Flow cytometric detection of cell-associated cytokines in alveolar macrophages

To elucidate the cytokine-producing capacity of alveolar macrophages (AMs), we have introduced a method of flow cytometry combined with saponin treatment to detect the cell-associated cytokines. We studied bronchoalveolar lavage fluid cells from six patients with sarcoidosis (SAR) and six control (CTL) subjects. Cells were either left uncultured, or cultured with and without lipopolysaccharide (LPS), then treated with paraformaldehyde and saponin and analysed for cell-associated interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) by flow cytometry. Cell-associated IL-1β and TNF-α were also analysed by immunoassays. The flow cytometric cytokine values were correlated with the immunoreactive cell-associated cytokines (IL- 1β: r=0.45, p<0.05; TNF-α: r=0.82, p<0.001). The histograms of cell- associated IL-1β yielded a single peak both in the patients and controls, whereas the histograms of cell-associated TNF-α exhibited two peaks in SAR patients, but just a single peak in the CTL subjects. The mean value of the cell-associated TNF-α in LPS(+) AMs was higher in the SAR patients than in the CTL subjects (p<0.001). In conclusion, the flow cytometric method can be applied to the semiquantitative detection of cell-associated cytokines in alveolar macrophages at the single cell level.