IMMUNOCYTOCHEMICAL AND FLOW CYTOFLUOROMETRIC DETECTION OF INTRACELLULAR IL-4, IL-5 AND IFN-GAMMA - APPLICATIONS USING BLOOD-DERIVED AND AIRWAY-DERIVED CELLS

We have compared an immunocytochemical and a flow cytofluorimetric met hod to detect intracellular IFN-gamma, IL-4 an IL-5 in T-cell clones, peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage f luid (BALF) cells, Intracellular bound cytokine-specific antibodies we re visualized either with amino-ethyl carbazole (for immunocytochemist ry), or with fluorescent antibodies (for flow cytofluorimetry). The st aining was inhibited with recombinant cytokines and corresponded quali tatively and quantitatively to cytokine levels in the supernatants of T-helper-0 (Th0), Th1 and Th2 clones. In analysing in vitro stimulated cells, sufficient signal in the fluorimetric assay was only obtained after the addition of monensin to the cultures, We then observed a goo d correlation between immunocytochemical (with no monensin added) and the flow cytofluorimetric staining for all three cytokines (PBMC, IFN- gamma and IL-4, rho = 0.9, no IL-5 detectable; clones, IL-5, rho = 0.8 1, all three p < 0.05). However, compared to flow cytometry, a greater percentage of positively stained cells was frequently observed using immunocytochemistry. In BALF cells, the immunocytochemical method was able to detect significant percentages of positive cells without in vi tro stimulation of the cells, in contrast to the flow cytofluorimetric method. In BALF cells from sarcoidosis patients, T-cells were mainly IFN-gamma-positive (immunocytochemically assessed), both with (mean +/ - SEM, 39.7 +/- 9.8%), and without (3.5 +/- 1.3%) in vitro stimulation . In BALF cells from allergic subjects, the immunocytochemical method showed lymphocytes positive for IFN-gamma (40.3 +/- 8.3%), IL-4 (19.1 +/- 0.49) and IL-5 (6.1 +/- 3.1). We conclude that both methods can be used to assess the production of IFN-gamma, IL-4 or IL-5 at the singl e-cell level in T-cell clones, PBMC and cells from the BALF. The high sensitivity and the low number of cells required for the immunocytoche mical method indicate that this method can provide detailed informatio n on cytokine production of airway-derived cells in diseases with airw ay inflammation such as sarcoidosis and asthma.