IL-4- and IL-5-positive T lymphocytes, eosinophils, and mast cells in allergen-induced late-phase cutaneous reactions in atopic subjects

It has previously been shown that cells mRNA(+) for T-H2-type cytokines (IL-4 and IL-5) infiltrate the site of allergen-induced cutaneous late-phase reactions (LPR) in atopic subjects. In this study we have used the same experimental model to identify the cell source of both IL-4 and IL-5 mRNA and protein product. Allergen-induced LPRs were provoked in the skin of atopic individuals and the sites microscopically examined at 6, 24, and 48 hours. Using single in situ hybridization and immunohistochemistry, we first showed that the numbers of IL-4 and IL-5 mRNA and protein product positive cells peaked at 24 hours. This coincided with the magnitude of the LPR. By double in situ hybridization/immunohistochemistry, we then established (in 24-hour biopsy specimens) that the percentage of CD3(+) T lymphocytes, EG2(+) eosinophils, and tryptase-positive mast cells that were either IL-4 or IL-5 mRNA(+) was 19%, 24%, and 5% and 19%, 20%, and 5%, respectively. Conversely, the percentage of EG2(+) and tryptase-positive cells that were IL-4 or IL-5 protein product positive were 62% and 53% and 72% and 29%, respectively. IL-4 and IL-5 protein did not colocalize to CD3(+) cells. CD68(+) macrophages were negative in both in situ hybridization and immunohistochemistry. With eosinophils we obtained direct evidence of time-dependent stimulus-induced IL-4 and IL-5 mRNA transcription by semiquantitative reverse transcription-polymerase chain reaction of cells incubated with either IgG- or sIgA-coated particles in vitro. Taken together, these experiments suggest that eosinophils, mast cells, and T cells all contribute in variable degrees to the expression of IL-4 and IL-5 in human cutaneous LPR. The failure to colocalize IL-4/IL-5 protein (as opposed to mRNA) to CD3(+) cells is attributed to the inability of T lymphocytes to store and concentrate sufficient intracellular amounts of these cytokines to produce positive immunostaining.