Ex vivo flow cytometry determination of intracytoplasmic expression of IL-2, IL-6, IFN-gamma, and TNF-alpha in monocytes and T lymphocytes, in chronic hemodialysis patients

Aims: To determine the intracytoplasmic expression of TNF-alpha, IL-2, IL-6 and IFN-gamma, ex vivo and in vitro, in both monocytes and T lymphocytes b y flow cytometry after appropriate stimulation using phorbol myristate acet ate (PMA)/ionomycin or lipopolysaccharides (LPS) in the presence of monensi n, in order to assess the bio(in)compatibility of different dialysis membra nes. Methods: We examined monocytes and T lymphocytes taken from chronic he modialysis patients (using either cuprophane (CUP), n = 6; polyacrylonitril e (AN 69), n = 6; or polysulfone (PS), n = 6 membranes), before and after a dialysis session. We compared the results with those obtained from end-sta ge chronic renal failure patients (n = 3) and healthy volunteers (n = 11). Results: Before any stimulation there was a statistically significant diffe rence in the percentages of TNF-alpha, IL-6, and IFN-gamma- expressing mono cytes with respect to the dialysis membrane used. The highest percentages w ere observed for CUP and AN69: patients with figures of around 30% for each cytokine; the lowest percentages were found in PS patients and healthy vol unteers. One hour after LPS stimulation the patterns remained unchanged for TNF-alpha and IFN-gamma, whereas the percentages of IL-6-expressing cells in PS patients and in healthy volunteers reached the figures obtained in th e other groups. When we examined the percentage of IFN-gamma-, TNF-alpha- a nd IL-6-expressing monocytes in patients before and after a dialysis sessio n, before any stimulation, we found that the results were significantly dif ferent for the three membranes (p = 0.01). Thus, a dialysis session with po lysulfone membranes had no significant effect on the precentages of IFN-gam ma-, TNF-alpha-, and IL-6-expressing monocytes, whereas percentages were si gnificantly lower after the dialysis session when using cuprophane or AN69 membranes, suggesting a release of these cytokines by the monocytes during dialysis. A significant number of IFN-gamma- and IL-2-expressing T lymphocy tes were only detected after 18 hours of PMA/ionomycin stimulation. The per centages of IFN-gamma-expressing T cells recorded for the different membran es were not statistically different from those recorded for healthy subject s or pre-dialysis patients, i.e., they were between 11.5 and 20%. However, the percentages of IL-2-expressing T lymphocytes were significantly differe nt between the 5 groups, i.e., 31.3, 30.5, 18.6, 13.9 and 7.6%, respectivel y, for CUP patients, pre-dialysis patients, healthy volunteers, PS and AN69 patients. This suggests that pre-dialysis and CUP patients have, at baseli ne, a stimulation of their T lymphocytes. Finally, a 4-hour dialysis sessio n had no impact on the percentages of IL-2-expressing T lymphocytes, wherea s it was associated with a significant decrease in the percentage of IFN-ga mma-expressing cells, but only when cuprophane membranes were used. Conclus ion: Cytokine flow cytometry enables one to study, ex vivo, i.e., without a ny stimulation of the cells, and in vitro after appropriate stimulation, th e bio(in)compatibility of dialysis membranes assessed by the intracytoplasm ic cytokine profiles of TNF-alpha, IFN-gamma, IL-6 and IL-2, evaluated at t he single cell level. Copyright (C) 2000 S. Karger AG, Basel.