Ralph Giorno

Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis.

Recent studies have suggested that T cells play a critical role in the pathogenesis of psoriasis. Guttate psoriasis is a well-defined form of psoriasis frequently associated with streptococcal throat infection. This study tested the hypothesis that T cells in acute guttate psoriasis skin lesions may be activated by streptococcal superantigens. Peripheral blood as well as lesional and perilesional skin biopsies were analyzed for T cell receptor V beta repertoire using monoclonal antibodies against 10 different V beta families. Skin biopsies from all patients with acute guttate psoriasis, but not skin biopsies from patients with acute atopic dermatitis or inflammatory skin lesions induced in normal subjects with sodium lauryl sulfate, demonstrated selective accumulation of V beta 2+ T cells (P < 0.05). The expansion of V beta 2+ T cells occurred in both the CD4+ and the CD8+ T cell subsets. Sequence analysis of T cell receptor beta chain genes of V beta 2-expressing T cells from skin biopsies of patients with guttate psoriasis showed extensive junctional region diversity that is more compatible with a superantigen rather than a conventional (nominal) antigen-driven T cell response. All streptococcal isolates from patients with guttate psoriasis secreted streptococcal pyrogenic exotoxin C, a superantigen known to stimulate marked V beta 2+ T cell expansion. These data support the concept that acute guttate psoriasis is associated with superantigenic stimulation of T cells triggered by streptococcal superantigen(s).

Epidermal HLA-DR and the enhancement of cutaneous reactivity to superantigenic toxins in psoriasis

Streptococcal and staphylococcal superantigens (SAgs) have been implicated in the pathogenesis of inflammatory skin diseases, but the mechanisms by which these toxins act are unknown. The present study assessed the ability of nanogram quantities of topically applied purified toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin type B, and streptococcal pyrogenic enterotoxin types A and C to induce inflammatory reactions in clinically uninvolved skin of normal controls and subjects with psoriasis, atopic dermatitis, and lichen planus. These SAgs triggered a significantly greater inflammatory skin response in psoriatics than in normal control subjects or in subjects with atopic dermatitis or lichen planus. Surprisingly, skin biopsies did not exhibit the T-cell receptor Vbeta stimulatory properties predicted for SAg-induced skin reactions. By 6 hours after patch testing with SAgs, TNF-alpha mRNA had increased in the epidermis (but not the dermis) in biopsies from psoriatics, compared with controls. Immunohistochemical studies revealed significantly higher HLA-DR expression in keratinocytes from psoriatics than from controls. However, a mutant TSST-1 protein that fails to bind HLA-DR did not elicit an inflammatory skin reaction. These results indicate that keratinocyte expression of HLA-DR enhances inflammatory skin responses to SAgs. They may also account for previous studies failing to demonstrate selective expansion of T-cell receptor Vbetas in psoriatics colonized with SAg-producing Staphylococcus aureus, and they identify a novel T cell-independent mechanism by which SAgs contribute to the pathogenesis of inflammatory skin diseases.

Simultaneous in situ detection of IgE receptors and cytoplasmic granules in murine cutaneous mast cells

A method is described for the simultaneous in situ detection of surface receptors and cytoplasmic granules in mast cells of frozen sections of mouse skin. Surface IgE receptors are detected after saturation of the receptors with a murine monoclonal antibody of IgE isotype. The latter is subsequently detected by monospecific rabbit anti-mouse IgE (purified on protein A-Sepharose) followed by FITC-conjugated goat anti-rabbit IgG. Cytoplasmic granules are localized by staining with TRITC-avidin conjugate. Normal cutaneous mast cells show green surface fluorescence and red intracellular granules. The method is specific for mast cells; other cells with Fc receptors for IgE are not seen. This method should be useful in the study of situations in which mast cells may have become degranulated.

Mast cell immunohistochemistry: non-immunological immunostaining mediated by non-specific F(ab′)2-mast cell secretory granule interaction

SummaryDuring investigations of murine and human mast cell immunoreactivity with potential anti-interleukin-4 antibodies, non-specific, non-immunological labelling of mouse and human mast cells became apparent. Non-specific, non-immunological labelling was identified by (i) immunolabelling of mast cells when using control isotype primary antibodies, (ii) ability of conjugated secondary antibodies to label mast cells without prior mast cell exposure to a primary antibody, (iii) extinction of the non-specific labelling and retention of specific labelling when the pH of the diluting and washing buffers is shifted from pH 7.2 to pH 6.0, and (iv) reduction/extinction of the labelling when the antibodies are pre-incubated with soluble heparin prior to immunostaining. The site of the reactivity on the electron microscope level was shown to be confined to the mast cell secretory granules. The results of this study support the hypothesis that non-specific labelling of mast cells results from an ionic interaction between the F(ab′)2 segments of antibodies and the heparin constituent of the mast cell secretory granules. This study points out the necessity of stringent controls when using immunohistochemistry to determine mast cell reactivity to various antibodies.

Analysis of Macrophages, Activated Cells and T Cell Subsets in Inflammatory Myopathies using Monoclonal Antibodies

The availability of monoclonal antibodies which react with lymphocytes and cells of the MPS permits the construction of panels of reagents for immunophenotypic analysis of infiltrating cells in IM. We used immunohistochemical methods to immunophenotype inflammatory cells in muscle biopsies of patients with PM, DM and IBM. The cases of PM and IBM contained an intense MPS cell infiltrate in perivascular and endomysial areas, closely surrounding muscle fibers and occasionally included within the muscle fiber itself; an infiltrate of Leu-6+ cells with a morphology consistent with MPS cells in perivascular and endomysial distribution and occasionally closely apposed to muscle fiber membranes; lymphocytes and MPS cells expressing the IL2-R, and approximately equal numbers of T cells of helper-inducer and suppressor-cytotoxic phenotypes. DM cases had fewer MPS cells and relatively more B cells than T cells.