Clinical effects of soluble interleukin-6 receptor detection on autoimmune rheumatic disease patients

Objective : This study aims to explore the effects of soluble interleukin-6 receptor (IL-6R) on the clinical diagnosis and treatment of autoimmune rheumatic disease (ARD) patients. Methods: Sixty-eight ARD patients enrolled in our hospital recently were selected, the IL-6R levels of whom were detected by double-antibody sandwich ELISA method and compared with the IL-6 levels of normal subjects. The expressions of IL-6R in joint tissues were detected by section staining. The mechanism regarding the effects of IL-6R was postulated by administering the patients with blocking agent. Results: The blood IL-6R level of ARD patients was 2-3 times higher than the IL-6 level of normal subjects with significant difference. The detection results of C-reactive protein and erythrocyte sedimentation rate show that both the indicators were significantly decreased (P=0.0098 and 0.0097 respectively). IL-6R was associated with autoimmunity based on the considerable expression in tissue sections, which was also verified by the alleviated symptoms after blocking IL-6R expression. Conclusion: Detecting soluble IL-6R is able to determine the patient's conditions and treatment effects. Meanwhile, soluble IL-6R detection can also serve as the inflammatory responses of ARD, as well as the determination index for abnormal immune responses and generated antibodies number, which is crucial for early diagnosis.

Our objective was to explore the effects of soluble interleukin-6 receptor (IL-6R) on the clinical diagnosis and treatment of autoimmune rheumatic disease (ARD) patients

METHODS
General Information: Sixty-eight ARD patients who had been treated in our hospital from 2010 to 2012 were selected in this study, including 39 males and 29 females.There were 16 cases of ankylosing spondylitis (AS), 23 cases of rheumatoid arthritis (RA), 19 cases of systemic lupus erythematosus (SLE) and 10 cases of polymyositis (PM).The IL-6R levels were analyzed and detected by doubleantibody sandwich ELISA method and compared with the IL-6 levels of normal subjects.Meanwhile, the microscopic results of leukocytes in the patients and normal subjects under the intervention of IL-6 were compared.Detection Methods: The levels of IL-6R were detected by double-antibody sandwich ELISA method.All apparatus and drugs were purchased from Immunotch Medical Products Co., Ltd.During the test, plasma sample and 150 μL of antibodies at different concentrations were added first to wells with alkaline phosphatase as the marker.After the addition of substrate (200 μL) to observe the reaction of CH-segment enzyme as well as staining, the absorbance was calculated at the wavelength of 405 nm to obtain the measurement value of IL-6R. 4 Immunohistochemical Staining: Pathological and healthy joint tissues were collected from ARD patients and normal subjects for section preparation, to which were then dropwise added known labeled antibody.The sections were observed by light microscopy after being rinsed, based on which results were obtained.
Treatment Methods: Blocking agent was used to study the correlative factors of IL-6R in autoimmunity.The patients were administered according to weights once (8 mg/kg) every four weeks.][7] Statistical Analysis: All data were analyzed by SPSS 15.0.The numerical data were expressed as (mean ± SD) and compared by paired t test.The correlations were calculated based on the experimental data of two groups by using the software package of SPSS15.0.P<0.05 was considered statistically significant.

RESULTS
According to the results of double-antibody sandwich ELISA, the blood IL-6R level of ARD patients was 2-3 times higher than that of normal subjects, between which the difference was statistically significant.The results are listed in Table-I.
Meanwhile, the correlations between soluble IL-6R levels and titers of ANA, DNA, ENA and RF were studied, revealing that IL-6R levels in the patients were positively correlated with titers (Fig. 1).
Immunohistochemical staining method was used for tissue section staining and microscopic   observation.We found that IL-6 and IL-6R existed in cells around the inflammatory tissues of RA patients, inferring that IL-6 was correlated with ARD joints, and played an essential role in its pathogenesis, the effect of which is evident in allergic reaction-related parts (Fig. 2 and 3).
At the same time, IL-6R blocker therapy was adopted for patients.The detection results of C-reactive protein and erythrocyte sedimentation rate show that both the indicators were somewhat decreased.The outcomes after treatment were significantly different from those before treatment (Table-II).

DISCUSSION
IL-6, which is a key substance that mediates inflammatory response in human body, is mainly manifested in regulating physiological activities of various immune cells and controlling immune response.[10] Glycoprotein attached to the membrane of IL-sensitive cells may be prone to falling off in metabolism, thus forming soluble IL-6R after changes for a period of time.This receptor consists of ligand and non-ligand binding receptor protein chains, the molecular weights of which are 80 kD and 130 kD respectively.2][13] In this study, the bloods of ARD patients and normal subjects were collected, which were reacted with same amounts of soluble IL-6 antibody detecting agents for comparison.
ARD patients had a higher proportion of soluble IL-6R than normal subjects, which was also positively correlated with the severity.It has previously been reported that some autoimmune diseases may be accompanied by an increase in IL-6 level probably related with the pathological changes in human body (e.g.tumorigenesis).5][16] The specific reason remains undefined, but it is estimated that the increase of soluble IL-6R can enhance the binding ability of non-ligand binding receptor protein involved in cytokine signal transduction to IL-6R, thus rendering the complex of soluble IL-6R and IL-6 to reach limit that engenders immune responses in human body.8][19][20] In other words, soluble IL-6R indicates the appearance of immune response, the changes of which have close relationship with disease and treatment progress, and the strength of immune response.Detecting the changes of IL-6 and soluble IL-6R levels in patients can more directly reflect the disease conditions, which can be used in clinic to auxiliarily determine the intensity of inflammatory response and post-treatment recovery.
Obviously, the rise of soluble IL-6R level is closely associated with the appearance of "acutephase protein" in inflammation, and soluble IL-6R can also affect the gene expression of albumin in liver and inhibit the production of albumin to be significantly lower than the normal level and to produce other adverse reactions in human body.Soluble IL-6R regulates the interaction between IL-6 and cells, while in turn, IL-6 controls the expression level of soluble IL-6R.Moreover, the interaction synergetically influences the physiological activities of immune cells and abnormal immune response, which can thus be applied in clinic to monitor the adverse reactions in patients as an auxiliary treatment protocol.

Fig. 2 :
Fig.2: Anti-IL-6 staining sections of RA.A: Staining section of patient; B: Staining section of normal subject.

Fig. 3 :
Fig.3: Anti-IL-6R staining sections of RA.A: Staining section of patient; B: Staining section of normal subject.

Table - I
: IL-6 and IL-6R results of ARD patients and normal subjects.

Table -
II: C-reactive protein levels and erythrocyte sedimentation rates before and after treatment.