[Cytokine assessed in the serum are denatured by calcium ion and resultantly activated protease].

When we assessed cytokine levels in both plasma and serum from the patients with atopic dermatitis and healthy volunteers, we found that IL-2, 5 and 10, and IFN-gamma were significantly elevated in the plasma from atopic dermatitis patients but not in their sera and that, in an average, each cytokine level, especially IL-2 level is far higher in plasma than in serum. In order to solve the cause for this dissociation. Calcium ion was dose-dependently added in the plasma in which calcium ion had been inactivated by citrate contained in the plasma. Protease inhibitors, PMSF, aprotinin and leupeptin were added in the plasma in which each cytokine was decreased in the presence of calcium ion. Finally, in RPMI medium where cytokine IL-2 or IL-10 is present, proteases, thrombin, trypsin and chymotrypsin themselves were introduced. Results revealed that addition of calcium ion dose-dependently decreased each cytokine level in the plasma, respectively. Protease inhibitors, PMSF, aprotinin and leupeptin significantly elevated each cytokine level in the plasma where cytokines had been decreased by the presence of calcium ion, respectively. These changes were comparable between plasma and blood cell-containing plasma, attesting that the effect of enzymes released from the calcium ion-activated neutrophil granules on the decrease in cytokine levels is negligible. Cytokines, IL-2 itself was also decreased in the presence of calcium ion alone, or in the presence of both proteases, thrombin, trypsin or chymotrypsin itself, and calcium, respectively. This study suggests that calcium ion itself or calcium ion-activated protease may denature the cytokine and that for this reason, cytokine should be, in general in our laboratory, assessed in not serum but plasma.