sq-23377 and Dermatitis--Atopic

The role of the type-2 T helper (Th2) cell-mediated immune response in the immunopathogenesis of atopic dermatitis (AD) is well documented. Whether polarized immunoresponse is confined to antigen-specific T cells or is distributed among all T cell subsets is still controversial. We investigated frequencies of interleukin-2 (IL-2), IL-4, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) producing CD3(+) and CD4(+) T cells in peripheral blood from children with atopic dermatitis and healthy subjects with and without in vitro stimulation. Children with severe AD had a significantly lower percentage of CD4(+) T cells spontaneously expressing IL-4 compared with healthy controls (p <0.01). Polyclonal stimulation significantly increased cytokine production in both AD patients and healthy individuals. Frequencies of CD3(+) and CD4(+) producing IL-2, IL-4, IFN-gamma, and TNF-alpha after in vitro stimulation with phorbol-12-myristate 13-acetate (PMA) + ionomycin were comparable in the AD and control groups. In response to PMA/ionomycin, children with AD and asthma symptoms had a significantly lower percentage of CD3(+) T cells producing TNF-alpha. We failed to demonstrate evidence of an imbalance with respect to type-2 cytokine productions in children with AD. Comparable induction of Th1 and Th2 cytokines in polyclonally stimulated peripheral CD3(+) and CD4(+)T cells from AD patients and controls puts into question the polarized Th2 immune response as a general characteristic of T cells in children with atopic dermatitis.

Topics: Adolescent; Asthma; CD3 Complex; CD4-Positive T-Lymphocytes; Child; Child, Preschool; Cytokines; Dermatitis, Atopic; Flow Cytometry; Humans; Ionomycin; Lymphocyte Count; Phorbol Esters; Severity of Illness Index; Statistics, Nonparametric; T-Lymphocytes

Imiquimod (IMQ), an activator of Toll-like receptor-7 (TLR-7), induces by several routes a profound anti-viral and anti-tumor effect in vivo. Physiologically, the immune system is using perforin-containing granules of cytotoxic T lymphocytes (CTL) towards the same biological purpose. This functional synergism prompted our current experiments addressing the question whether IMQ may influence perforin-release and/or induce perforin in CTLs in vitro. In peripheral lymphocytes of healthy and diseased subjects, IMQ induced a significant increase of perforin(+) CTLs within 12h in all experiments performed. This effect was most pronounced in CTLs of patients suffering from atopic dermatitis, a model disorder for subnormal perforin expression: as compared to perforin(+) CTLs detected at time point zero (100%), up to 270% of perforin(+) CTLs were induced by 2.5 microg/ml [corrected] IMQ. Perforin release from peripheral blood CTLs after PMA/ionomycin-stimulation was not influenced significantly by IMQ. Thus, the biological activity of IMQ appears to exceed its previously known functions, inasmuch as it boosts up significantly the perforin-granule system.

Topics: Adjuvants, Immunologic; Adult; Aminoquinolines; Antineoplastic Agents; Antiviral Agents; Cells, Cultured; Cytoplasmic Granules; Dermatitis, Atopic; Flow Cytometry; Humans; Imiquimod; Ionomycin; Membrane Glycoproteins; Perforin; Pore Forming Cytotoxic Proteins; Receptors, Cell Surface; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Toll-Like Receptor 7; Toll-Like Receptors

As a plasma membrane pore-forming protein, perforin is essential for T-cell cytotoxicity mediated by lytic granules. Recent studies on the immune system of perforin knockout mice demonstrated striking similarities to the immunopathology of atopic diseases.. We sought to investigate the perforin system of atopic patients.. Monoclonal antibodies were used to characterize perforin-positive PBMCs of patients with exacerbated atopic dermatitis (AD) and asymptomatic rhinoconjunctivitis allergica (RCA) by means of immunoflow cytometry. In addition, a perforin release assay was developed to quantify the velocity of ionomycin and phorbol 12-myristate 13-acetate-induced secretion of lytic granules.. In atopic patients significantly fewer lymphocytes contained perforin-positive lytic granules compared with those of healthy control subjects (patients with AD: 14% +/- 5%, n = 13, P <.0001; patients with RCA: 24% +/- 5%, n = 9, P <.01; healthy control subjects: 33% +/- 11%, n = 13). Of all CD8(hi+) cytotoxic T lymphocytes (CTLs), only 18% +/- 9% and 17% +/- 12% were perforin-positive in patients with AD and RCA, respectively, compared with 44% +/- 13% in control subjects (P <.0001). In addition, perforin-positive CD8(hi+) CTLs of atopic patients released their perforin twice as fast and more completely than control CTLs. This means that 50% of initially perforin-positive CD8(hi+) CTLs from patients with AD and RCA released their perforin completely within 32 +/- 16 and 36 +/- 19 minutes, respectively, and an over 85% release was reached within 113 +/- 41 and 118 +/- 60 minutes, respectively. In CTLs of healthy control subjects, however, it took 64 +/- 40 minutes to achieve a 50% release of lytic granules, and an 85% depletion was not reached in 60% of healthy control subjects, even after 180 minutes.. The perforin hyperreleasability explains, at least in part, the decreased percentage of perforin-positive CD8(hi+) CTLs in atopic patients. These distortions in the system of lytic granules of atopic patients may contribute to the functional defects observed in T-cell cytotoxicity in vivo and in vitro in patients with AD and RCA.

Topics: Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; Antigens, Neoplasm; CD4-CD8 Ratio; Conjunctivitis, Allergic; Cytoplasmic Granules; Cytotoxicity, Immunologic; Dermatitis, Atopic; Flow Cytometry; Humans; Hypersensitivity, Immediate; Immunoglobulin E; Ionomycin; Killer Cells, Natural; Membrane Glycoproteins; Perforin; Pore Forming Cytotoxic Proteins; Rhinitis, Allergic, Perennial; Rhinitis, Allergic, Seasonal; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Th1 Cells; Th2 Cells; Time Factors

Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of patients with hyperimmunoglobulin E (IgE) atopic dermatitis (AD) and expanded in vitro in the presence of IL-2 in combination with IL-4. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T helper/inducer phenotype in SIL populations. In 3H-thymidine incorporation assays, SIL showed proliferation in response to IL-2, IL-3, IL-4, ionomycin (Io) + 12-o-tetradecanoyl-phorbol-13-acetate (TPA) and OKT3 + TPA. OKT4 with and without TPA did not induce proliferation. Tumour necrosis factor alpha (TNF-alpha) did not block proliferative responses of SIL to IL-2 and IL-4. Cultured SIL showed no cytotoxic activity against K562 and Jurkat target cells. Expanded skin-derived T cells were tested for their capacity to secrete several cytokines in vitro. SIL secreted significant amounts of IL-4, GM-CSF and TNF-alpha upon stimulation with mitogens but failed to secrete IFN-gamma. Io in combination with phorbol-ester induced the secretion of larger amounts of IL-4, GM-CSF, TNF-alpha and low amounts of IFN-gamma. The data indicate that SIL derived from AD lesions were defective in their capacity to secrete IFN-gamma but were enriched in T cells capable of producing IL-4 upon stimulation. The results support the possibility of a predominant 'TH2-like' cell-mediated immune response in lesional skin of AD patients.

Topics: Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; CD3 Complex; CD4 Antigens; CD56 Antigen; CD8 Antigens; Cell Division; Dermatitis, Atopic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunophenotyping; Interferon-gamma; Interleukin-2; Interleukin-3; Interleukin-4; Ionomycin; Lymphocytes; Receptors, Antigen, T-Cell; Receptors, Antigen, T-Cell, gamma-delta; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha