. In contrast, OKT 8 + cells were present mainly in the peripheral region of the granuloma. A small proportion of OKT6 + cells were also seen in these granulomas. Ia like antigens and T6 antigens were not discernible on the epithelioid cells. No difference in the number of OKT6 + epidermal langerhan cells was observed in the various types of reactions.     

Immunological effects of stress in psoriasis

Background Psychological stress causes phenotypic changes in circulating lymphocytes and is regarded as an important trigger of the Th1‐polarized inflammatory skin disease psoriasis. Objective To study the effects of psychological stress on immunological parameters, i.e. membrane molecules relevant to the pathophysiology of psoriasis, especially cutaneous lymphocyte‐associated antigens (CLA) involved in T and natural killer (NK) cells homing in on the skin. Methods The severity of psoriasis was assessed in patients using the Psoriasis Area and Severity Index. Patients with psoriasis (n = 15) and healthy volunteers (n = 15) were exposed to brief psychological stress in the laboratory. In vitro analyses were conducted 1 h before, immediately following and 1 h after stress exposure. Peripheral T‐ and NK‐cell subsets including CD8+ T lymphocytes, CLA+ lymphocytes and lymphocyte function‐associated antigen type 1 (LFA‐1)+ lymphocytes were analysed by flow cytometry. Results We found a significant stress‐induced increase of CD3+ T lymphocytes in patients with psoriasis only. Analyses of T‐cell subsets revealed that this increase was observable for cytotoxic CD8+ T lymphocytes and CLA+ CD3+ lymphocytes. The total number of circulating NK cells (CD16+, CD56+) increased immediately after stress in both groups whereas only patients with psoriasis showed a significant increase in CLA+ NK cells. Conclusions A higher stress‐induced increase of CLA+ T and CLA+ NK cells in the circulation of patients with psoriasis might point to an increased ability of T and NK cells in the presence of psoriasis to home in on the skin during mental stress. Further studies are needed to verify these relationships in more detail and to investigate the time point at which these cells accumulate within lesional skin, and whether or not psychotherapy improves the quality of life of patients with psoriasis and influences stress‐dependent parameters.     

Epidermal lymphocyte chemotactic factor specifically attracts OKT4-positive lymphocytes

Summary Epidermal lymphocyte chemotactic factor (ELCF) from skin overlying a positive tuberculin reaction was compared with the chemoattractants leukotriene B₄ (LTB₄), N-formyl-methionyl-leukylphenylalanine (FMLP), and complement split product C5a (C5a). The chemotactic assay used is a modified Boyden chamber technique. The lymphocytes were subsets of T lymphocytes from healthy young individuals first separated by flotation of E rosettes on Isopaque Ficoll followed by incubation of T cells with anti-CD4 and anti-CD8 monoclonal antibodies and further separation using fluorescence-activated cell sorting. ELCF specifically attracted OKT4+ lymphocytes, while LTB₄, FMLP, and C5a induced significant migration in both OKT4+ and OKT8+ lymphocytes without any clear difference between the various chemoattractants or cell populations. We found no blocking of the chemotactic capacity of ELCF when we added antibodies towards IL-1 and IL-1 to the chemotactic assay. Further recombinant IL-1 and IL-1 did not induce any chemotactic response. Our observations may be of significance in explaining the predominance of OKT4+ cells in allergic contact dermatitis and certain other skin diseases.This work was supported by the Danish Medical Research Council grants no 12-6132 and 12-5691     

Reduction of the fraction of circulating helper-inducer T cells identified by monoclonal antibodies in psoriatic patients treated with long-term psoralen/ultraviolet-A radiation (PUVA)

Ultraviolet radiation has been found to alter the distribution and function of human lymphocytes. To determine whether photochemotherapy (PUVA) alters circulating levels of T cell subset marker-bearing lymphocytes, cells from 9 patients with psoriasis undergoing PUVA therapy for several years (mean 4.6 +/- 1.4 yr), 17 patients with active untreated psoriasis, and 20 healthy volunteers were reacted with monoclonal antibodies to T cell surface markers, including OKT3 (all peripheral blood T cells), OKT4 (helper/inducer T cells), OKT6 (common thymocytes), and OKT8 (suppressor/cytotoxic T cells), and analyzed by flow cytometry. There were no differences in the distribution of T cell subsets between healthy volunteers and patients with active psoriasis. In contrast, the percentages of lymphocytes reacting with OKT3 and OKT4 were lower (by 16% and 12% percent respectively, p less than 0.0025) in the PUVA-treated patients compared to healthy volunteers or patients with active psoriasis that had not received PUVA therapy. There was no difference in the percentage of OKT8 and OKT6 bearing cells. Squamous cell carcinoma of the skin subsequently developed in 2 of 3 PUVA-treated patients with the lowest percentages of T4-bearing cells. These findings indicate that long-term PUVA therapy is associated with a reduction in circulating helper/inducer T cells. This reduction may have a role in the altered immune function reported in PUVA-treated patients.     

Immunoperoxidase examination of cutaneous infiltrates of mycosis fungoides and large-plaque atrophic parapsoriasis with OKT10

A monoclonal antibody (OKT10), which was developed recently, reacts with pro-thymocytes, T cell acute lymphoblastic leukemia (ALL) cells, cells in normal bone marrow (including plasma cells), and activated T cells. Tissues from patients with cutaneous T cell lymphoma were studied for the presence of OKT10-reactive cells with the use of an indirect immunoperoxidase technic. OKT10-reactive cells were identified in three of eight cases of mycosis fungoides, one of two cases of Sézary syndrome, with an equivocal reaction in one of ten cases of large-plaque parapsoriasis and in one of seven positive patch tests (allergic contact dermatitis). The biologic and possible clinical implications are discussed.     

Mononuclear cell subpopulations in the skin defined by monoclonal antibodies after HLA-identical sibling marrow transplantation

Mononuclear cell subpopulations present in the skin of 36 recipients of HLA-identical sibling marrow transplants were defined by immunoperoxidase using a battery of monoclonal antibodies to cell surface differentiation antigens. The T4-positive (T4+) (helper-inducer T cells), T8+ (cytotoxic-suppressor T cells) and the T6+ (Langerhans cells) decreased in number early post transplant and returned towards normal numbers from day 42 onwards. There was no evidence that either the T4+ or the T8+ subset was involved in cell-to-cell contact damage in acute graft-versus-host disease (GVHD). The paucity of lymphoid cell infiltration of the epidermis in acute GVHD suggested the possibility of a soluble factor being responsible for basal layer damage. In patients with chronic GVHD there was no evidence of T4+ lymphocyte involvement, but T8+ lymphocytes were present in increased numbers, suggesting a role for the T8+ population in the skin lesions of chronic GVHD, or possibly a reflection of the pattern of T4+ and T8+ cell reconstitution in the blood post-transplant. Finally, our study provided no evidence that BI+ (B cells), Leu 7+ (natural killer cells), OKMI+ (histiocytes) or OKT1O+ cells were involved in cell-to-cell contact damage in either acute or chronic GVHD.     

Immunopathologic features of allergic contact dermatitis in humans: participation of plasmacytoid dendritic cells in the pathogenesis of the disease?

Contrary to our abundant knowledge about the sensitization phase of human contact hypersensitivity, little is known about the cell types orchestrating the effector phase. In order to address this issue, we phenotypically analyzed biopsies from 72 h epicutaneous patch test reactions (n=10) and normal human skin (n=5) for the presence of various leukocyte differentiation antigens. The inflammatory infiltrate was dominated by CD3+/CD4+ T cells with approximately 30% of the cells coexpressing CD25 and CTLA-4, a phenotype consistent with either activated effector or regulatory T cells. In our search for professional antigen-presenting cells, we were surprised to find not only sizeable numbers of CD1a+ dendritic cells and CD1c+ dendritic cells, but also of CD123+, CD45RA+, BDCA-2+, CLA+, and CD62L+ plasmacytoid dendritic cells. Although virtually absent in normal human skin, these cells were detectable already 6 h after hapten challenge and were often found in close proximity to CD56+ natural killer cells, indicative of a functional interaction between these cell types. The detailed knowledge of the cellular composition of the inflammatory infiltrate in allergic contact dermatitis and its kinetics should form the basis for the investigation of the immunologic and molecular events operative in the perpetuation and resolution of the eczematous response.     

CD4+ CD8+ (thymocyte-like) T lymphocytes present in blood and skin from patients with atopic dermatitis suggest immune dysregulation

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease expressed early in life. Disease development is primarily determined by as yet unknown genetic factors, leading to the accumulation of activated T lymphocytes in the skin. OBJECTIVES: To investigate the nature of these T cells. METHODS: T-cell lines could be established from AD skin biopsies, but not from normal skin or AD peripheral blood, when placed in RPMI 1640 medium with 10% human AB serum, antibiotics, and the T-lymphocyte growth factors interleukins 2 and 4. The cell lines were subjected to phenotypic analysis using a fluorescence-activated cell sorter and compared with lymphocytes from AD and normal control peripheral blood. RESULTS: T-cell lines from 22 of 24 consecutive skin biopsies taken from 24 adult patients with AD were established. All cells were T lymphocytes expressing several activation markers. A significant proportion of the lymphocytes had stable expression of a CD4+ CD8+ phenotype (26% +/- 6%; mean +/- SEM). Such double-positive T lymphocytes are normally only seen in the thymus and not in the peripheral immune system. CD4+ CD8+ cells in peripheral blood of the patients (12.5% +/- 3.3%) were also detected. CONCLUSIONS: We suggest that a basic pathophysiological change in AD may be a faulty maturation of the T-lymphocyte system, leading to skin inflammation with CD4+ CD8+ T lymphocytes resembling immature T cells. This is likely to lead to skewing of many immune reactions in the patients.     

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.     

Recent developments in the pathogenesis of allergic contact dermatitis

Allergic contact dermatitis is both an important clinical problem and a model system for lymphocyte-mediated pathologic changes. Elicitation of allergic contact dermatitis requires interaction of antigen with epidermal Langerhans cells, followed by migration of the Langerhans cells to the lymph nodes to present antigen to T lymphocytes. These activated T lymphocytes must then home to the antigen-exposed skin. Adhesion molecules such as LFA-1 and ICAM-1 have a role in this homing. Only a small proportion of the T lymphocytes in the skin lesion are specific for the inducing antigen. Studies of poison ivy (urushiol dermatitis) have determined this fraction to be less than one per 100 infiltrating lymphocytes. By a variety of amplification mechanisms, it is possible for this small number of antigen-specific T lymphocytes to induce the pathologic changes of allergic contact dermatitis. Improved understanding of this condition should result in increased knowledge of the pathogenesis of a variety of T lymphocyte-mediated skin conditions.     

Macrophage marker 27E10 on human keratinocytes helps to differentiate discoid lupus erythematosus and Jessner's lymphocytic infiltration of the skin

Chronic inflammatory discoid lupus erythematosus (DLE) and Jessner's lymphocytic infiltration of the skin (LIS) are both characterized by dermal infiltrates of activated T lymphocytes. However, an inflammatory involvement of the epidermis is only found in DLE. We therefore compared the phenotypic properties of the keratinocytes using immunohistochemical stainings of biopsies from typical DLE and LIS. Keratinocytes failed to express HLA-DR in LIS and surprisingly also in DLE. The adhesion molecule ICAM-1 was only expressed in DLE, with focal staining of the basal keratinocytes in close association with intraepidermal lymphocytes. The monoclonal antibody 27E10, a distinct marker for macrophage activation and differentiation, revealed a strong band-like labelling of the suprabasal and upper keratinocytes in DLE. In contrast, no epidermal expression of this biologically active heterodimer of the calcium-binding proteins MRP-8 and MRP-14 was found in LIS. The staining patterns provide a new method to differentiate DLE and LIS by immunohistochemistry and suggest a distinct type of keratinocyte activation and differentiation in DLE which would in turn mediate epidermal T cell infiltration.     

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.     

Establishment of T and B cell lines from patients with mycosis fungoides

Eighteen patients with mycosis fungoides were studied in order to establish cell lines that might be associated with the human T cell leukaemia-lymphoma virus (HTLV). Three T cell lines were established, two from affected skin and one from a lymph node showing dermatopathic lymphadenopathy. The T cells expressed OKT3 and OKT4 antigens. They temporarily expressed an HTLV p19-like antigen in up to 5% of the cells during culture. None of our patients had lymphocytosis or abnormal lymphocytes, except one patient with Sézary's syndrome. We could not establish T cell lines from peripheral blood, but five B lymphoblastoid cell lines were obtained, all positive for the Epstein-Barr virus nuclear antigen. Our finding that T cell lines can be established from skin biopsies and lymph nodes of patients with mycosis fungoides, but not from the blood, supports the concept of a malignant T lymphocyte primarily localized in the skin. The temporary expression of HTLV p19 antigen may indicate the presence of retrovirus, but further studies are needed.     

The skin immune system (SIS): distribution and immunophenotype of lymphocyte subpopulations in normal human skin

The complexity of immune response-associated cells present in normal human skin was recently redefined as the skin immune system (SIS). In the present study, the exact immunophenotypes of lymphocyte subpopulations with their localizations in normal human skin were determined quantitatively. B cells were not found to be present in normal human skin. Lymphocytes were always of T-cell type, and 90% of these T cells were clustered in 1-3 rows around postcapillary venules of the papillary vascular plexus or adjacent to cutaneous appendages. In such perivascular localizations, they were found to differ from their circulating counterparts in three ways. First, skin perivascular cells were found to be approximately evenly distributed over CD4+ inducer and CD8+ suppressor-cytotoxic T-cell subsets (mean CD4/CD8 ratio: papillary layer 0.96, reticular layer 0.99). Second, within the category of CD4+ inducer T cells, most were phenotyped as CD4+, 4B4+ helper inducer T lymphocytes, whereas CD4+, 2H4+ suppressor inducer T lymphocytes were found to be relatively rare (less than 5%). Third, the majority of skin perivascular T cells were activated as they expressed HLA-DR and interleukin 2 receptors. Intraepidermal, directly subepidermal, and other ("free") lymphocytes were mostly of the CD8+ suppressor-cytotoxic T-cell subset but accounted for less than 10% of the total number of lymphocytes. Intraepidermally localized T cells accounted for less than 2% of the total number of lymphocytes present in normal skin. Our results indicate that preferential perivascular localization of activated T lymphocytes is the characteristic of normal human skin. This might be a reflection of continuous antigen recognition upon endothelial cell presentation and/or continuous T cell-mediated endothelial cell activation thereby inducing enhanced antigen clearing by the skin's endothelium.     

Characterization of cytotoxic immune response in skin and mucosal lesions of paracoccidioidomycosis

Background: CD8+ T cells and natural killer (NK) cells are involved in the immune response against some pathogens. For this purpose, we investigated the in situ paracoccidioidomycosis (PCM) immune response addressing the participation of NK cells, CD8+ T cells, perforin and granzyme B expression. Methods: Sixty biopsies of PCM skin and mucosa were classified according to the presence of compact granulomas (G1), poorly organized granulomas (G2) and both kinds in the same lesion (G3). CD8+ T cells, NK cells, perforin and granzyme B were showed by immunohistochemistry. Results: CD8+ T cells were increased over NK cells in cutaneous G1 and G2 lesions. There was no difference regarding such cells in G3 lesions, although they were abundant in such lesions. In mucosa, CD8+ T cells were increased in number over NK cells in all groups. Granzyme B in skin increased in G2 and G3. The number of granzyme did not differ in mucosal lesions in the three groups. Conclusions: CD8+ T cells and NK cells play a role in PCM cutaneous and mucosal lesions. The predominance of CD8+ T cells over NK cells may represent an effective response against the fungi. Moreover, the high number of granzyme B expressing cells corroborates this possibility. Pagliari C, Pereira NV, Kanashiro L, Stegun FW, Croda J, Seixas Duarte MI, Sotto MN. Characterization of cytotoxic immune response in skin and mucosal lesions of paracoccidioidomycosis.     

Different in situ distribution patterns of dendritic cells having Langerhans (T6+) and interdigitating (RFD1+) cell immunophenotype in psoriasis, atopic dermatitis, and other inflammatory dermatoses

Dendritic cells bearing Langerhans cell (OKT6+) or interdigitating cell (RFD1+) immunophenotype may be regularly detected within the dermis of chronic skin diseases characterized by a lymphohistiocytic (lymphoreticular) infiltrate. These 2 subsets of antigen-presenting cells within the dermis of lesions of exacerbating chronic plaque psoriasis, exacerbating nummular dermatitis (discoid eczema), atopic dermatitis, allergic contact dermatitis, pityriasis rosea, lichen ruber planus, and cutaneous lupus erythematosus were quantified using computer-assisted morphometry. The mean dendrite length per dermal dendritic cell was significantly higher for RFD1 than for OKT6 (74.4 +/- 0.98 microns vs 70.0 +/- 1.26 microns: p = 0.0023). The mean dendrite length per dermal dendritic cell was remarkably constant for each marker in the various diagnostic categories studied. Disease-specific patterns of total dendrite length and number (expressed per 100 infiltrating mononuclear cells) of these 2 dendritic cell types within the subepidermal infiltrates were obtained. Pityriasis rosea was characterized by its unique high percentage of OKT6+ Langerhans cells. Atopic dermatitis and psoriasis had relatively high percentages of both RFD1+ interdigitating cells and OKT6+ Langerhans cells. Nummular dermatitis had an intermediate number and total dendrite length for OKT6, but was relatively low in RFD1+ cells. Allergic contact dermatitis, lichen planus, and lupus erythematosus had low numbers and dendrite lengths for both dendritic cell subsets. It is suggested that pityriasis rosea is characterized by an abnormal migration pattern of Langerhans cells. Psoriasis and atopic dermatitis may be examples of diseases in which skin-localized antigen-presenting and T-cell-inducing events are continuously taking place. The other diseases may reflect inflammatory processes in which local antigen presentation is less relevant to the tissue reaction.