Immunocytochemical and flow cytofluorimetric detection of intracellular IL-4, IL-5 and IFN-γ: applications using blood- and airway-derived cells

Abstract

We have compared an immunocytochemical and a flow cytofluorimetric method to detect intracellular IFN-γ, IL-4 and IL-5 in T-cell clones, peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage fluid (BALF) cells. Intracellular bound cytokine-specific antibodies were visualized either with amino-ethyl carbazole (for immunocytochemistry), or with fluorescent antibodies (for flow cytofluorimetry). The staining was inhibited with recombinant cytokines and corresponded qualitatively 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 good correlation between immunocytochemical (with no monensin added) and the flow cytofluorimetric staining for all three cytokines (PBMC, IFN-γ and IL-4, ρ=0.9, no IL-5 detectable; clones, IL-5, ρ=0.81, 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 vitro stimulation of the cells, in contrast to the flow cytofluorimetric method. In BALF cells from sarcoidosis patients, T-cells were mainly IFN-γ-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-γ (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-γ, IL-4 or IL-5 at the single-cell level in T-cell clones, PBMC and cells from the BALF. The high sensitivity and the low number of cells required for the immunocytochemical method indicate that this method can provide detailed information on cytokine production of airway-derived cells in diseases with airway inflammation such as sarcoidosis and asthma.