Cytokine Profiles Of Human Whole Blood For Monitoring The Status Of Immune Suppression

BackgroundImmunosuppressants have been applied in patients with transplant rejection, or autoimmune disorders, and most of the tumor patients have to receive radiotherapy or chemotherapy. All of these therapies can inhibit the patients' immune system, which will unavoidably bring a critical concern:how to use the immunosuppressants, radiotherapy or chemotherapy in a proper way? On one hand, insufficient dose of immunosuppressants is likely to result in the rejection of transplanted organ or aggravation of autoimmune disease. Without effective radiotherapy or chemotherapy, tumor cells may not be effectively eliminated. However, overdose of the immunosuppressants, radiotherapy or chemotherapy will lead to excessive inhibition of the immune system, which might lead to severe infection. At present, deaths of most posttransplantation patients are caused by severe infection due to the overuse of immunosuppressants. Therefore, it is a clinical dilemma for the proper application of immunosuppressants and rational medication of radiotherapy or chemotherapy. And the main reason for the problem is the lack of fast and effective methods to evaluate the patients' immune status.Cytokine, secreted by the immune cells, are important mediators during the functioning of immune system, which lead us to hypothesize that cytokine profiles can be used to evaluate the immune status.For cytokine detection, we chose the advanced Luminex xMAP technique which is able to simultaneously detect up to a hundred of cytokines in a single sample.Objective1. To find proper immunostimulants that can stimulate the immune cells in whole blood to secrete cytokines rapidly and effectively2. To investigate the inhibitory effect of different immunosuppressants on cytokine secretion in vitro and to compare specific cytokine profiles of distinct immunosuppressant.3. To collect arid detect the samples of renal transplant patients to find out the differences of cytokine changes between different immune statuses, such as rational immunosuppression group, graft rejection group, infection group, and to establish the cytokine profiles that can evaluate the immune statuses of patients.Methods:1. Sample selectionAlthough diluted whole blood or peripheral blood mononuclear cells have been used widely, they could not meet the clinical requirement of immune state evaluation as too many artificial factors are applied. In order to maximally mimic the clinical situation, we chosed heparinized undiluted whole blood as our test sample.2. The selection of immunologic stimulantsAlthough PHA has been proved to be the most effective stimulatant for cytokine production in whole blood as compared to LIPS or Con A stimulation, PHA still needs at least 12hrs to maximize the production of most cytokines, which is difficult to meet the clinical application. Therefore, we compared the secretions of cytokines under the stimulation of PHA and combination of PMA and IONO in whole blood, in order to know whether PMA+IONO could maximize cytokine production in shorter time intervals.3. The variation of cytokine values under different concentrations of immunosupprants.Whole blood samples were firstly co-cultured with different concentrations of immunosuppressants for 6 hours, then stimulated with PMA+|IONO for another 6 hrs, in order to understand whether there were dose dependent inhibitory effects of different immunosuppressive drugs on cytokines.4. The accumulative effect of combined immunosuppressive protocols on the cytokine profiles.Since steroids, calcineurin inhibitors such as CsA or FK506 (FK) and mycophenic acid (MMF) are common immunosuppressants in clinical transplantation, we investigated the combined effects of DEX, MPA (the active motablite of MMF in vivo) and CsA or FK on cytokine production. Therefore, we tested two protocols consisting of three immunosuppressive agents, MPA+DEX+FK or MPA+DEX+CsA, on cytokine secretion in whole blood and compared the effects of combined treatments with that of single immunosuppressant.5. The Luminex technique to detect cytokines.In order to detect multiple cytokines in tiny volume of whole blood, we selected the Luminex technique which combines flow cytometry and flow chip techniques together. Theoretically, Luminex can detecte up to 100 cytokines in only 15ul samples within 2 hours.6. StatisticsData were analyzed with SPSS13.0 software and comparisons between groups were made with analysis of variance. And LSD method (homogeneity of variance) or Dunnett's T (uneven variance) were used in multiple comparisons.Results:1. PMA+IONO was able to maximize the secretion of multiple cytokines within 6 hrs, with PMA(0.15μg/ml)+IONO(2.5μg/ml)as the best concentrations.2. DEX (0.1,1 and 10μg/ml) were able to dose dependently inhibit multiple cytokines, including IL-2, IL-4, IL-5, IL-6, IL-13, IFN-y, TNF-a, G-CSF, and GM-CSF. However, the lowest concentration of DEX (0.1μg/ml) could not inhibit G-CSF secretion.3. Similarly, CsA (0.05,0.25 and 1.25μg/ml) dose dependently inhibited the secretion of some cytokines, including IL-17, IFN-y, GM-CSF and G-CSF. While only higher concentrations (0.25 and 1.25μg/ml) could inhibit other cytokines like IL-2, IL-4, IL-5, IL-6, IL-10, IL-13 and TNF-a.4. Importantly, among three groups of different FK concentrations (1,5 or 20ng/ml), only the highest concentration of FK(20ng/ml) were able to inhibit the secretion of most cytokines, such as IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, IFN-y, TNF-a and G-CSF. Whereas FK (5 or 20ng/ml) could inhibt GM-CSF secretion.5. MPA of any concentrations showed no inhibition on the secretion of cytokines.6. Since combination treatment with different immunosuppressants is a common practice, we tested the combination treatment of DEX (1μg/ml), FK (5ng/ml) and MPA (10μg/ml) on cytokine profiles and compared their effect with no treatment control group, as well as groups with single treatment of DEX, MPA or FK, respectively. Combination treatment inhibited the secretion of IL-2, IL-4, IL-5, IL-6, IL-13, IFN-y, TNF-a, GM-CSF and G-CSF as compared with no treatment control. Interestingly the production of some cytokines like IL-4, IL-5, IL-6, IL-13, IFN-γ, TNF-a and G-CSF is significantly decreased in combination group as compared with that of MPA or FK single treatment, indicating that those cytokines reflected the inhibitory effect of DEX. And GM-CSF displayed significantly decreased in combination group as compared with no treatment control or MPA single treatment group, indicating the combined effect of FK and DEX.We also tested the combination treatment of DEX (1μg/ml), CsA (0.25μg/ml) and MPA (10μg/ml) on cytokine profiles and compared their effect with no treatment control group, as well as groups with single treatment of DEX, MPA or CsA, respectively. The results showed that combination treatment inhibited the secretion of IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IFN-γ, TNF-a, GM-CSF and G-CSF as compared with no treatment control group. Importantly, the production of some cytokines like IL-2, IFN-y and TNF-a is significantly decreased as compared with that of DEX, MPA or CsA single treatment, indicating that there were the combining effects of the three immunosuppressants on those cytokines. Other cytokines, such as IL-4 and GM-CSF displayed significantly decreased secretion as compared with control and MPA single treatment group, but their secretion was not different as compared with DEX or CsA single treatment groups, thus specifically reflecting the combining effects between DEX and CsA. Furthermore, IL-5, IL-6 and IL-13 seem to specifically reflect the inhibitory effect by DEX, as its secretion in the combined treatment is only significantly decreased as compared with that in control, MPA or CsA single treatment groups, but not different from that in DEX single treatment. Similarly, IL-10 and G-CSF may specifically reflect the effect of CsA, in that its level in combined treatment is only significantly decreased as compared with control, MPA or DEX single treatment groups, but not different from CsA single treatment group.Conclusion:1. PMA+IONO were able to maximize the secretion of multiple cytokines within 6 hrs.2. All the immunosuppressants except MPA could effectively inhibit the secretion of cytokines in whole blood under the stimulation of PMA+IONO. Different immunosuppressants showed dose-dependently unique inhibitory cytokine profiles.3. Cytokine profiles in combination treatment were different with those in groups of single immunosuppressants. In the combination of FK(5ng/ml), DEX(1μg/ml) and MPA(10μg/ml), it was found that IL-2 mainly reflected the combining effects of FK, DEX and MPA; That IL-4, IL-5, IL-6, IL-13, IFN-y, TNF-a and G-CSF mainly served as the indicators of inhibitory effects of DEX; and that GM-CSF was a major indicator of the combining effects of DEX and FK. In the combination of CsA(0.25μg/ml), DEX(1μg/ml) and MPA(10μg/ml), it was found that IL-2, IFN-y and TNF-a mainly reflected the synergistic effects of three immunosuppressants; that IL-4 and GM-CSF mainly reflected the synergistic effects of CsA and DEX; that IL-5, IL-6 and IL-13 mainly served as the indicator of inhibitory effects of DEX; and that IL-10 and G-SCF served as the effect of CsA. Therefore, we conclude that in the combination treatment of different immunosuppressants, som cytokines specifically reflected the inhibition effect of a certain kind of single drug on immune system. In this way, we can provide more accurate information for clinicians to better use immunosuppressants.