Rapid determination of cell-associated tumor necrosis factor production by flow cytometry

Tumor necrosis factor (TNF) is a cytokine implicated in many disease states, including septic shock and transplant rejection. Regulation of TNF production is complex, and under certain conditions TNF is expressed in cells, but not released. Determination of TNF, particularly on cells, may allow more rapid and specific diagnosis of diseases, and provide a better guide to therapy. We developed a method to quickly measure TNF associated with cells using flow cytometric techniques.

RAW cells, a murine macrophage cell line, were stimulated with lipopolysaccharide (LPS) and examined by staining with an anti-murine TNF antibody followed by a fluorescein isothiocyanate FITC-labeled secondary antibody.

Different permeabilization protocols showed that treating cells with 0.1% Triton X-100 for 5 minutes provided staining of cells comparable to 0.005% digitonin and 0.1% saponin; however, it was no better than untreated cells. Comparison of fluorescein isothiocyanate, phycoerythrin, and Red 613 fluorochromes showed that fluorescein isothiocyanate was comparable to phycoeryrthrin, but better than Red 613. Multiple experiments demonstrated that LPS-stimulated cells exhibited a 1.3- to 6.8-fold increase in the number of cells staining positively for TNF, as compared with unstimulated cells. Kinetic studies showed that LPS induced cell-associated TNF within 30 minutes, which plateaued between 2 and 4 hours. TNF staining correlated with the appearance of TNF biologic activity associated with the cells and immunoperoxidase staining of cytospin preparations. Cyclosporin A has been reported to inhibit secretion of TNF. LPS-stimulated cells treated with cyclosporin A showed no decrease in TNF staining by either flow cytometry analysis or immunohistochemistry, but there was a reduction of TNF in the culture supernatant. The human macrophage cell line, THP-1 was also tested and showed statistically significant results when comparing control versus anti-TNF antibodies.

Flow cytometric detection of cell-associated TNF represents a relatively rapid and simple method with potential applications in the study of TNF gene expression. This method may be used to detect early TNF production in disease states such as septic shock and transplant rejection.