Evaluating Immune Status Of Renal Allograft Recipients By Cytokine Profiles Of Whole Blood

Back groundKidney transplantation is an effective therapy for ESRD (end-stage renal disease). Lifelong application of immunosuppressants is indispensable for the control of allograft rejection. Chemical drugs such as CsA, FK506 and MPA etc. and antibodies such as anti-CD3 and anti-CD25 etc. reduced the incidences of rejections and greatly prolonged the survival of allograft. However, anti-rejection is a double-edged sword which can not only protect kidney from rejection but may also kill the patient due to severe infection. Therefore, it is very essential to use immunosuppressants and to prevent both rejection and infection in kidney and other organ transplantation.Accurate immune evaluation is necessary to find out the balance point and solve the dilemma between rejection and infection. The immune status of patients are fluctuant along with the variation of administration of immunosuppressants. Patients can maintain basic immunity to protect themselves from infection but fail to induce rejection if the dose of immunosuppressant is appropriate, we define this condition as disired immunosuppression. Clinicians judge the immunosuppressive degree mainly according to drug concentrations, clinical signs and symptoms, UV (urine volume), serum creatine, imaging examination and clinical experiences. However, the method is subjective and hysteretic to some exent, as the kidney must have been severely damaged when clinical changes occur. So, an accurate and rapid immune evaluation method which can indicate the immune status and predict the occurrence of rejection and infection is urgently needed. Therefore, clinician are able to adjust the dose of drugs ahead of the occurrence of infection and rejection.Although immune evaluation is important, no effective method is available in clinic. The detection of cytokines secreted by lymphocytes stimulated ex vivo is a potential strategy. Accurate and rapid test which can reflect patient's immune status in a short term was needed. The detection of cytokines in whole blood stimulated in vitro is thought to serve as a good method to reflect patient's immune status, firstly, cytokines can be secreted quickly, secondly, cytokines can reflect the activity of immune cells after stimulation, thirdly, the change of cytokine profile is related to the immunosuppressants. Immune state specific cytokine profiles can be acquired by comparation between patient's clinical symptoms and cytokine profiles. At last, diagnostic criteria of immune status will be established and clinical immune evaluation system will be provided for both patients and clinicians.The liquid chip guarantees fast test for multible cytokines. Single cytokine test can hardly reflects the whole situation in vivo because of the complexity of immune responses, drugs and alloantigens. Thus, we proposed a rapid, micro-volume and multi-cytokine test, so that we can get enough information for analysis. The liquid chip technique developed by Luminex Company can detect multi-cytokine simultaneously, sensitively and quickly in micro-volume serum with the same mechanism with ELISA. The technique was awarded medal for technical innovation in 2005. Bio-rad company further developed this technique by subbing standard board and special software, extended full detectble range and sensitivity (0.2pg to 3200pg) and validity. Furthermore, X-plex provides us with more choices by composing antibodies to interesting cytokines.We proposed to evaluate kidney recipient's immune status by cytokine profile in whole blood stimulated in vitro. The following is a summary of our explorations in this field.ⅠExploration of parameters for immune evaluationby cytokine profileMany stimulators especially mitogens can be used to activate immune. We compared different stimulators and their combinations, and found PHA can activate immune cells more effectively and induce higher concentrations for most cytokines as compared to other common stimulators such as LPS and ConA. So, PHA was chosen as special stimulator in the research. We know that the activation effects of PHA varies a lot in different concentrations, but the cytokine concentration is blented gradually along with the increase of the PHA concentrations. We compared the effect of PHA at 5μg/ml and 10μg/ml for different time interval and found that the levels of some cytokines were different and some other cytokines are similar and the curves nearly overlap. So, we chosed 10μg/ml as the proper concentration in the research. Cytokine increased gradually after stimulation and then reached a peak value at 6-12h. So we chosed 12h as proper stimulation time in the research. In addition, we determined the vitality of PBMC to confirm that the 12culture in undiluted whole blood has no damage to immune cells. We found that the alive PBMCs from groups with or without PHA stimulation were similarly over 90%. At last, we decised to use PHA as the optimal stimulation condition as PHA at the concentration of 10μg/ml for 12h. ⅡThe effect of immunosuppressants on cytokine profileCalcineruin inhibitors (CsA and FK506), MMF and steroids (methylprednisolone and prednisone) are sommon immunosuppressants in clinic. Immunosuppressants may have different effects on the secretion of cytokines in whole blood due to different mechanisms. So, we compared the effects of FK506, MPA and DEX (dexmethasone) on the secretion of cytokines at the concentrations comparable to clinical drug concentration in vitro. We added PHA and immunosuppressant into whole blood and cultured for 12h, then detected the concentration of cytokines. We found that all the three immunosuppressants would influence the secretion of cytokines but in different ways. DEX had a wide inhibitory profile and inhibited the secretion of IL-2, IFN-y, IL-6, IL-8, IL-17, IL-5, IL-10, IL-13, IL-1βand G-CSF (P<0.05) significantly. FK506 only decreased IL-2 and IL-13 (P<0.05) and MPA also decreased IL-2 and IL-13 (P<0.05) but increased IL-1β(P<0.05) significantly. So, IL-1βcan be used as the signature of MPA. The results indicate that immunosuppressants can influence the secretion of cytokines and had specific profile.We know that the immune state is dependent on the dose of immunosuppressants, and we speculated that cytokine concentrations also rely on the concentations of immunosuppressants. We compared the effect of four concentrations of DEX, FK506 and MPA on the secretion of cytokines in whole blood in vitro. Dose dependent style was found in the three drugs. So, cytokine profile can reflect different immune status induced by immunosuppressants.Individual difference of the effect of immunosuppressants was frequently found in clinic. Simmilar concentrations of immunosuppressants were found in patients with infection or rejection. Whether there was an individual difference on the secretion of cytokines remains unknown. We compared the effects of DEX on cytokine secretion among different volunteers and analyzed the accuracy by three repetitive experiments. We found that the method was accurate (P>0.05) with the intra-individual CV 16.05%±10.84%. But the inhibitory rates were different among three volunteers (P<0.05) with the inter-individual CV was 23.95%±10.87%. So, cytokine profile can reflect individual difference of the effect of immunosuppressants.Kidney recipients have to receive multi-drugs to prevent rejection. Thus, to understand the effect of combination of common inhibitory drugs on cytokine secretion is important. We compared the effects of FK506+DEX, FK506+MPA, FK506+DEX+MPA groups at concentrations comparable to clinical drug concentration. We found that the characteristic of single drug can be maintained and the effects were accumulated in the combination groups. The groups containing DEX can inhibit the secretion of most cytokines, but groups containing MPA increase IL-1β. So, cytokine profile can reflect the effect of the combination of immunosuppressants.ⅢThe dynamic cytokine profile of kidney recipientCytokine profile can reflect the potency of immune cells from volunteers in vitro. This suggests that we can evaluate immune status of kidney recipients by cytokine profile. However, the in vivo condition of recipient is more complicated because many factors such as drugs, alloantigens and immune responses were mixed together. Whether cytokine profile can reflect the actual immune status of kidney recipient is not clear. We chosed two patients to monitor cytokine profiles. They were both male, one patient received first transplant with negative PRA, the other one received second transplant with positive PRA. The two kidneys were from one donor. After transplantation, the patient who received first transplant had a good postoperative recovery, kidney function became normal quickly. In contrast, the patient with second transplantation suffered from rejection, showing increased serum Cr and urine volume decreased again. We found that the basic level of cytokines of patient with rejection was higher, and cytokines increased ahead of clinical symptoms and the changes of cytokines such as IL-2, IL-4, IL-6, GM-CSF, IFN-γand TNF-αwere consistent with clinical symptoms. Cytokine concentration of patient with normal kidney function maintained very low level all the time. These results suggest that cytokine profile can serve as an indication of immune status of recipients, which is more sensitive than symptoms. However, in terms of the complexity of immune response in vivo, more clinical experiments are needed to assess and optimize the method.Innovation1. In clinic, no method was available to evaluate immune status, our research may provide a strategy.2. We detect the concentrations of cytokines in undiluted whole blood to evaluate the potency of immune cells and then judge the immune status of recipients.3. The sample, undiluted whole blood, maintains the complexity of in vivo condition as great as possible. Meanwhile, the testing time cost in the procedure was also reduced.4. We detected 17 cytokines simultaneously and analyzed the significance of cytokine profile in the immune system. The result can reflect the actual condition in the body.