A rapid measuring technique for allergen-induced IL2 responsiveness of lymphocytes by the propidium iodide-staining method. Detection of the etiological antigen in patients with allergic diseases]

A method for rapidly measuring the interleukin 2 (IL-2)-responsiveness of allergen-stimulated lymphocytes has been newly developed using propidium iodide (PI) staining and drawing ink quenching of the fluid medium fluorescence. There was a linear correlation between the number of PI-stained cells and the fluorescence intensity. The background was less than 5 percent. This fluorochromasia assay reflected the cell number for the quantitative measurement of lymphocyte proliferation. Antigen-activated patient cells added to IL2, showed greater increase in 3H-TdR uptake than IL-2-untreated cells, and were capable of acquiring IL2 responsiveness. Increased numbers of the cells were observed by both the PI- and trypan blue-staining methods. In contrast, unstimulated cells also showed increased 3H-TdR uptake response without increased cell numbers on stimulation with r-IL2 by a 6.8 to 19.3 fold stimulation index compared to the antigen-activated cells. The results indicated that the unstimulated cells in addition with r-IL2 still remained in the initial phase of the DNA-synthetic (S) period through the cell cycle, whereas the activated cells had passed through the post-synthetic gap (C2) and/or cell division at mitosis (M). 3H-TdR uptake of cultured cells usually demonstrates the presence of antigen-sensitized lymphocytes by in vitro proliferative response, and shows an increased stimulation index in 4 or 7 day cultured cells on stimulation with allergens. However, proliferation of some cell populations decreased; furthermore no distinct differences in cell proliferation between allergic and normal lymphocytes were observed, as is usual in this assay, although the present method was capable of measuring increased-IL2 responsiveness of patient lymphocytes. The results indicate that the 3H-TdR uptake method can not be substituted for cell enumeration in the evaluation of the antigen-specificity of induced-IL2 responsiveness of activated cells. Therefore, cell enumeration using the PI-staining method may be preferable in this system. The induced response was observed in lymphocytes from patients with atopic dermatitis, bronchial asthma and/or allergic rhinitis specifically on stimulation with the antigen causing clinical symptoms. The results obtained using the PI-staining method were very similar to those in previous reports where the trypan blue staining method was employed. The present method appears to be capable of rapidly screening etiological antigens for disease and easily monitoring clinical activity.