Candida albicans mannan- and protein-induced humoral, cellular and cytokine responses in atopic dermatitis patients

BACKGROUND: The cytokine observed most often in atopic dermatitis (AD) is IL-4, but a role for IL-5 and IFN-gamma in the late and delayed phase reactions has been suggested. In AD with head, neck and shoulder distribution, hypersensitivity to saprophytic yeasts is an important pathogenetic factor. The yeast allergens include both the mannan polysaccharides and the proteins. Mannans are major cross-reacting allergens likely to be involved in the pathogenesis of AD. OBJECTIVE: To characterize the humoral, lymphoproliferative and cytokine (IL-2, 4, 5 and IFN-gamma) responses of peripheral blood mononuclear cells (PBMCs) induced by Candida albicans mannan and protein antigens in AD. METHODS: Fifteen AD patients and seven healthy controls were included. Ficoll-isolated PBMCs were stimulated by PHA and laboratory-generated mannan and protein extracts of C. albicans. Lymphocyte proliferation was measured and cytokine production was studied by ELISA. The antigen-specific IgG and IgE antibodies were analysed by ELISA and nitrocellulose RAST. RESULTS: In AD mannan (P < 0.005) and protein (P < 0.002), specific IgE levels were higher than in healthy controls. Both mannan and protein-specific lymphoproliferations (both: P < 0.02) were higher in AD than in healthy controls. Mannan, but not protein, induced long lasting IL-2 and IL-4 productions from 24 h lasting up to 66-96 h and IL-5 and IFN-gamma productions with elevated levels at 66 and 96 h. The mannan-induced IL-2 (P = 0.015) and IFN-gamma (P < 0.005) were increased in AD as compared with healthy controls. Significant correlations were seen between the protein-induced proliferation responses and both serum total IgE (r = 0.59, P < 0.01) and protein-specific IgE (r = 0.65, P < 0.005). The mannan-induced IL-2 responses correlated with the specific IgE (r = 0.62, P < 0.01) and proliferation (r = 0.51, P < 0.02) and S-IgE level (r = 0.71, P < 0. 002). Mannan-induced IL-4 and IFN-gamma productions also correlated (r = 0.43, P < 0.05). CONCLUSIONS: C. albicans mannan induced elevated IL-2 and IFN-gamma responses in AD patients. The correlations of the cytokine responses with mannan-induced IgE and proliferation responses suggest that C. albicans mannan induced TH1 type cytokine responses are involved in AD.     

Inflammatory cell numbers and cytokine expression in atopic dermatitis after topical pimecrolimus treatment

BACKGROUND: In several clinical trials the topical application of pimecrolimus was shown to be effective in the treatment of atopic dermatitis (AD). By targeting calcineurin-dependent signaling pathways, pimecrolimus controls cytokine gene expression. The purpose of this study was to investigate the effect of pimecrolimus on the inflammatory infiltrate and cytokine expression pattern in AD upon topical therapy. METHODS: From 10 patients with acute AD, skin biopsies as well as immunophenotype and cytokine production of peripheral blood mononuclear cells (PBMC) were examined before and 3 weeks after therapy. RESULTS: The clinical improvement was associated with a marked regression of histopathological features. In particular, the density of the inflammatory infiltrate mostly containing lymphocytes and eosinophils declined. By double immunofluorescent staining, a reduced expression of the T helper (Th) 2 cytokines interleukin (IL)-5, IL-10, and IL-13 in both CD4+ and CD8+ T cells was demonstrated after therapy. Pimecrolimus therapy was also associated with a reduced expression of the Th1 cytokine interferon (IFN)-gamma. Interestingly, the numbers of epidermal CD1a+ dendritic cells increased following treatment. In the peripheral blood, a decrease of lymphocytes and eosinophils was noticed, but the distribution of lymphocyte subpopulations and their capacity of cytokine production did not change. CONCLUSIONS: Topical pimecrolimus exhibits anti-inflammatory effects in AD by reducing the inflammatory cell infiltrate and cytokine expression in the dermis.     

Interleukin-13: Targeting an underestimated cytokine in atopic dermatitis

Atopic dermatitis (AD) is a common inflammatory skin condition that has traditionally been considered a paradigmatic type 2 immunity (T2)-driven disease. Interleukin (IL)-4 and IL-13 are both pivotal cytokines involved in the generation of allergic diseases. Currently, besides dupilumab, which blocks the binding of both cytokines to their receptors, a number of new pharmacologic entities have been designed to target both T2 cytokines and/or their receptors and/or receptor-associated signal transduction machinery such as Janus kinases. Recently, IL-13 has been suggested to be the key T2 cytokine driving inflammation in the periphery, while IL-4 may merely have a central effect. There is increasing evidence that this concept holds true for the inflammatory reaction underlying AD, where IL-13 is overexpressed locally and has a significant impact on skin biology, including the recruitment of inflammatory cells, the alteration of the skin microbiome, and the decrease in the epidermal barrier function. This review provides an update on the role of IL-13 in AD and discusses the different strategies aimed at interfering with its biologic activity as well as their potential in a precision medicine approach in the management of AD.     

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8⁻ T cells, IL-2 and interferon-gamma (IFN-γ) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8⁺ T cells IL-2 was optimally induced after 10 h and IFN-γ after 6 h. The levels of IL-2 and IFN-γ in CD8⁺ and CD8⁻ T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2⁺ (range 9.7–41.3) and CD3/IFN-γ⁺ cells (10.1–44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3⁺/CD8⁺ peripheral blood T cells expressing IFN-γ and an increased percentage of CD3⁺/CD8⁻ T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.     

PPARgamma expression profile and its cytokine driven regulation in atopic dermatitis

BACKGROUND: Recent studies point to the pathophysiological role of the peroxisome proliferators-activated receptor gamma (PPARgamma) in the inflammatory immune response. We have showed that activation of PPARgamma by specific ligands attenuates the allergic immune response via monocytes and lymphocytes. The objective of this study was to analyse the PPARgamma expression and its regulation via inflammatory cytokines. METHODS: We examined the PPARgamma expression in the lesional and nonlesional skin of atopic patients by immunohistochemistry. The expression patterns of PPARgamma mRNA and its isoforms were investigated in peripheral mononuclear blood cells of atopic and nonatopic donors and in cytokine-stimulated populations by quantitative real-time RT-PCR. RESULTS: Our data show an increased PPARgamma expression in lesional skin from atopic dermatitis patients. The analysis of PPARgamma mRNA reveals a significantly up-regulated expression of PPARgamma1 but not of PPARgamma2 in monocytes and CD4(+) T-cells from atopic dermatitis patients. Furthermore, we demonstrate that Th-cytokines, like IL-4, IL-13 and IFNgamma, which regulate the biphasic atopic immune response, directly regulate the expression of PPARgamma1. CONCLUSION: Taken together, these data demonstrate that PPARgamma isoforms are differently expressed and regulated by the local cytokine-milieu. Whether the increased expression of the PPARgamma1 receptor may be beneficial or not for a PPARgamma ligand-based treatment of atopic dermatitis, is currently under investigation.     

Peripheral blood mononuclear cells from IgE- and non-IgE-associated allergic atopic eczema/dermatitis syndrome (AEDS) demonstrate increased capacity of generating interleukin-13 but differ in their potential of synthesizing interferon-gamma

BACKGROUND: A subgroup of patients with allergic atopic eczema/dermatitis syndrome (AEDS) are known to have normal total and specific IgE levels and negative skin prick tests towards common environmental allergens. This form of the disease has been termed non-IgE-associated allergic AEDS. Although allergic mechanisms appear to be important, the pathogenesis of both IgE- and non-IgE-associated forms of the disease is unknown. METHODS: We have compared the cytokine production pattern of peripheral blood mononuclear cells (PBMC) from IgE-associated AEDS, non-IgE-associated AEDS, and normal control individuals. PBMC were stimulated with anti-CD3 and/or anti-CD28 monoclonal antibodies (mAb) and cytokine production was measured by immunoassays in supernatants of 24-h cultures. RESULTS: Compared to healthy subjects and non-IgE-associated AEDS patients, stimulated PBMC from IgE-associated AEDS patients produced less interferon (IFN)-gamma. However, stimulated PBMC from both IgE-associated AEDS and non-IgE-associated AEDS patients produced more interleukin (IL)-13 than PBMC from control individuals. Moreover, IL-5 production was significantly increased in non-IgE-associated AEDS but not in IgE-associated AEDS patients. CONCLUSIONS: The underlying mechanism leading to increased differentiation of T helper (Th) 2 cells may involve a deficient capacity in producing IFN-gamma in IgE-associated AEDS but not in non-IgE-associated AEDS patients. IL-13 may be a key cytokine in the pathogenesis of both allergic forms of AEDS.     

CD30 expression on circulating memory CD4+ T cells as a Th2-dominated situation in patients with atopic dermatitis

Th2 clones have been reported to express CD30 preferentially, but whether T cells producing Th2-type cytokines may favor CD30 expression in the in vivo state remains unknown. We investigated the expression of CD30 on circulating T cells in atopic dermatitis (AD) as a Th2-dominated disorder. Peripheral blood mononuclear cells were prepared from 51 AD patients and 14 nonatopic controls, and their phenotypes were analyzed with flow cytometry. Cytokine production by stimulated CD4+ T cells was also assessed by the single-cell-staining method. Flow cytometric analysis clearly revealed that CD30+ T cells were identifiable in the blood of AD patients with greater frequency compared to controls. The important finding was that CD30 expression was restricted to a small but substantial population of memory (CD45RO+) CD4+ T cells, but not CD8+ ones. In AD patients, it was demonstrated that the percentages of CD30+ cells within CD45RO+ CD4+ T cells correlated well with the disease severity, serum IgE levels, peripheral eosinophil counts, and tendency toward Th2-dominant cytokine pattern as determined by the ratio of interleukin-4 to interferon-gamma production. This study suggests that CD30 expression in circulating T cells might serve as an in vivo marker for the Th2-dominated condition.     

Impaired TLR-2 expression and TLR-2-mediated cytokine secretion in macrophages from patients with atopic dermatitis

Background:  In many patients with atopic dermatitis (AD), the disease is complicated by their enhanced susceptibility to bacterial skin infections, especially with Staphylococcus aureus . The pattern recognition receptor toll-like receptor (TLR)-2 recognizes components of S. aureus , for example, lipoteichoic acid (LTA) and peptidoglycan (PGN) and, therefore, might be crucial in the pathogenesis and flare-ups of AD.
Objective:  To investigate TLR-2 expression and cytokine secretion in macrophages from patients with AD compared to healthy controls upon TLR-2 stimulation with PGN, LTA and Pam3Cys.
Methods:  Macrophages were cultivated from highly purified peripheral blood monocytes of AD patients and nonatopic healthy controls and stimulated with PGN, LTA and Pam3Cys in a time and dose–dependent manner. Afterwards, TLR-2 expression and cytokine secretion were measured on protein and mRNA level. TLR-1 and TLR-6 expression were investigated on the mRNA level. Immunohistochemical stainings from punch biopsies were performed to investigate TLR-2 expression in skin macrophages .
Results:  We could clearly show that macrophages from patients with AD expressed significantly less TLR-2, whereas the expression pattern of TLR-1 and TLR-6 were not altered. Macrophages had a reduced capacity to produce pro-inflammatory cytokines such as IL-6, IL-8 and IL-1β after stimulation with TLR-2 ligands.
Conclusion:  Our findings clearly show an impaired TLR-2 expression and functional differences of TLR-2-mediated effects on macrophages of AD patients compared to healthy controls which might contribute to the enhanced susceptibility to skin infections with S. aureus in AD.     

In vivo relevance of CD30 in atopic dermatitis

CD30 expression was evaluated by immunohistochemistry in lesional skin biopsies of eight patients with active atopic dermatitis (AD) and three patients with allergic contact (nickel-induced) dermatitis (ACD). CD30 expression was also assessed in a large panel of CD4 + and CDS + T-cell clones generated from the skin biopsies of four patients with AD. Finally, the levels of soluble CD30 (sCD30) were measured in the serum of 41 patients with AD, 19 patients with ACD, and 60 healthy controls. In all specimens of lesional AD skin, where the great majority of infiltrating cells were CD4+ T cells, remarkable numbers of cells were CD30+, whereas virtually no CD30 + cells were found in the skin of patients with ACD. In CD4+ T-cell clones generated from the lesional AD skin, most of which produced both interleukin (IL)-4 and interferon-gamma (IFN-γ) (Th0–like cells) or IL-4 and 1L-5, but not IFN-γ (Th2–like cells), CD30 expression directly correlated with the ability to produce IL-4 and IL-5, but was inversely related to IFN-γ production. High levels of sCD30 (correlated with disease activity: r = 0.618) were detected in the serum of most AD patients, whereas there was no increase of sCD30 levels in the serum of patients with ACD. These data support the view that Th0/Th2–type responses predominate in the skin of patients with AD and suggest that the presence of CD30 + T cells in tissues and/or increased levels of sCD30 in biologic fluids are indicative of Th2–dominated responses.     

The skin fungus-induced Th1- and Th2-related cytokine, chemokine and prostaglandin E2 production in peripheral blood mononuclear cells from patients with atopic dermatitis and psoriasis vulgaris

BACKGROUND: It is suggested that skin fungi may be involved in the development of atopic dermatitis (AD) and psoriasis vulgaris (PV). OBJECTIVE: We studied skin fungus-induced Th1- or Th2-related cytokine, chemokine and prostaglandin E2 (PGE2) secretion in peripheral blood mononuclear cells (PBMC) from patients with AD and PV and normal subjects. METHODS: PBMC were cultured with the extracts of Malassezia furfur (MF), Candida albicans (CA) and Trichophyton rubrum (TR). The cytokine, chemokine and PGE2 amounts in the supernatants were measured by enzyme-linked immunosorbent assays. RESULTS: MF induced IL-4 and macrophage-derived chemokine (MDC) secretion in AD patients, while induced IFN-gamma and interferon-inducible protein of 10 kDa (IP-10) secretion in PV patients, however, did not induce either secretion in normal subjects. CA induced IL-4, MDC, IFN-gamma and IP-10 secretion in AD and PV patients and normal subjects. In AD patients, the magnitude of IL-4 and MDC responses to CA was higher than that to MF. Compared with PV patients and normal subjects, the magnitude of IL-4 and MDC responses to CA was higher while that of IFN-gamma and IP-10 responses to CA was lower in AD patients. TR induced moderate IL-4 and MDC secretion only in AD patients. The three fungi induced higher levels of PGE2 secretion in AD patients than in PV patients and normal subjects. Cyclooxygenase-2 inhibitor NS-398 suppressed PGE2 responses to MF, CA and TR, and partially suppressed IL-4 and MDC responses to MF, CA and TR, while enhanced IFN-gamma and IP-10 responses to CA in AD patients, and these effects of NS-398 were reversed by cyclic AMP analogue. CONCLUSION: AD patients manifest Th2-skewed responses to MF, CA and TR, which may be partially attributable to the enhanced PGE2 responses to these fungi. PV patients manifest Th1-skewed responses to MF.     

Different cytokine production and activation marker profiles in circulating cutaneous-lymphocyte-associated antigen+ T cells from patients with acute or chronic atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is an inflammatory skin disease whose lesions can have two stages: acute and chronic. In skin biopsies a biphasic pattern of cytokine expression has been shown, Th2 in acute lesions and Th1 in chronic AD lesions. OBJECTIVE: We investigated the expression of an activation marker and a homing receptor, as well as cytokine production, in different peripheral blood T cell subpopulations from AD patients with chronic (Group A) and acute lesions (Group B) and controls. METHODS: We evaluated 26 adult AD patients (12 Group A, 14 Group B) and 14 non-atopic controls. IgE was measured by immunoassay. CD4, CD8, cutaneous-lymphocyte-associated antigen (CLA) and human leucocyte antigen (HLA)-DR expression, and cytokine production (IL-2, IL-13, IFN-gamma, TNF-alpha, IL-10, IL-4) were analysed in mononuclear cells by flow cytometry. RESULTS: In Group B there was a significant increase in eosinophil levels and a non-significant increase in IgE. In Group A we found an increase in CLA(+)CD4(+) cells (8.19+/-1.84) compared with controls (4.83+/-0.53) (P<0.05) and CD4(+)HLA-DR(+) cells in the CLA(+) subpopulation (45.54+/-15.40) compared with controls (30.49+/-6.07) (P<0.05). In the CLA(+)CD4(+) subpopulation, there was a significant increase in IL-4, IL-13 and TNF-alpha production in Group B (12.46+/-7.7, 11.26+/-5.97, 43.92+/-15.55) compared with controls (5.34+/-3.50, 4.54+/-1.78, 19.29+/-9.97) with no differences in Group A. CONCLUSION: Greater immunological differences were detected in peripheral blood from patients with acute compared with chronic lesions, especially in the circulating T cell-subset with skin tropism that preferentially responded to cutaneous allergens. This is the first demonstration of phenotypic changes in circulating CLA(+) T cells between AD patients with acute and chronic lesions.     

CD4(+)IL-13(+) cells in peripheral blood well correlates with the severity of atopic dermatitis in children

BACKGROUND: In atopic dermatitis (AD) a Th1/Th2 imbalance has been reported, and interleukin (IL)-13 seems to play a pivotal role in the inflammatory network. We tried to assess the correlation between the immunological marker CD4(+)IL-13(+) and the clinical phase of extrinsic AD in children. METHODS: Twenty children with AD were studied. Assessed parameters were: clinical severity (SCORAD index), total serum immunoglobulin E (IgE), blood eosinophil count, and percentage of CD4(+)IFNgamma(+), CD4(+)IL-4(+), CD4(+)IL-13(+) T cells. Determinations were carried out in the acute phase and after clinical remission were achieved. Ten nonatopic-matched children served as controls. RESULTS: At baseline, AD was mild in 25%, moderate in 50% and severe in 25% of children. In the acute phase a significant relationship between the eosinophil count and the SCORAD index was found (P = 0.0001). Blood CD4(+)IL-4(+) were significantly higher in the AD group (median 17.0, range: 13.7-21.4) than in controls (12.6, 6.4-17.2, P < 0.0001). CD4(+)IL-13(+) cells in the AD group well correlated (P = 0.0007) with SCORAD index. At remission, a significant correlation between SCORAD index and eosinophil count was found (P < 0.03) and the percentage of CD4(+)IL-13(+) cells globally decreased (P < 0.0001), while no difference was found among SCORAD classes. CONCLUSION: This study confirms the Th2 profile predominance in the peripheral blood of children with AD, and evidences close relationship between the number of CD4(+)IL-13(+) T cells and the disease's severity.     

Time-course studies of immediate and delayed immune reactivity in patients with atopic dermatitis treated with herbal drugs

We followed a number of in vitro parameters in nine adult patients with atopic dermatitis for a period of 3 weeks during which the patients took herbal drugs that are claimed to improve chronic eczema. The following investigations were performed weekly: blood leucocytes, circulating eosinophils, serum IgE, mitogen responsiveness of lymphocytes, interleukin 1 release, soluble interleukin 2 receptor levels in serum, and in vitro histamine release from basophils in blood. The study demonstrated increased levels of IgE in six patients, increased levels of soluble interleukin 2 receptor in six patients, and a significant correlation between the amount of IgE in serum and the maximal release of histamine from basophils in blood. In this open study five patients experienced a clinical worsening of their disease during the intake of herbal medicine. All other parameters were within normal levels. There was no significant change in the in vitro parameters during the observation period.     

Vaccinia Virus Pathogenicity in Atopic Dermatitis is Caused by Allergen-Induced Immune Response that Prevents the Antiviral Cellular and Humoral Immunity

Atopic dermatitis (AD) serves as a contraindication for the immunization of AD patients with a live vaccinia virus (VV) vaccine. The antiallergen IgE interacts with the Fc receptors (FcRI) on dendritic cell (DC) membranes and with allergen molecules. The immunological events that lead to AD disease, the activation of the T-helper 2 (Th2) immune response, the synthesis of the cytokines IL-4, IL-5, IL-13, and the inhibition of the T-helper 1 (Th1) damage the capacity of the host to develop anti-VV cytotoxic cells (CTLs). In the presence of Th2-derived cytokine IL-4 in the AD skin and the synthesis of VV proteins that interfere recruitment of DCs by host cytokines, the VV can cause a generalized infection.Conceptually, new VV recombinants may be needed for human immunization. Such VV recombinants should lack the genes that interfere with the host immune system and express a mutated human IL-4 cytokine gene that will prevent negative regulatory mechanisms. Such improved VV recombinants may be used to express genes from pathogenic viruses.