mRNA Expression of Chemokine Receptors on Peripheral Blood Mononuclear Cells and Correlation with Clinical Features in Systemic Lupus Erythematosus Patients△

Yu-mei Li ,Zhi-qiang Chen *,Xu Yao ,Ai-zhen Yang ,An-sheng Li ,Dong-ming Liu ,and Juan-qin Gong

1Department of Dermatology,4Department of Medical Imaging and Radiology,the Affiliated Hospital of Jiangsu University,Zhenjiang 212001,China

2Hospital for Skin Diseases,Chinese Academy of Medical Sciences &Peking Union Medical College,Nanjing 210042,China

3Central Lab of People’s Liberation Army Cancer Center,the 81st Military Hospital,Nanjing 210002,China

SYSTEMIC lupus erythematosus (SLE) is a chronic,inflammatory autoimmune disease characterized by antinuclear autoantibodies,immune complex formation,and systemic vasculitis.1The involved organs and systems in SLE include the skin,joints,lungs,peripheral blood cells,kidneys,and the central and peripheral nervous systems.The disease course of SLE is characterized by unpredictable flare and remission.Thus,biomarkers need to be developed for more accurate diagnosis and better assessment of the disease activity and organ involvement.

The efficient operation of the immune system is critically dependent on a complex series of cellular interactions and migrations to specific locations.Chemokines are small chemotactic cytokines characterized by critically positioned cysteine residues,which recruit distinct leukocyte subsets to the sites of inflammation and specific microenvironments within secondary lymphoid tissues.2,3Chemokine receptor expression is exquisitely regulated depending on the stage of activation and differentiation of T cells,and coordinates tissue localization and encounters with antigen presenting cells.4–6We propose that this receptor interaction also promotes the internalization,processing,and presentation of the autoantigens,thus enhancing their immunogenic capabilities.Over 40 chemokines have been identified as major traffic directors of leukocytes engaged in innate and adaptive immune responses.7The subsets of leukocytes responding to particular chemokines are determined based on their interaction with selected G protein-coupled receptors.8

In response to the accumulation of immune complexes at the kidney in SLE,monocytes,T lymphocytes,and neutrophils infiltrate the kidney and mediate tissue injury and renal dysfunction.Chemotactic factors induced by immune complexes are responsible for recruiting inflammatory cells to the kidney.Considerable attention has focused on the role of the chemokine network in regulating renal leukocyte recruitment in autoimmune glomerular diseases.Intervention studies directed at chemokines or chemokine receptors in animal models of lupus nephritis have provided definitive proof that specific chemokines are involved in the pathogenesis of renal inflammation.These chemokines and chemokine receptors are expressed in human kidney during lupus nephritis,and correlate with the markers of renal injury and inflammation.CCR5+cells were detected in the interstitial tissue in specimens from lupus nephritis patients,meanwhile no CCR5+cells were documented in normal controls.A strong positive correlation was found between tubulointerstitial immunoexpression of regulated upon activation,normal T cell expressed and secreted protein(RANTES) and the number of interstitial CCR5+cells as well as between immunoexpression of RANTES and the immunoexpression of transfer growth factor beta1,alpha-smooth muscle actin,renal cortical volume,and serum creatinine in patients with lupus nephritis.9Lupus erythematosus profundus skin lesions were characterized by lobular panniculitis,dominated by cytotoxic CXCR3+lymphocytes.10

These studies showed that chemokines and chemokine receptors play an important role in the pathogenesis and tissue damage of SLE.In the present study,we extended the analysis of chemokine receptor expression profiles on the peripheral blood mononuclear cells (PBMCs) of SLE patients.We also analyzed the correlation between expression level of chemokine receptors and the scores of SLE disease activity index (SLEDAI) or clinical features.

PATIENTS AND METHODS

Patients

After the approval of Medical Ethics Committee of Chinese Academy of Medical Sciences,93 patients (84 females and 9 males) who met at least four of the revised American Rheumatism Association (ARA) criteria11were enrolled into the study from the cohort of Chinese SLE patients in Hospital for Skin Diseases.The average age was 31.73±1.88 years (range,12-59).SLE disease activity was assessed by SLEDAI.12Inactive disease was defined as SLEDAI score ≤7 (40 cases),and active disease as SLEDAI score ＞7(53 cases).Thirty healthy volunteers (4 males and 26 females),whose average age was 30.63±1.05 years(range,18-48),were also enrolled.The clinical manifestations and the results of laboratory tests including complete blood count,routine urine test,erythrocyte sedimentation rate,lupus erythematosus cell test,serum level of complements (C3,C4,and CH50),circulating immune complex,antinuclear antibody (ANA),anti-ENA mutipeptide,anti-dsDNA,etc,were recorded for analyzing the clinical features.All the enrolled volunteers signed informed consent forms approved by the Ethics Committee.

Sampling peripheral blood

Blood was collected into sterile tubes containing 1.5%EDTA.PBMCs were isolated with lymphocyte-separation medium by the density gradient separation.

Protocol for reverse transcription-polymerase chain reaction (RT-PCR)

mRNA was extracted from PBMCs with TRIzol agent(GIBICO/BRL,Paisley,Scotland,UK).After precipitation with ethanol,the first-strand cDNA was reversely transcribed using SuperScriptTMFirst-strand Synthesis System(GIBICO/BRL).Amplification of cDNA was performed as described in protocol (Tag polymerase,Promega,USA).cDNAs of chemokine receptors and interleukin (IL) receptors were amplified by PCR on GeneAmp PCR System 2700 (Applera Corporation-Applied Biosystems,CA,USA).The sequences of sense and antisense primers used are listed in Table 1.13,14The PCR conditions included preheating at 94°C for 5 minutes followed by 38 cycles of denaturation at 94°C for 30 seconds,annealing at 55°C-66°C for 30 seconds,elongation at 72°C for 1 minute,and the final elongation at 72°C for 7 minutes.PCR products were electrophoresed on 2% agarose gel and visualized with ethidium bromide.The densities of target gene mRNA bands were semi-quantified with β-actin mRNA by Quantity One 1-D (BioRad Corporation,Sundbyberg,Sweden).

Statistical analysis

All data were expressed as means±SEM.The differences in mRNA expressions of chemokine receptors and IL receptors among active SLE group,inactive SLE group,and healthy control group were analyzed byt-test.The correlations of clinical data including SLEDAI with chemokine receptors and IL receptors mRNA expression levels were investigated using linear regression analysis (Pearson’s correlation).To determine the statistical significance of each comparison,random permutation was performed to define theP-value thresholds.Differences were considered significant ifPvalues were lower than 0.05.Analysis was performed with SPSS 16.0.

Table 1.Primer sequences for PCR reactions

To assess the possible role of chemokine receptors in the organ-specific disease involvement,we identified those patients who showed evidence of active disease as determined by the organ specific features of Physician’s Global Assessment,SLEDAI,or laboratory testing.The patients were accordingly divided into positive and negative groups,and Student’s unpairedt-tests were used to compare the difference in chemokine receptor levels between the two groups.Some cases without sufficient clinical data were excluded from the analyses.

RESULTS

mRNA expression levels of chemokine and IL receptors on PBMCs from SLE patients

The mean expression levels of CCR2,CCR3,CCR4,CCR6,CX3CR1,CXCR5,XCR1,and IL-4R mRNAs in active SLE group were significantly higher than those in the other two groups (allP＜0.05).The mean level of CCR5 mRNA in SLE patients (including active and inactive) was significantly higher than that in healthy control (P＜0.05),and there was no difference between active and inactive patients(P＞0.05).CX3CR1 mRNA in inactive group was also significantly higher than that in controls (P＜0.05).The levels of CXCR3 and IL-10R mRNAs in active SLE group were significantly higher than those in inactive SLE group(P＜0.05),while there was no statistical difference in the mean expression levels of CCR8,CXCR3,and IL-10R between active SLE group and healthy control group (allP＞0.05) (Fig.1,Table 2).

Correlation between SLEDAI and mRNA expression levels of chemokine receptors and IL receptors

There were positive correlations between SLEDAI and mRNA expression levels of some chemokine receptors,including CCR2 (r=0.424,t=4.313,P＜0.001),CCR3 (r=0.518,t=5.410,P＜0.001),CCR4 (r=0.376,t=3.851,P＜0.001),CCR6 (r=0.457,t=4.513,P＜0.001),CXCR5 (r=0.455,t=4.629,P＜0.001),CX3CR1 (r=0.445,t=4.523,P＜0.001),XCR1 (r=0.540,t=5.445,P＜0.001),except for CCR5 (r=0.062,t=-0.589,P>0.05).No significant correlation was found between SLEDAI and IL-4R (r=0.085,t=-0.611,P>0.05) or IL-10R mRNA expression levels (r=0.066,t=0.473,P>0.05).

Correlation between mRNA expression levels of chemokine receptors and IL receptors

Between chemokine receptors and IL receptors,there was a positive correlation between CCR5 and IL-4R mRNA expression levels (r=0.313,t=2.353,P＜0.05).

Associations between chemokine receptor expression and clinical features

CCR2 mRNA expression significantly lowered in patients with myositis (95%CI0.48-2.61,P＜0.01) or thrombocytopenia (95%CI0.46-3.12,P＜0.01) than that in patients without these two features.The other similar links between the lower mRNA levels of chemokine receptors and clinical features were as follows:CCR3 associated with myositis(95%CI1.85-4.91,P＜0.001);CCR4 with cutaneous vasculitis (95%CI0.01-2.10,P＜0.05);CCR5 with myositis (95%CI1.84-5.34,P＜0.001),cutaneous vasculitis(95%CI0.88-5.72,P＜0.01),and photosensitivity (95%CI1.26-5.69,P＜0.05);CXCR3 with renal disorder (95%CI0.13-1.73,P＜0.05) and C3 decrease (95%CI0.32-2.09,P＜0.001);CXCR5 with myositis (95%CI1.67-4.42,P＜0.001),hemolytic anemia (95%CI2.23-5.31,P＜0.001),leukopenia (95%CI0.72-4.69,P＜0.01),elevated serum IgA (95%CI1.54-4.74,P＜0.001),and positive anti-Ro/SSA (Sj?gren’s syndrome antigen) antibody (95%CI0.18-4.36,P＜0.05);CX3CR1 with myositis (95%CI1.02-3.89,P＜0.01),cutaneous vasculitis (95%CI0.63-4.69,P＜0.05),renal disorder (95%CI0.13-1.73,P＜0.05),and thrombocytopenia (95%CI0.86-4.69,P＜ 0.01);and XCR1 with positive anti-Ro/SSA antibody (95%CI0.59-3.60,P＜0.01)(Table 3).

Figure 1.RT-PCR products for chemokine receptor and interleukin receptor mRNAs on PBMCs of SLE patients detected with ethidium bromide-stained agarose gel.

Correlation between the mRNA level of chemokine receptor with clinical features and SLEDAI

We found that only the lowered level of CCR5 expression was negatively correlated with the scores of SLEDAI in the SLE cases with photosensitivity (r=0.426,t=-2.155,P＜0.05).

DISCUSSION

Many autoimmune diseases are mainly characterized by lymphocyte and monocyte infiltration in target tissues,leading to the damage of these tissues characterized by accumulation and activation of specific leucocytes attracted by chemokines.The composition of infiltrating cells mainly depends on the chemokine receptors expressed on these cells and the kind of chemokine produced at the location,which is somewhat associated with the activity of the disease.The levels of serum chemokines were proposed to be convenient biomarkers for disease activity in lupus.15In our research,the chemokine receptor expression profiles were found associated with the disease activity in SLE patients.

To compare the expression levels of these markers,we applied semi-quantitative RT-PCR since it is a more precise tool to quantify gene expression.First,this study discovered over-expressions of CCR2,CCR3,CCR4,CCR6,CX3CR1,CXCR5,XCR1,and IL-4R in active SLE patients compared with inactive patients and controls.The increased mRNA expressions of CCR2,CCR3,CCR4,CCR6,andCXCR5 on PBMCs in active SLE patients were positively correlated with disease activity.These data conform with the previously reported studies and suggest that Th2 cells play a predominant role in SLE.

Table 2.Comparison of chemokine receptor and IL receptor mRNA expressions on PBMCs among active SLE,inactive SLE,and healthy control groups§

Table 3.Correlation of chemokine receptor expression with clinical parameters in SLE patients§

Second,the mRNA levels of CXCR3 and IL-10R in active patients were significantly higher than those in inactive patients,but there were no significant difference between active patients and controls or between inactive patients and controls.The reason might be the limited size of samples in this study.The expressions of CXCR3 are possibly enriched in kidney-infiltrating cells,as reported in previous studies,16,17but there were only 19 cases with renal disorder in our study.

Last,the level of CCR5 mRNA in both active and inactive SLE patients was significantly higher than that in controls.This result is slightly different from Al-Saleh’s report in which patients in active SLE group had significantly elevated expression of CCR5 on the surface of CD4+T lymphocytes compared with SLE patients in remission and healthy controls.18In this study,CX3CR1 mRNA expression was the highest in active group,and it showed positive correlation with SLEDAI.CX3CR1 is the receptor of CX3CL1 (fractalkine),which is released by activated endothelial cells,activated platelets,injured subendothelial parenchymal cells,and dendritic cells.19-21Upregulated in inflammatory tissues,CX3CL1 not only serves as an adhesion molecule and chemokine,but also participates in an amplification circuit of polarized Th1 responses.22The up-regulation of CX3CR1 in our study suggests its involvement in the Th1/Th2 imbalance of SLE,and in multi-organ injury except for vascular damage.

In order to observe whether there were abnormal IL expressions in the same SLE patients,we analyzed the correlation between IL-4R and IL-10R with chemokine receptors.There was positive correlation between the mRNA expression of CCR5 and that of IL-4R.We previously reported a positive correlation between IL-4R Ile50/Ile50 genotype and susceptibility to SLE (P=0.022).23It was showed that IL-4R gene dysfunction led to its abnormal over-expression.Although IL-10R expression in active patients was significantly higher than that in inactive patients,there was no significant difference between SLE patients and controls in this respect.This revealed that IL-4R was more related to the pathogenesis of SLE than IL-10R.Interestingly,in the present study,there was a positive correlation between CCR5 and IL-4R mRNA levels,suggesting some possible linkage between these two receptors.The up-regulated expression of CCR5 mRNA in PBMCs of SLE patients suggests its roles in the pathogenesis of SLE,which may be involved in newly activated CD4+T cells although there is no correlation between CCR5 level and SLEDAI.Further study is still needed.

Furthermore,the lowered expression levels of some chemokine receptors in SLE patients were found associated with some clinical features,suggesting that these receptors probably play some roles in preventing or interdicting the development of these symptoms or signs.Each individual clinical parameter is linked to one or more declining chemokine receptor,but their clinical significance is unclear and needs further investigation.Among these receptors,only CCR5 and CX3CR1 showed over-expressions in both active and inactive SLE patients,and down-expressions with both cutaneous vasculitis and myositis in SLE patients;the lowered level of CCR5 expression was negatively correlated with the scores of SLEDAI in cases accompanied by photosensitivity.We therefore propose the following scenario:injury induces the release of selfantigen,sending a tissue-specific signal to cells of innate and adaptive immune system to migrate to the site of injury and eliminate pathogens and cellular debris,inducing molecular marker expression on lymphocytes.The CCR5 HHE/HHG*2 genotype was associated with the maximal risk of developing SLE.These findings implicate a key role of CCR5 and CX3CR1 in the pathogenesis of SLE.24The levels of CCR5 and CX3CR1 mRNAs might act as indicators of cutaneous vasculitis and myositis in SLE.

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