Cytokine signatures of human whole blood for monitoring immunosuppression

Abstract

How to evaluate status of the immune system is extremely critical for clinical immunosuppressive treatment. In this study, we tested the secretion of cytokines in undiluted whole blood samples stimulated with Phorbol 12-myristate 13-acetate (PMA) and ionomycin (IONO), and compared the effects of dexamethasone (DEX), cyclosporine A (CsA) or mycophenolic acid (MPA), either alone or in combination, on cytokine profiles. The results showed that both DEX and CsA dose-dependently inhibited the production of eleven cytokines: interleukin (IL)-2, IL-4, IL-5, IL-6, IL-13, IL-17, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). Unexpectedly, MPA showed no obvious influences except for the mild inhibition on GM-CSF production. In combination treatment, cytokine profiles reflect not only the synergistic effects among drugs, but also the specific effect of the individual drug. Thus, the effects of different immunosuppressants could be reflected through their specific cytokine signatures, which can be applied to maximize immunosuppressive effects, while to minimize risk of infections and help physicians to reasonably apply immunosuppressants.

Keywords: autoimmune immune diseases; cytokine; immune status; immunosuppression; infection; rejection.

Fig. 1

DEX dose-dependently inhibits cytokine secretion. Whole blood (200 µl) was treated with DEX (0.1, 1 and 10 µg/ml) for 6 h followed by stimulation with PMA (0.15 µg/ml) + IONO (2.5 µg/ml) for another 6 h. Supernatants were collected for cytokine detection using the Bio-Plex system. Values are presented as Mean ± SD (n = 3) (linear trend test *p < 0.05; ***p < 0.001)

Fig. 2

CsA dose-dependently inhibits cytokine secretion. Whole blood (200 µl) was treated with CsA (0.05, 0.25 and 1.25 µg/ml) for 6 h followed by stimulation with PMA (0.15 µg/ml) + IONO (2.5 µg/ml) for another 6 h. Supernatants were collected for cytokine detection using the Bio-Plex system. Values are presented as mean ± SD (n = 3) (linear trend test **p < 0.01; ***p < 0.001)

Fig. 3

MPA dose-dependently inhibits cytokine secretion. Whole blood (200 µl) was treated with MPA (1, 10 and 30 µg/ml) for 6 h followed by stimulation with PMA (0.15 µg/ml) + IONO (2.5 µg/ml) for another 6 h. Supernatants were collected for cytokine detection using the Bio-Plex system. Values are presented as mean ± SD (n = 3) (linear trend test *p < 0.05)

Fig. 4

Cytokine profiles reflect the synergistic and specific effects of immunosuppressants. Whole blood was treated with either DEX (1 µg/ml), CsA (0.25 µg/ml), MPA (10 µg/ml) alone or a combination of three drugs for 6 h followed by stimulation with PMA (0.15 µg/ml) + IONO (2.5 µg/ml) for another 6 h. The results show that cytokine profiles can reflect the synergistic effects among immunosuppressants and distinctive effect of individual drug (b, c, d, m: the indicated groups were compared with a group of blank control, or single treatment groups with CsA, DEX or MPA, respectively). Values are presented as mean ± SD (n = 3, One-way ANOVA analysis *p < 0.05; **p < 0.01; ***p < 0.001)