Impaired responses of peripheral blood mononuclear cells to staphylococcal superantigen in patients with severe atopic dermatitis: a role of T cell apoptosis

Staphylococcus aureus colonization is an almost universal feature of atopic dermatitis. In order to investigate the role of staphylococcal enterotoxin B in the pathogenesis of atopic dermatitis, we assessed the correlation between clinical disease severity and proliferative response of peripheral blood mononuclear cells to staphylococcal enterotoxin B in patients with atopic dermatitis. Peripheral blood mononuclear cells from patients with mild atopic dermatitis showed significantly increased proliferative responses to staphylococcal enterotoxin B compared to controls. In contrast, peripheral blood mononuclear cells from patients with severe atopic dermatitis showed markedly suppressed proliferative responses. Additionally, longitudinal evaluation of peripheral blood mononuclear cell samples from the same patient demonstrated that proliferative responses were suppressed only at times of severe disease exacerbation. Mixing experiments, using autologous T cells and antigen presenting cells that were isolated at different time points from the same patient, demonstrated that T cells of severe atopic dermatitis patients were dysfunctional, but their antigen presenting cell function remained intact. We found no significant differences of interleukin-2 levels in the culture supernatants between healthy controls and atopic dermatitis groups. Fluorescence-activated cell sorter analysis for APO2.7 antigen, an early apoptosis cell marker, demonstrated that approximately 60% of staphylococcal-enterotoxin-B-stimulated T cells expressed APO2.7 antigen in severe atopic dermatitis cases. By contrast, 5%-20% of T cells expressed APO2.7 after staphylococcal enterotoxin B stimulation in cases of mild atopic dermatitis and in healthy controls. Nuclear staining with Hoechst 33258 also showed approximately 40% apoptotic cells in the CD19-CD16-PBMC of severe atopic dermatitis patients, compared with only 5%-10% in the mild atopic dermatitis group and in healthy controls. Blocking monoclonal antibody to Fas ligand partially prevented the staphylococcal-enterotoxin-B-induced apoptosis detected by APO2.7 expression and Hoechst 33258 staining. Suppressed proliferation of peripheral blood mononuclear cells in severe atopic dermatitis patients may be secondary to T cell death by apoptosis. These results suggest that an infection of S. aureus producing staphylococcal enterotoxin B may play a role in aggravation of atopic dermatitis by inducing apoptosis in T cells.     

Production of interleukin-2 by mononuclear cells in vitro in patients with atopic dermatitis and psoriasis. Comparison with serum interleukin-2 receptor levels.

Atopic dermatitis is associated with profound immunological alterations, in particular decreased lymphoproliferative responses upon stimulation with T-cell mitogens. T-cell blastogenesis involves the production of the soluble cytokine interleukin-2 (IL-2), which in turn upregulates the expression of its own receptor. To investigate the potential role of this cytokine for the pathomechanisms present in atopic dermatitis, 24-h supernatants of PHA-stimulated peripheral blood mononuclear cells from patients with atopic dermatitis (n = 30) of a moderate to severe disease activity were tested for IL-2 activity. In addition, serum concentrations of soluble interleukin-2 receptor (IL-2R) were measured. Non-atopic healthy controls (n = 19) and patients with psoriasis (n = 20), an inflammatory skin disorder with distinct pathogenesis, served as controls. In comparison with psoriasis patients and normal controls, PHA-stimulated mononuclear cells of atopic dermatitis patients released significantly less IL-2 into supernatants. Moreover, there was an inverse correlation between IL-2 concentrations and body surface involvement or serum IgE levels. In contrast, serum IL-2R levels were significantly elevated in both atopic dermatitis and psoriasis, as compared with healthy controls. Furthermore, IL-2R levels in atopic dermatitis patients showed a significant correlation with IgE levels and body surface involvement. The data indicate that T cell activation may occur in both skin diseases. Atopic dermatitis, however, is further characterized by the decreased capacity of mononuclear cells to release IL-2 upon stimulation in vitro.     

异位性皮炎的记忆性T细胞浸润及ICAM－1表达

为了解粘连分子在异位性皮炎（ＡＤ）炎症及免疫反应过程中的作用，对ＡＤ皮损部位细胞间粘连分子－１（ＩＣＡＭ－１）的表达作了研究。结果虽然正常皮肤表皮不表达ＩＣＡＭ－１，但ＡＤ皮损处角朊细胞则局灶性表达ＩＣＡＭ－１，尤其在有严重单个核细胞浸润及表皮内淋巴细胞移入的部位。免疫表型研究表明，ＡＤ真皮浸润中ＣＤ４＋／ＣＤｗ２９＋／ＣＤ４５ＲＡ－记忆性Ｔ细胞占主导，推测它们可能通过分泌某些细胞因子而诱导角朊细胞表达ＩＣＡＭ－１。ＩＣＡＭ－１与淋巴细胞表面的淋巴细胞功能相关抗原－１（ＬＦＡ－１）之间相互作用可能对淋巴细胞在皮肤内的运行起调控作用。     

CC chemokine receptor 4 expression on peripheral blood CD4+ T cells reflects disease activity of atopic dermatitis

Recent studies indicate that Th1 and Th2 cells differ in their chemokine receptor expression and their responsiveness to various chemokines. Therefore, selective Th2 cell recruitment in Th2-predominant inflammatory diseases such as atopic dermatitis may be under the influence of some chemokines. It is reported that CC chemokine receptor (CCR) 4 is selectively expressed on Th2 cells whereas CXC chemokine receptor (CXCR) 3 is selectively expressed on Th1 cells. In this study we examined CCR4 and CXCR3 expression on peripheral blood CD4+ and CD8+ T cells obtained from adult atopic dermatitis subjects, and compared the results with those from patients with psoriasis vulgaris and healthy controls. CCR4 was preferentially expressed on CD4+ T cells from atopic dermatitis subjects and CXCR3 was preferentially expressed on CD4+ T cells from psoriasis vulgaris subjects. This CCR4 expression was prominent especially in severe atopic dermatitis subjects. CCR4 expression on CD4+ T cells in severe atopic dermatitis subjects decreased on improvement of disease activity. CD25 was preferentially expressed on CCR4+CD4+ T cells but not on CXCR3+CD4+ T cells in atopic dermatitis subjects. Cutaneous lymphocyte-associated antigen was also preferentially expressed on CCR4+CD4+ T cells but not on CXCR3+CD4+ T cells in atopic dermatitis subjects. CD4+ T cells in atopic dermatitis skin lesions were predominantly CCR4+ cells. Taken together, this study strongly indicates that CCR4+CD4+ T cells reflect disease activity and suggests that CCR4 expression is important for T cell infiltration into atopic dermatitis lesions. Thus, CCR4 may be a possible target for therapy of atopic dermatitis in the future.     

Cytokine expression of skin T-lymphocytes from patients with atopic dermatitis.

We analysed the cytokine profile of skin T cells by establishing 11 T-cell lines from adult patients with moderate-to-severe atopic eczema using T-cell growth factors interleukin-2 and interleukin-4. We compared T-cell lines from lesional skin of atopic dermatitis patients with those from non-atopic skin of patients with other skin diseases, observing that T-cell lines of patients with atopic dermatitis unstimulated cultures expressed a Th1 profile. After stimulation with anti-CD3 and anti-CD28 monoclonal antibodies, the cytokine expression showed rapid initial upregulation of Th2 followed by a Th1 profile. Furthermore, strong upregulation of interleukin-10 was observed after 24 h stimulation. Our findings suggest that skin T-lymphocytes from atopic dermatitis patients seem to consist of a heterogenous population of Th1 and Th2 or Th0 cells and the results for secreted cytokines indicate that T-cell lines from each inflammatory skin disease showed the corresponding disease-specific original cytokine profile.     

The presence of surface CD30 on T cells in atopic dermatitis

Atopic dermatitis (AD) has cellular immunohistochemical features similar to those of allergic contact dermatitis (ACD) and there is plenty of evidence for T-cell activation in this disease. The involvement of CD30+ T cells in acute stages of atopic dermatitis might establish CD30 as a helpful marker in differentiating those two diseases. Tissue sections from the skin of 12 patients with active atopic dermatitis and 13 with allergic contact (nickel-induced) dermatitis were immunohistochemically analyzed for cell-surface antigens, including CD30, CD3, CD4, and CD45RO. The severity of the disease was graded by the SCORAD clinical scoring system. The analysis of CD30+, CD45RO+, CD3+, and CD4+ cells in the dermis and epidermis showed a much wider range of values and statistically higher median (p<0.01) in the inflammatory infiltrate of acute atopic dermatitis compared with that of allergic contact dermatitis. Our results showed an association of CD30 expression with atopic dermatitis, but not allergic contact dermatitis. CD30 expression in AD might be helpful in histologic differentiation of these disorders and further characterization of atopy patch testing. The results suggested a specific regulatory function of CD30+ T cells in acute AD. Abundant CD45R0+ cells were detected in both AD and ACD lesions.     

Efalizumab therapy for atopic dermatitis causes marked increases in circulating effector memory CD4+ T cells that express cutaneous lymphocyte antigen

Efalizumab is an mAb directed against CD11a, a molecule involved in T-cell activation and extravasation from blood into tissue. Ten patients with severe atopic dermatitis were treated with efalizumab for 84 days, and peripheral blood mononuclear cells were analyzed for expression of activation and adhesion markers. Efalizumab treatment led to decreases in CD11a mean fluorescence intensity (MFI) on naive, central memory, and effector memory CD4+ and CD8+ T cell subsets. MFI for CD18 was decreased in both CD4+ and CD8+ T cells. Percentages of cells positive for cutaneous lymphocyte antigen (CLA) were increased fourfold in all CD4+ and CD8+ T cell subsets. Increases in the percentages of CD4+ and CD8+ T cells expressing beta7 and CD49d were also observed. No significant changes were observed in the percentages of CD4+ and CD8+ T cells that produced either IFN-gamma or IL-4. In summary, efalizumab treatment resulted in (i) decreases in CD11a and CD18 expression in all circulating T-cell subsets and (ii) increases in the percentages of blood T cells expressing tissue homing markers (CLA, beta7, CD49d). These data suggest that blockade of T-cell extravasation into tissue is the major pathway by which efalizumab leads to improvement in cutaneous inflammation.     

趋化性细胞因子受体CCR4和CXCR3在特应性皮炎患者外周血的表达

目的为研究特应性皮炎患者外周血趋化性细胞因子受体CCR4和CXCR3在特应性皮炎的发病过程中的作用。方法采用三色流式细胞仪测定20例特应性皮炎患者和30例健康对照者外周血趋化性细胞因子受体CCR4和CXCR3的表达水平。结果特应性皮炎患者外周血CCR4+CD4+T细胞的水平明显高于对照组(P<0.01);特应性皮炎患者外周血CCR4/CXCR3比率明显高于对照组P<0.01);特应性皮炎患者外周血CXCR3+CD4+T细胞的水平与对照组差异无统计学意义。结论趋化性细胞因子受体CCR4可能促进了Th2细胞从血液进入特应性皮炎患者炎症皮损。     

Plaque psoriasis vs. atopic dermatitis and lichen planus: a comparison for lesional T-cell subsets, epidermal proliferation and differentiation

BACKGROUND: T-cell infiltration in plaque psoriasis has recently been an important subject of investigation. Interestingly, comparative analyses of the disease-specific composition of the lesional T-cell infiltrate in plaque psoriasis and other inflammatory dermatoses have only sparsely been performed. OBJECTIVES: To compare plaque psoriasis vs. atopic dermatitis and lichen ruber planus with respect to T-cell subsets, epidermal proliferation and keratinization. PATIENTS AND METHODS: Biopsies were taken from untreated lesional skin of patients, six with psoriasis, six with atopic dermatitis and six with lichen planus. T-cell subsets (CD4+, CD8+, CD45RO+, CD45RA+, CD2+, CD25+), an epidermal proliferation (Ki-67) and a keratinization marker (K10) were stained immunohistochemically and quantified using image analysis. RESULTS: The high number of CD8+ T cells (52 +/- 13 cells mm(-1)) found in the psoriatic epidermis was not found in the epidermis of atopic dermatitis (9 +/- 4), nor in the epidermis of lichen planus (34 +/- 10). The other T-cell subsets in the epidermis and dermis showed no statistically significant differences between psoriasis and atopic dermatitis. In contrast to the limited presence of CD4+, CD8+ and CD2+ in the psoriatic dermis (110 +/- 19, 27 +/- 9, 127 +/- 41, cells mm(-1), respectively), more impressive numbers of these cells were observed in the dermis of lichen planus (300 +/- 53, 144 +/- 38, 272 +/- 48, respectively). CD45RO+ memory effector T-cell counts were significantly higher in the epidermis of lichen planus (39 +/- 10) than in psoriasis (19 +/- 5). Psoriatic epidermis proved to have major keratinocyte hyperproliferation (247 +/- 26 cells mm(-1) lamina basalis), as compared with atopic dermatitis (134 +/- 15) and lichen planus (128 +/- 20). Furthermore, a marked decreased expression of keratin 10 was observed in psoriasis (41% of epidermal area) contrary to atopic dermatitis (70%). CONCLUSIONS: Psoriatic epidermis exhibits a pronounced CD8+ epidermotropism with accompanying epidermal hyperproliferation and abnormal keratinization, which changes are only minimally expressed in atopic dermatitis and lichen planus. In plaque psoriasis, substantially fewer activated CD4+ and CD8+ T cells in the dermis and less CD45RO+ T cells in the epidermis are present in comparison with lichen ruber planus.     

Expression of dipeptidyl-peptidase IV (CD26) on CD8+ T cells is significantly decreased in patients with psoriasis vulgaris and atopic dermatitis

T cells play a major role in inflammatory skin disorders such as psoriasis vulgaris and atopic dermatitis. They are both active on the level of cell-to-cell interaction and by the secretion of pro-inflammatory mediators. CD26 is a lymphocyte membrane-associated dipeptidyl peptidase IV (DPP IV), which is able to inactivate chemokines such as RANTES or eotaxin by cleaving dipeptides from the NH2-terminus of proteins. We investigated the expression of CD26 on CD4+ and CD8+ peripheral blood T cells in patients with psoriasis and atopic dermatitis. In addition PASI and SCORAD as a measure of disease severity were determined in each patient at the time of blood drawing. Thirty patients with psoriasis, 15 with atopic dermatitis and 17 age- and sex-matched healthy persons were investigated by two-colour flow cytometry using epitope-specific monoclonal antibodies. Our results revealed, that there is a significant decrease (P<0.05) of CD26 expression on CD8+ T cells in both psoriasis (7.7%+/-3.3, mean and SD, n=30) and atopic dermatitis patients (7.9%+/-3.7, mean and SD, n=15) compared to the control population (11.58%+/-5.0, mean and SD, n=17). However, there was no correlation to disease severity as determined by PASI and SCORAD, respectively. Since CD26 can be regarded as an anti-inflammatory principle the decreased expression in psoriasis and atopic dermatitis patients may lead to a dysbalance in favour of pro-inflammatory mediators in both clinical conditions.     

Peripheral natural killer cells exhibit qualitative and quantitative changes in patients with psoriasis and atopic dermatitis

Background Psoriasis and atopic dermatitis are the most recurrent skin inflammatory disorders. Despite their distinct aetiology and clinical aspects, these diseases share several immunological features. Besides the largely documented role of T cells, emerging literature supports a potential involvement of innate immune effectors, the natural killer (NK) cells, in both pathologies. In the peripheral blood, NK cells consist of CD3? CD56dim and CD3? CD56bright cell subsets, harbouring a distinct cell surface phenotype, but both endowed with the main NK‐cell effector functions: cytotoxicity and cytokine production. Objectives To determine whether the frequency, the cell surface phenotype and the functional properties of peripheral NK cells were affected in patients with psoriasis or atopic dermatitis. Methods Peripheral blood mononuclear cells were isolated from 11 patients with psoriasis, nine patients with atopic dermatitis and 16 healthy individuals. By using flow cytometry, we analysed the following parameters of peripheral NK cells: the frequency, the cell surface expression of several NK‐cell receptors (NKR) and the activation of the effector functions upon various in vitro stimuli. Results Peripheral NK cells were significantly reduced in both skin diseases. The cell surface expression of various NKR was differently modified in peripheral NK cells of the two cohorts of patients. Finally, NK‐cell natural cytotoxicity was affected only in atopic dermatitis, while interferon‐γ production was defective in both groups of patients. Conclusion Psoriasis and atopic dermatitis are associated with quantitative and qualitative changes of peripheral NK cells, mostly shared by both diseases, supporting a common process implicating these innate effectors in skin inflammation.     

UM4D4+ (CDw60) T cells are compartmentalized into psoriatic skin and release lymphokines that induce a keratinocyte phenotype expressed in psoriatic lesions

UM4D4 (CDw60), the surface molecule of a novel antigen-independent T-cell activation pathway, was found to be highly expressed on lesional psoriatic T cells. To examine whether UM4D4 represents a T-cell activation pathway for psoriatic T cells, a T-cell line was initiated from an acute skin lesion and cloned by limiting dilution. Clonality was verified by analysis of T-cell receptor gene rearrangement. All T-cell clones tested, whether CD4+2H4+CD8-, CD4+2H4-CD8-, or CD4-CD8+CD11b-, expressed UM4D4 and were activated by the monoclonal antibody anti-UM4D4. Lesional psoriatic T-cell clones were heterogeneous in the degree of anti-UM4D4-induced proliferation and in their production of IL-2 and gamma-interferon. Lymphokines released by anti-UM4D4 activation were capable of inducing ICAM-1 and HLA-DR expression on cultured normal keratinocytes. Thus, the high expression of UM4D4 on T-cells in psoriatic skin provides an alternative mechanism for T-cell activation that may be operative in the psoriatic lesional milieu. Indeed, activation of lesional T-cells through the UM4D4 molecule resulted in release of lymphokines that directly induced keratinocytes to express a phenotype displayed in psoriatic skin lesions.     

Expression and functional role of co-stimulatory molecules in CD40+IL-4-stimulated B cells from atopic and non-atopic donors

As immunological dysregulation is a possible key defect in atopic diseases, we have studied the expression and function of costimulatory molecules in atopic dermatitis (AD) patients compared with normal controls. Using flow cytometry, we showed that CD80 and CD86 are expressed at increased levels on human peripheral B cells in both groups after stimulation with anti-CD40 and interleukin 4 (IL-4), but to a significantly higher extent in the AD group. Furthermore, baseline expression of CD80 and CD86 on peripheral B cells was low in normal donors and increased in AD donors. To study the functional role of the costimulatory molecules in CD40+IL-4-stimulated peripheral mononuclear cells from normal and atopic donors, proliferation and IgE production were analysed in the presence of antibodies against the receptors of the costimulatory molecules. In the presence of either anti-CD28 or anti-CTLA-4, cell proliferation and IgE synthesis were significantly enhanced in the atopic group in anti-CD40+IL-4-stimulated peripheral mononuclear cells. These findings suggest that interaction of CD80 and CD86 with their receptors CD28 and CTLA-4 selectively promotes cell activation, including proliferation and IgE production in CD40+IL-4-stimulated peripheral blood mononuclear cells from atopic donors. It remains to be elucidated whether these changes are primary, based on the genetic background of atopics, or whether they are induced secondarily in the context of atopic pathology.     

T cells and T cell-derived cytokines as pathogenic factors in the nonallergic form of atopic dermatitis.

A subgroup of patients with atopic dermatitis are known to have normal serum total immunoglobulin E levels, undetectable specific immunoglobulin E, and negative skin prick tests towards allergens. This form of the disease has been termed nonallergic atopic dermatitis. In this study, we found that, among 1151 chronic atopic dermatitis patients, about 10% had normal serum immunoglobulin E levels with no evidence for immunoglobulin E sensitization. We investigated immunologic mechanisms of patients with "allergic" and "nonallergic" atopic dermatitis using peripheral blood and skin biopsy samples. Our data suggest that T cells are likely involved in the pathogenesis of both forms of atopic dermatitis. Skin T cells equally responded to superantigen, staphylococcal enterotoxin B, and produced interleukin-2, interleukin-5, interleukin-13, and interferon-gamma in both forms of the disease. Interleukin-4, however, was not detectable in the skin biopsies of both atopic dermatitis types and was secreted in very low amounts by T cells cultured from the skin biopsies. Moreover, skin T cells from nonallergic atopic dermatitis patients expressed lower interleukin-5 and interleukin-13 levels compared with allergic atopic dermatitis patients. Accordingly, T cells isolated from skin biopsies of atopic dermatitis, but not from the nonallergic atopic dermatitis, induced high immunoglobulin E production in cocultures with normal B cells that was mediated by interleukin-13. In addition, B cell activation with high CD23 expression was observed in the peripheral blood of atopic dermatitis, but not nonallergic atopic dermatitis patients. These data suggest, although high numbers of T cells are present in lesional skin of both types, a lack of interleukin-13-induced B cell activation and consequent immunoglobulin E production in nonallergic atopic dermatitis.     

Elevated serum levels of soluble CD30 are associated with atopic dermatitis, but not with respiratory atopic disorders and allergic contact dermatitis

Type 2 helper T-cell immune responses can be demonstrated in the human atopic disorders atopic dermatitis and allergic asthma/rhinoconjunctivitis. The CD30 (Ki-1) antigen, originally described on Hodgkin and Reed-Sternberg cells, has recently been proposed as a marker of T cells with potent B-cell helper activity producing IL-5 and γ-IFN, as well as on CD4+ and CD8+ T cells with a Th2 cytokine profile. As a soluble form of CD30 (sCD30) is released by CD30+ cells in vivo , we studied its clinical significance in atopic disorders compared with allergic contact dermatitis and healthy controls. Elevated sCD30 levels were associated with atopic dermatitis ( P  < 0.0001), but not with respiratory atopic disorders or allergic contact dermatitis. sCD30 levels in patients with atopic dermatitis were independent of serum IgE. The particular occurrence of serum sCD30 in patients with atopic dermatitis indicates a special regulatory function of CD30+ cells in this disease.     

Nickel-responding T cells are CD4+ CLA+ CD45RO+ and express chemokine receptors CXCR3, CCR4 and CCR10

BACKGROUND: Whereas T lymphocytes are widely accepted as effector cells determining the pathogenesis of allergic contact dermatitis, contradictory results have been found regarding the roles of different T-cell subsets. The use of various experimental models, involving long-term cultured T-cell lines or clones, may explain these contradictory results. OBJECTIVE: To investigate the involvement of distinct T-cell subsets in patients with nickel contact allergy. METHODS: Different T-cell subsets were directly isolated from peripheral blood mononuclear cells (PBMCs) of nickel-allergic patients, and their proliferative capacity, type-1 or type-2 cytokine secretion [measured by interferon (IFN)-gamma or interleukin (IL)-5 release] and phenotypical marker expression were analysed after stimulation with nickel. RESULTS: Only CD4+ CLA+ CD45RO+ and not CD8+ T cells proliferate and produce both type-1 (IFN-gamma) and type-2 (IL-5) cytokines in response to nickel. Moreover, cells expressing the marker CLA in combination with CD4, CD45RO or CD69 are increased after nickel-specific stimulation. Interestingly, in addition, CD45RA+ CLA+ cells showed an increased frequency after allergen-specific stimulation. Analysis of nickel-reactive T cells for expression of distinct chemokine receptors showed that both proliferative capacity and cytokine production are restricted to subsets expressing CXCR3, CCR4 but not CCR6. Fluorescence-activated cell sorting analysis of chemokine receptors expressed on nickel-stimulated T cells confirmed these results; a subset of T cells expressing CLA and CXCR3, CCR4 and, most importantly, CCR10 increased in response to allergen, while these CLA+ nickel-reactive T cells were all negative for CCR6. CONCLUSIONS: These findings demonstrate that freshly isolated nickel-reactive T cells can be characterized as CD4+ CLA+ memory T cells which express the chemokine receptors CXCR3, CCR4 and CCR10, but not CCR6.     

A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA+CCR4+ lymphocytes into lesional atopic dermatitis skin

Atopic dermatitis is an inflammatory skin disease in which the inflammation is characterized by the influx of lymphocytes into the dermis. It is generally believed that atopic dermatitis is a Th2-type disease, i.e., the T lymphocytes produce interleukin-4, interleukin-5, interleukin-10, and interleukin-13, although it has become evident in recent years that the cytokine profile in the skin changes during the course of the disease towards a Th1-Th2 mixed cytokine profile (interferon-gamma, tumor necrosis factor alpha, and interleukin-2). The lymphocytes that home into the skin express cutaneous lymphocyte-associated antigen, and it has recently been shown that most of the lymphocytes in this population express the chemokine receptor CCR4. CCR4 is the receptor for the CC chemokine TARC (thymus and activation regulated chemokine), and this chemokine is expressed predominantly by keratinocytes in the basal layer of the epidermis of lesional atopic dermatitis skin in mice. In humans, however, it was shown to be expressed in the endothelial cells of the dermis. We have examined the peripheral blood mononuclear cells of atopic dermatitis patients for the expression of cutaneous lymphocyte-associated antigen and CCR4 and compared them with peripheral blood mononuclear cells from normal controls. We found that the proportion of CLA+CCR4+ lymphocytes is upregulated in atopic dermatitis patients. In addition we have examined skin biopsies of lesional and non-lesional skin from atopic dermatitis patients and found that the keratinocytes, but not the endothelial cells, produce TARC in the lesional but not in the nonlesional skin. To gain insight in the stimulatory mechanisms for TARC production in keratinocytes, as previously observed in mice, we cultured HaCaT cells and found that interferon-gamma and tumor necrosis factor alpha work synergistically to induce TARC production. These observations suggest that the induction of TARC production in keratinocytes plays an important role in the late phase skin invasion by CCR4+CLA+ Th2-type lymphocytes in atopic dermatitis.     

Interleukin-4 promotes the expansion of skin-infiltrating lymphocytes from atopic dermatitis in vitro

Functional studies of lymphocytes in atopic dermatitis (AD) have so far focused on peripheral blood mononuclear cells (PBMC), whereas cells at the involved site, the skin, have not been examined. Accordingly, we have developed methods to generate lymphocyte cultures from biopsies of inflammatory skin areas. Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of 6 patients with severe AD and expanded in vitro in the presence of interleukin-2 (IL-2) without additional antigens. After 6-10 d in culture, outgrowth of mononuclear cells from biopsy tissue was observed in all cases. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T-helper/inducer phenotype in SIL populations. Parallel cultures of SIL and PBMC showed an increase and expansion of CD8+ T cells in cultured PBMC, whereas the CD4+ phenotype was predominant in SIL cultures. As indicated by their expression of HLA-DR and CD25 antigens, most of the SIL were activated and the cells mainly expressed T-cell receptors (TCR) composed of alpha and beta chains. Different strategies for expansion of SIL in vitro were examined. High levels of IL-4 (1,000 U/ml) in combination with IL-2 (50 U/ml or 1,000 U/ml) preferentially promoted growth of SIL derived from AD and were much more effective than IL-2 alone. No cells expanded in cultures with IL-4 alone. SIL grown with high concentrations of IL-4 contained a significant proportion of double-positive CD4+8+ cells. No other marked differences were observed in the distribution of T cell subsets in cultures propagated under different conditions for 21 d. Our results demonstrate the feasibility of growing infiltrating T lymphocytes from inflammatory skin of AD patients. The use of high concentrations of IL-2 in combination with high levels of IL-4 allows a large expansion of these cells and thus represents a useful strategy to expand cells for further functional and molecular biologic studies.     

Proliferation of T lymphocytes from atopic dermatitis skin is enhanced upon anti-CD3, reduced upon mitogen and superantigen, and negligible upon tuberculin stimulation

Knowledge about the nature of lymphocytes infiltrating atopic dermatitis skin is restricted to allergen-specific T cells. We investigated the proliferative capacities of T lymphocytes cultured in an antigen-independent way from biopsies of atopic dermatitis skin. When compared with peripheral blood mononuclear cells (PBMC) from healthy donors or atopic dermatitis patients, the skin-homing lymphocytes proliferated more vigorously in response to stimulation with anti-CD3 antibodies (1 microglml), reflecting their high response capacity. When stimulated with phytohemagglutinin (10 microg/ml) or staphylococcal enterotoxin A (0.1 microg/ml) the skin-homing lymphocytes achieved significantly lower proliferation levels than PBMC. In contrast to normal and atopic PBMC the skin-homing lymphocytes did not respond to tuberculin purified protein derivative (10 microg/ml). In the mixed lymphocyte reaction the skin-homing lymphocytes did not stimulate autologous PBMC to proliferate. We conclude that skin-homing lymphocytes have more pronounced immune deviations than PBMC in patients with atopic dermatitis. They represent a valuable approach for further investigating the pathogenesis of the disease.