Double staining of intracellular cytokine proteins and T-lymphocyte subsets. Evaluation of the method in blood and bronchoalveolar lavage fluid

An immunocytochemical staining method has been developed for simultaneous s taining of both cell surface markers (CD4 and CD8) and intracellular cytoki ne proteins IFN-gamma, IL-4 and IL-5. Cell surface molecules were visualize d with alkaline phosphatase, which was developed by Fast Blue BB. Intracell ular cytokine proteins were detected by amino-ethyl carbazole. We applied t his technique to T cells from T-cell lines and T-cell clones, peripheral bl ood mononuclear cells and broncho-alveolar lavage fluid cells. Cells were u sed either unstimulated or stimulated for 4 h with 1 ng/ml PMA and 1 mu g/m l ionomycin, which proved to be an optimal stimulus taking cytokine stainin g, cell recovery and cell viability into account. We studied peripheral blo od mononuclear cells from healthy subjects and found that without in vitro stimulation on average 0.4% of the cells were IFN-gamma positive cells. In unstimulated broncho-alveolar lavage fluid cells of the 2 allergic asthmati c subjects studied so far we found higher numbers of cytokine-positive cell s (up to 22% of the lymphocytes being IL-4(+) cells). By in vitro stimulati on, the numbers of cytokine-positive peripheral blood mononuclear cells fro m the healthy subjects were increased to maximally 5% IFN-gamma(+) cells. I n stimulated lavage fluid cells from allergic asthmatic subjects maximally 34% of the lymphocytes became IFN-gamma(+). We conclude that this method al lows detection of intracellular cytokine proteins in both CD4(+) and CD8(+) T cells without the need for stimulating the cells in vitro. In vitro stim ulation may change the cytokine profile detected.