The Role Of MDC In The Development Of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a kind of auto-immune disease that can violate multiple systems with the characteristics of production of large amounts of antibodies and precipitation of immune complex. The kidney is usually involved in SLE by the manifestation of different degrees of lesions on renal parenchyma and interstitium.. The precipitation of antibodies and immune complex may result in the inflammation and lesions of multiple organs, in which complex interaction of many inflammatory mediators and cells are involved. Among them the chemotaxis and activation of specific leukocytes by chemotactic factors is one of the most crucial steps in starting the process of auto immune reactions.As one of Th2 type CC subclass chemotactic factors, macrophage derived chemokine (MDC) can evoke the chemotaxis of dendritic cells, monocytes, T cells, especially Th2 cells. MDC can combine with CCR4 receptors specifically and has close relationship with the onset and development of auto immune diseases. In order to evaluate the position and contribution of MDC in the proceeding of SLE, we detected and analyzed MDC level in plasma of patients with SLE.Some researches have discovered that there is infiltration of large amounts of CD4+T cells and monocytes in renal tissues of patients suffered from LN, which suggests the recognition ligand MDC of CCR4 and thymus and activation-regulated chemokine (TARC) may play an important role in the process of LN. During the investigation of antibasement membrane nephritis, the use of MDC antibodies decreased the infiltration of monocytes in renal tissues and delayed the productioin of crescent lesions obviously. By detecting the expression of MDC in renal tissues of MRL/lpr mice (SLE animal model), blocking the activity of MDC specifically by neutralization reaction in lupus mice, observing the monocyte cells distribution and changes of tissues damage in renal tissues and the expression and distribution of MDC, TARC and CCR4, we planed to discuss the position and contribution of MDC in the process of SLE accompanied with renal lesions, and thus to make further investigation to its function and discuss the value and mechanism of MDC antibody in renal lesions of lupus.Methods1. Detecting MDC level in plasma by ELIS A method26 SLE patients who just received initial treatment,20 patients who had received therapy,16 rheumatoid arthritis patients and 16 healthy individuals as normal control were involved in the experiment.2 ml anticoagulated blood of every patient was collected in the morning with an empty stomach. After centrifugalization the samples were detected by ELISA method, standard curves and blank tubes were established adding in every hole 100μl test samples, the first antibody fluid, enzyme labelled antibody fluid, substrate fluid and stop buffer sequentially, measuring the absorbance values at 492nm by enzyme labeled meter, standard curve was drawn to calculate MDC concentration values.2. Clinical dataclinical and laboratory examinations of patients were collected and arranged to analyze the results of pathological typing of renal biopsy and score every SLE patient according to SLEDAI scoring criteria.3. Animal samplesEight 8-week-old MRL/lpr mice were obtained and divided into two groups by random number table, each group of mice were injected 300μlMDC antibodies and PBS respectively from caudal vein once every other day from 12 weeks old.4 8-week-age BALB/c mice were used as normal control, they were administrated with the same volumes of PBS from 12 weeks old. The mice were executed by spinal cord transaction when they were 16 weeks old. Renal tissues were took out from one side and fixed in formalin for histopathology examination. Renal tissues on the other side were used for extraction of RNA and protein.24 hours' urine volumes of mice were collected accurately by metabolic cage and the blood was obtained from posterior orbital vena plexus.4. Renal pathological analysisRenal tissues of mice fixed in formalin were dehydrated, imbedded by paraffin, sliced and stained with HE and PAS, then the stained cells were visualized with 400×high power field light microscope to exhibit the pathological changes of renal glomerulus and tubules.5. Immunohistochemical stainAfter deparaffinage the section were sealed off by goat sera for 20 minutes, added in 1:100 diluted mouse anti-mouse MDC and incubated at 37℃for 1 hour, biotinylation goat anti-mouse IgG for 20 minutes and peroxidase labeled streptavidin fluid for 20 minutes, followed by stained with DAB and afterstained with hematoxylin. Pictures were taken with 400×light microscope and image analysis, calculation of MDC expression intensity and the account of macrophage cells.6. Determination the expression of MDC, TARC and CCR4mRNA in renal tissues of mice by RT-PCR methodTotal RNA was extracted from renal tissues by trizol method. cDNA was synthesized by reverse transcription from 1μg sample RNA. The reverse transcript underwent PCR amplification. Results of electrophoresis were analyzed by image analysis software, grey scale values of every DNA amplification zone were recorded, the ratio of grey levels of every target DNA amplification fragment to that of inner controlβ-actin was calculated as the semi-quantitative index of every target gene mRNA. 7. Western blotting of expression of MDC, TARC and CCR4 protein in renal tissues of miceGet some tissues and sheared them into pieces, adding in managed tissue fluid and made them into homogenate by homogenizer. The supernatant was obtained by centrifugalization at 12000rpm at 4℃and undergone protein quantitative analysis by lowry method. Adding in the 20μl samples obtained from every supernatant, after protein isolation by SDS-PAGE gelatum, they were transferred to nitrocellulose filter and sealed off with buffer under room temperature for 2 hours, subsequently incubated overnight with the first antibody at 4℃, incubated with the second antibody for 1 hour. Ultimately MDC protein zones were exhibited by alkaline phosphatase coloration and analyzed by gelation image analysator.8. Statistical analysisThe statistical results were demonstrated by x±s, Mann-Whitney test, Kruskal-Wallis test, rank sum test and Pearson coefficient analysis were used to analyze data. A P-value less than 0.05 was considered statistically significant.Results1. Detection and analysis of the plasma level of MDCThe plasma level of MDC in systemic lupus erythematosus (SLE) group with initial treatment was obviously higher than that in the treated group, rheumatoid arthritis group and normal controls. But there wasn't statistical difference among the treated groups, rheumatoid arthritis group and normal controls. When analyzing the pathological types of the fifteen lupus nephritis patients, we found that the plasma level of MDC in typeⅡand typeⅢlupus nephritis patients were higher than that in the typeⅣlupus nephritis patients. The plasma level of MDC in forty-four SLE patients was positive correlated with the 24-hours urine protein, the creatine and SLE disease activity index (SLEDAI). 2. Detection of the mice 24-hour urine protein quantization and serum creatinineThere wasn't statistical difference of the urine protein and serum creatinine in the mice at 8 weeks and 12 weeks. The urine protein was significantly decreased and the serum creatinine was stable at 16 weeks in the mice group which was treated with antibodies. There was statistical difference between the treated group and the MRL/lpr mice, but with no difference once compared with the normal controls.3. The results the mice renal tissue with RT-PCRWe detected the gray scale of the RT-PCR electrophoresis graph and found that the MDC, TARC and CCR4 didn't express in the normal BALB/c mice renal tissue but could express in the MRL/lpr mice renal tissue. The MDC and CCR4 mRNA level in the antibodies treated group was lower than that in the MRL/lpr mice. There was no difference of TARC/β-actin in mice renal tissue between the MRL/lpr mice and antibodies treated group.4. The results of the mice renal tissue with Western BlotThe MDC, TRAC and CCR4 didn't express in the BALB/c mice renal tissue. The MDC and CCR4 protein level in the antibodies treated group was lower than that in the MRL/lpr mice renal tissue. There was no difference of TARC gray scale in mice renal tissue between MRL/lpr mice and antibodies treated group.5. Routine pathological detection of the mice renal tissueIn the BALB/c mice, the glomerular capillary loop was thin and clear and the number of endothelial cells and intercapillary cells and the peripheral nephric tubules was normal. As well, in the MRL/lpr mice the endothelial cells and intercapillary cells were hyperplasy and the glomcrulus enlarged accompany with the capsular space stenosis, inflammatory cells infiltrated in the interstitial of the nephric tubules and the granucrulus. The infiltration of the inflammatory cells in the interstitial of the granucrulus and the nephric tubules in the antibodies treated group was evidently decreased than that in the MRL/lpr mice.6. The analysis of mice renal tissue with MDC immunol histochemistryIn the MRL/lpr mice renal tissues it showed that MDC expressed in the intercapillary cells of glomcrulus, epithelial cells of nephric tubule, endothelial cells and the cytoplasm and interstitial matrix of the renal interstitial infiltrated inflammatory cells, especially in the area of renal tubules. The MDC side optical density in the antibodies treated group was lower than that in the MRL/lpr mice and was positive correlated with the account of macrophage cells. There was nearly no expression of MDC in the BALB/C mice renal tissue.ConclusionsThe MDC level in the initial treated SLE patients was higher than that in the treated SLE patients, rheumatoid arthritis patients and the normal controls. The MDC level of SLE patients was positive correlated with 24hours urine protein, the creatine and SLEDAI score. The plasma MDC level of the LN patients was related with the pathological type. The MDC level of typeⅡand typeⅢLN group was higher than that of typeⅣLN group. The disease process was closely related with the plasma MDC level, which indicated that MDC might involve in the development of SLE and the pathological injury of lupus nephritis.The MRL/lpr mice showed some abnormal manifestations about renal function and morphology which were related with the LN at 16 weeks. The MDCmRNA was evidently expressed in MRL/lpr mice renal tissue. The protein level and mRNA level of MDC was coincident in MRL/lpr mice renal tissue. Not only the MDC expressed highly in the peripheral blood of the SLE patients, but also in the renal tissue of the lupus mice model. Then it could demonstrate that MDC might involve in the develop of LN in MRL/lpr mice.The lupus mice which were treated with anti-MDC antibodies got better prognosis in the renal function and morphology than the untreated lupus mice. And the expression of MDC and CCR4mRNA and protein in the treated group was lower than that of normal controls. The high expression of MDC which led to monocyte infiltration in the kidney may play an important role in the development of LN. And the anti-MDC antibodies could relieve the renal injury by inhibiting the monocytes infiltration in the renal tissue of the lupus mice.