Evidence for degradation of cytokines in the serum of patients with atonicdermatitis by calcium-dependent protease

Changes in the levels of cytokines in the circulating blood and skin have b een reported in patients with atopic dermatitis (AD), We determined IFN-gam ma, IL-2, IL-4, IL-5 and IL-10 in both the serum and plasma of 45 AD patien ts and 20 healthy donors. Since differences in the levels of these cytokine s between serum and plasma were found, the roles of Ca2+ and proteolytic en zymes were examined. Levels of IL-2 and IL-10 were measured in citrated pla sma to which various amounts of CaCl2, protease inhibitors, and proteases h ad been added. All cytokine determinations were carried out using a standar d ELISA, The plasma levels of IFN-gamma, IL-2, and IL-5 were significantly elevated, but the serum levels of these cytokines were not significantly ch anged. The levels of IL-2 in the plasma of the AD patients averaged 4.25-fo ld higher than in the serum of the AD patients, and 2.5-fold higher than in the plasma of healthy controls (P < 0.001), CaCl2 produced a dose-dependen t decrease in IL-2 and IL-10 in citrated plasma. The protease inhibitors PM SF, aprotinin and leupeptin produced a dose-dependent increase in measurabl e levels of IL-2 and IL-10 in plasma. A decrease in IL-2 levels was also se en in CaCl2-supplemented serum-free medium, and this was accentuated by the addition of the proteases thrombin, trypsin, chymotrypsin and elastase, Th ese findings suggest that although significant changes in cytokine levels h ave been reported not to occur in circulating blood but have been reported to occur in the skin of AD patients both in vivo and in vitro, cytokines ca n indeed also be found to be elevated in circulating blood when assessed ca refully by statistically valid methods. Further, it is suggested that durin g the preparation of serum, some circulating cytokines are degraded by calc ium-dependent proteases, and that Ca2+ itself can also affect the measureme nt of cytokines, The measurement of circulating cytokines needs to be caref ully reassessed.