Identification and Quantitation of Interferon-

Abstract

Interferon-γ (IFN-γ) produced by lesional T cell clones is critical for the induction into G1 of the cell cycle by psoriatic keratinocyte stem cells; however, direct data demonstrating psoriatic lesional T cell subset IFN-γ expression, and quantitation at a single cell level to calculate in vivo proportions, are lacking. In this study, using flow cytometry of freshly isolated normal and psoriatic lesional T cells from keratome biopsies, we found elevated CD3?, CD4?, and CD8? T cells in all compartments of psoriatic skin, compared with normals. Using Brefeldin A to induce short-term intracellular accumulation of IFN-γ in T cells capable of IFN-γ production, we found that 90% of psoriatic patients have IFN-γ-producing T cells at a greater proportion of their CD3? cells than normals, with a mean of 16% ? 3%, as compared with 4%?2% in normal epidermis (p?0.01). Expressed as density in the tissue, the IFN-γ? CD3? cell number in psoriatic epidermis was 97 ? 22 per mm2 surface area, as compared with 4.4 ? 1.8 per mm2 of normal epidermis (p ? 0.002). Thus, the total number of IFN-γ?CD3? T cells in the skin of a patient with 20% involvement is estimated to be 3.9 ? 108. CD4? and CD8? IFN-γ? T cells were both elevated in psoriatic epidermis (p?0.04 and p?0.008, respectively) relative to normal skin. In the dermis, only 44% of patients demonstrated a higher percentage of IFN-γ-producing T cells than did normals (p ? 0.1), possibly indicating dilution, in some patients, by fresh infiltrating T cells. Interleukin-4 was not found by a combination of flow cytometry, reverse transcriptase-polymerase chain reac- tion, western blot, and immunoprecipitation. In conclu- sion, a significant portion of lesionalTcells in psoriasis are IFN-γ producing, without interleukin-4. The increased numbers of both IFN-γ?CD4? and IFN-γ?CD8? T cells indicate that both CD4? and CD8? IFN-γ? T cells are present in appropriate anatomic locations to sustain the lesional pathology. Key