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Plasma and Synovial Fluid CXCL12 Levels Are Correlated With Disease Severity in Patients With Knee Osteoarthritis
Plasma and synovial fluid CXCL12 levels are correlated with disease severity in patients with knee osteoarthritis Wei He, Mingxin Wang, Yang Wang, Qian wang, Bin Luo PII: DOI: Reference:
S0883-5403(15)00772-X doi: 10.1016/j.arth.2015.08.018 YARTH 54670
To appear in:
Journal of Arthroplasty
Received date: Revised date: Accepted date:
2 July 2015 11 August 2015 18 August 2015
Please cite this article as: He Wei, Wang Mingxin, Wang Yang, wang Qian, Luo Bin, Plasma and synovial fluid CXCL12 levels are correlated with disease severity in patients with knee osteoarthritis, Journal of Arthroplasty (2015), doi: 10.1016/j.arth.2015.08.018
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ACCEPTED MANUSCRIPT Plasma and synovial fluid CXCL12 levels are correlated with disease severity in patients with knee osteoarthritis Wei He1, Mingxin Wang2*, Yang Wang3, Qian wang2, Bin Luo4
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1. Department of Orthopedics, the 305 Hospital of Chinese PLA, Beijing 100017, People’s Republic of China
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2.Department of Orthopedics, General Hospital of Chinese Armed Police Forces, Beijing 100039, People’s Republic of China
ai 200003, People’s Republic of China.
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3.Department of Orthopedics, Changzheng Hospital of Second Miliary Medical University, Shangh
4. Department of Orthopedics, People's Hospital of Poyang Country, Jiangxi Province, 333100, People’s Republic of China
Fax: +86- 010-57976688
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*Corresponding Author: Mingxin Wang; Email: [email protected]; Tel: +86- 010-57976688;
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Address: NO. 69, Yongding Road, Beijing 100039, People’s Republic of China
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Articles type：Original Article
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Running title：CXCL12 are correlated with OA severity
ACCEPTED MANUSCRIPT Plasma and synovial fluid CXCL12 levels are correlated with disease severity in patients with knee osteoarthritis Abstract
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Background
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The aim is to determine whether CXC chemokine ligand-12 (CXCL12) levels in plasma and synovial fluid (SF) of patients with knee osteoarthritis (OA) are correlated with the disease
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aspirating patients to determine CXCL12 levels.
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severity. In addition, we set out to investigate whether a peripheral blood test can avoid
Methods
This study consisted of 244 patients with knee OA and 244 age and sex–matched healthy controls. OA progression was classified based on Kellgren–Lawrence (KL) by evaluating x-ray changes observed in anteroposterior knee radiography. CXCL12 levels in the plasma and SF were
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measured by a quantitative sandwich ELISA.
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Results
Plasma CXCL12 levels were higher in OA patients as compared to controls (P<0.0001). There was
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a positive correlation between levels of CXCL12 and grade (P<0.0001). Base on the ROC curve, the optimal cutoff value of plasma CXCL12 levels as an indicator for screening of OA was
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estimated to be 5.5ng/ml, which yielded a sensitivity of 78.4 % and a specificity of 80.2%, with the area under the curve at 0.850 (95%CI, 0.816-0.889; P<0.0001). In multivariate analysis, there was an increased risk of active OA associated with plasma CXCL12 levels ≥10.5ng/ml (OR 6.76, 95% CI: 3.88–12.53; P<0.0001) after adjusting for possible confounders. Similarly, there was an increased risk of active OA associated with SF CXCL12 levels ≥15.0 ng/ml (OR 8.45, 95% CI: 3.23–18.22; P<0.0001) after adjusting for possible confounders. Conclusion The CXCL12 levels in the plasma and SF may serve as effective biomarkers for the severity of OA. Keywords: CXC chemokine ligand-12, knee osteoarthritis, radiographic severity, plasma, synovial fluid
ACCEPTED MANUSCRIPT Introduction Osteoarthritis (OA) is a chronic degenerative joint disease leading to pain, stiffness, reduced motion, swelling, crepitus, and disability [1]. The knee is the most clinically significant site of
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primary OA involvement [2]. It is likely that numerous etiologic factors, such as aging, obesity, being female, smoking, genetics, joint injury, inflammation, and some mechanical and metabolic
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factors, participate in the pathogenesis and are well accepted risk factors for OA [3]. Although OA is generally considered as a non-inflammatory disease, recent studies have
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indicated that Inflammation involved in the pathogenesis of OA [4]. A series of inflammatory factors in the serum and synovial fluid (SF) have been indicated to be associated with the radiographic severity in patients with knee OA [3]. Recent studies provide evidence that inflammation is involved in the pathophysiological process of OA at both its early and end stage
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[5-7].
Chemokines are small chemoattractant cytokines that play key roles in the accumulation of
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inflammatory cells at the site of inflammation [8]. Based on the conserved cysteine motifs, chemokines are classified into C, CC, CXC and CX3C chemokines [9]. Recently, chemokines such
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as CXCL10 [10], CCL13 [11], and CCL3 [12] have been shown to be involved in the mechanism of OA. Zou et al. [13] found that the CX3CL1 concentrations in serum and SF may serve as an
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effective biomarker for the severity of OA. The CXC chemokine ligand-12 (CXCL12) is a member of the CXC chemokine subfamily that is constitutively expressed in the bone marrow and other tissues [3]. CXCL12 levels in SF had been reported as an alternative biomarker for the progression of osteoarthritis (OA) [4]. However, it is difficult to obtain SF samples. Thus, this study aims to determine whether CXC chemokine ligand-12 (CXCL12) levels in plasma and synovial fluid (SF) of patients with knee Osteoarthritis (OA) are correlated with the disease severity in a cohort of Chinese patients. In addition, we also set out to investigate whether a peripheral blood test can avoid aspirating patients to determine CXCL12 levels.
ACCEPTED MANUSCRIPT Patients and methods Patients From January 2013 to May 2014, a total of 244 patients diagnosed with first ever knee OA
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according to the criteria of the American College of Rheumatology [14] were enrolled in the present study. All patients fulfilled established classification criteria and were followed up for at
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least 1 year to allow confirmation of the accuracy of the diagnosis. Patients with histories of corticosteroids, infectious or inflammatory arthritis, knee injury, aseptic osteonecrosis and
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congenital abnormality, receiving various drugs including analgesics and non-steroidal anti-inflammatory drugs (NSAIDs) were excluded from this study. The patients did not receive any treatment for knee OA before enrolling this study.
Two hundred forty-four age and sex-matched subjects with no clinical and radiological
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evidence of OA served as the control group. The control subjects were enrolled from the Health Physical Examination Center of our hospital. The present study has been approved by the ethics
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committee of xxxx. All participants or their relatives were informed of the study protocol and their written informed consent was obtained.
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Radiographic assessment of OA
Radiographic severity was assessed according to the Kellgren and Lawrence (KL) grading system
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[15](grade 1, doubtful narrowing of joint space and possible osteophytic lipping; grade 2, definite osteophytes and possible narrowing of joint space; grade 3, moderate multiple osteophytes, definite narrowing of joint space, some sclerosis, and possible deformity of bone contour; grade 4, large osteophytes, marked narrowing of joint space, severe sclerosis, and definite deformity of bone contour). The radiographs were taken with the patients standing. The grading of radiographs was performed by an experienced observer who was blinded to the source of subjects. OA patients were defined as having radiographic knee OA of KL grade ≥2 in at least one knee. In our study, patients with grade 4 were considered as active OA. Subjects who had KL grades of 0 for both knees were defined as healthy controls. The grading scale used for analysis was the one found higher upon comparison between both knees.
ACCEPTED MANUSCRIPT Laboratory test Fasting venous blood was collected at 8:00 AM after overnight fasting from all participants in vacutainer tubes and quickly centrifuged to avoid glycolysis. Plasma samples were kept at
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−80°C until assay. Before any treatment on OA, SF was obtained from OA patients. CXCL12 levels of patients and normal controls were blindly assessed simultaneously with a quantitative
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sandwich ELISA kit (Quantikine; R&D Systems, Minneapolis, Minn., USA). The inter-assay and intra-assay coefficients of variation for CXCL12 were shown to be: 3.5-5.5% and 4.2%-6.4%. In
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addition, the other biomarkers, such as white blood cells (WBC), C-reactive protein (CRP), Antistreptolysin-O(ASO) and Rheumatoid factor(RF) were also tested using standard detection methods. Statistical Analysis
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The results were expressed as percentages for categorical variables and as medians (interquartile ranges，IQRs) for continuous variables. The Mann-Whitney U test and chi-square test were used
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to compare the two groups. Correlations among laboratory parameters were analyzed by using
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Spearman’s rank correlation test. Associations between the levels of CXCL12 and OA and radiographic severity were assessed by using ordered logistic regression models with
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multivariate adjustment for possible confounders, for instance, age, gender, BMI, smoking habit, levels of WBC, CRP, ASO and RF. The results were expressed as adjusted OR (odds ratios), with the corresponding 95% confidence interval (CI). Receiver operating characteristic (ROC) curve was used to evaluate the accuracy of CXCL12 in diagnosing OA and radiographic severity. The area under the curve (AUC) was calculated as a measure of the accuracy of the test. All calculations were performed using SPSS for Windows, version 19.0 (SPSS Inc., Chicago, IL, USA). Differences were considered significant at P < 0.05 Results Baseline Clinical Parameters In the study population, 105 (43.0%) were male and the median age was 55 years (IQR, 42–63). 97 patients were defined as Grade 2, 85 were as Grade 3 and 62 were as Grade 4. The baseline
ACCEPTED MANUSCRIPT clinical parameters of patients with knee OA and control subjects are presented in Table 1. The CXCL12 Levels and knee OA risk The results indicated that the plasma CXCL12 levels were significantly higher in OA patients as
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compared to normal controls [7.7(IQR, 4.5-12.4) ng/mL vs. 2.2(IQR, 1.3-4.1)ng/mL; Z=15.136, P<0.0001; Figure 1]. Based on the ROC curve, the optimal cutoff value of plasma CXCL12 levels as
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an indicator for screening of OA was estimated to be 5.5ng/ml, which yielded a sensitivity of 78.4 % and a specificity of 80.2%, with the area under the curve at 0.850 (95%CI, 0.816-0.889;
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P<0.0001; Figure 2A). With an AUC of 0.850, CXCL12 showed a significantly greater discriminatory ability as compared with CPR (AUC, 0.730; 95% CI, 0.685–0.774; P<0.01), ASO (AUC, 0.597; 95% CI, 0.546–0.678; P< 0.001) and RF (AUC, 0.753; 95% CI, 0.707–0.802; P<0.001). Interestingly, the combination model (CXCL12/CRP/ASO/RF) had a higher discriminatory accuracy (AUC, 0.912; 95%
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CI, 0.848-0.947) than those markers alone.
The CXCL12 Levels and radiographic severity
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Plasma CXCL12 levels increased with increasing severity of OA as defined by the KL grades. There was a positive correlation between levels of CXCL12 and KL grade(r=0.461, P<0.0001). Knee OA
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patients with KL grade 4 had higher CXCL12 levels in serum than did those with KL grades 3 and 2[13.9(IQR, 10.4-16.2) ng/mL vs. 7.5(IQR, 4.2-10.0)ng/mL vs. 6.1(IQR, 4.0-8.5)ng/mL; P<0.0001,
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respectively; Figure 3A]. However, no differences in the CXCL12 serum levels were observed between patients with KL grades 2 and 3 (P= 0.144; figure 3A). Similarly, Knee OA patients with KL grade 4 had higher CXCL12 levels in SF than did those with KL grades 3 and 2[21.9(IQR, 16.2-25.3) ng/mL vs. 12.0(IQR, 6.6-16.0)ng/mL vs. 11.7(IQR, 6.0-15.2)ng/mL; P<0.0001, respectively; Figure 3B]. Again, no differences in the CXCL12 levels of SF were observed between patients with KL grades 2 and 3 (P= 0.123; Figure 3B). In addition, there was a correlation between levels of CXCL12 in plasma and in SF(r=0.344, P<0.0001; Figure 4). We also found that there was a positive correlation between plasma levels of CXCL12 levels and CRP(r=0.244, P<0.001), age(r=0.168, P=0.015). Similarly correlation was found between SF levels of CXCL12 levels and CRP(r=0.268, P<0.001), age (r=0.176, P=0.012). Statistical analysis here also revealed no influence of sex, BMI,
ACCEPTED MANUSCRIPT ASO, RF and WBC on CXCL12 in OA patients (P>0.05, respectively). Based on the ROC curve, the optimal cutoff value of plasma CXCL12 levels as an indicator for screening of active OA was estimated to be 10.5 ng/ml, which yielded a sensitivity of 78.4 % and
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a specificity of 80.2%, with the area under the curve at 0.839 (95%CI, 0.707-0.913; P<0.0001; Figure 2B). With an AUC of 0.839, CXCL12 showed a significantly greater discriminatory ability as
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compared with CPR (AUC, 0.722; 95% CI, 0.676–0.771; P<0.01), ASO (AUC, 0.603; 95% CI, 0.547–0.685; P<0.001) and RF (AUC, 0.744; 95% CI, 0.712–0.807; P<0.01). Interestingly, the
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combination model (CXCL12/CRP/ASO/RF) had a higher discriminatory accuracy (AUC, 0.902; 95% CI, 0.837-0.942) than those markers alone. Further, in our study, we found that an increased risk of active OA was associated with plasma CXCL12 levels ≥10.5ng/ml (unadjusted OR 12.32, 95% CI: 5.22–27.15). In multivariate analysis, there was an increased risk of active OA associated with
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plasma CXCL12 levels ≥10.5ng/ml (OR 6.76, 95% CI: 3.88–12.53; P<0.0001) after adjusting for possible confounders.
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Similarly, based on the ROC curve, the optimal cutoff value of SF CXCL12 levels as an indicator for screening of active OA was estimated to be 15.0 ng/ml, which yielded a sensitivity of
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81.3 % and a specificity of 78.4%, with the area under the curve at 0.855 (95%CI, 0.721-0.919; P<0.0001). With an AUC of 0.855, CXCL12 showed a significantly greater discriminatory ability as
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compared with CPR (AUC, 0.754; 95% CI, 0.695–0.802; P<0.01), ASO (AUC, 0.643; 95% CI, 0.589–0.732; P<0.001) and RF (AUC, 0.766; 95% CI, 0.710–0.813; P<0.01). Interestingly, the combination model (CXCL12/CRP/ASO/RF) had a higher discriminatory accuracy (AUC, 0.915; 95% CI, 0.845-0.99) than those markers alone. In multivariate analysis, there was an increased risk of active OA associated with SF CXCL12 levels ≥15.0 ng/ml (OR 8.45, 95% CI: 3.23–18.22; P<0.0001) after adjusting for possible confounders. Discussion Recently, chemokines have been shown to be involved in the mechanism of OA [11]. In ours study, we determined whether CXCL12 levels in plasma and synovial fluid (SF) of patients with knee OA are correlated with the disease severity. We firstly found that, in our cohort, elevated plasma
ACCEPTED MANUSCRIPT levels of CXCL12 was a diagnose marker of knee OA. Elevated plasma and SF levels of CXCL12 may be useful in evaluating the radiographic severity of OA, which were better than traditional biomarkers. Similarly, in another study, stronger response in samples with higher diseases activity
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suggested that CXCL12 may be useful in evaluating the progression of RA as well as in elucidating the mechanisms of disease pathogenesis [16]. In addition, spearman correlation analysis showed
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that CXCL12 levels in plasma and SF were correlated with KL grades in patients with OA (P<0.0001).
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Consistent with our findings, chemokines such as CXCL10 [17], CCL13 [11], and macrophage inflammatory protein 1> (CCL3) [18] were all indicated to be associated with the severity of OA. Xu et al. [4] demonstrate d that the CXCL12 levels in SF of knee OA patients are correlated with the radiographic severity of OA. In our study, the plasma and SF sample were collected. We found
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that there was a correlation between levels of CXCL12 in plasma and in SF. We suggested that plasma levels of CXCL12 had a better prospect for clinical application than SF levels. In addition,
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in our study, plasma and SF levels of ASO, CRP and RF were tested, and we were able to control for those multiple variables.
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Inflammatory factors have been shown to be associated with pathogenesis of OA [5]. CXCL12 may contribute to the development and progression of OA indirectly by enhancing
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inflammation response. In our study, we found that there a positive trend between plasma levels of CXCL12 levels and CRP (P<0.001). However, in multivariate analysis, there was an increased the risk of active OA associated with plasma CXCL12 levels ≥10.5ng/ml (OR: 6.76) after adjusting for possible confounders, such as, CRP, RF and ASO, suggesting the different underlying mechanism of CXCL12 production and consumption from the acute reactive protein. Whether higher circulating CXCL12 level is an accelerator or only be a marker of OA remains uncertain. It is important to discuss whether CXCL12 in patients with OA have pathological roles or just was as indicator. In addition to inflammation, some other roles should be considered. Firstly, recent evidences have implicated that CXCL12 could induce the expression or release of different MMPs. During OA conditions, chondrocytes in the joint cartilage release increased
ACCEPTED MANUSCRIPT amount of MMPs, which leads to cartilage destruction [19]. Second, CXCL12 may lead to joint cartilage destruction indirectly by upregulating the production of collagen type I [20]. One study indicated that synoviocyte-derived CXCL12 accumulates and it was immobilized on heparan
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sulfate molecules of endothelial cells, where it can promote angiogenesis and inflammatory cell infiltration [21].
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This study has several potential limitations. First, the sample size was not large enough to reach definitive conclusions. Further studies with great numbers are warranted. Second, our
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study is of cross-sectional design, and the causative relation must be confirmed by future longitudinal studies. Third, we only tested the levels of CXCL12 at admission. Without serial measurement of the circulating CXCl12 levels, this study yielded no data regarding when and how long biomarkers were elevated in these patients. Fourth, we did not test other chemokines and
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pre-inflammation markers, thus we did not obtain the correlation between those markers and OA. Last, we did not assess the differences of CXCL12 levels in SF between knee OA patients and
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control subjects because of ethical concerns. Conclusions
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In conclusion, CXCL12 levels in plasma and SF were positively correlated with the severity of knee OA. CXCL12 levels in plasma may serve as a new biomarker in addition of the traditional methods
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for assessing the risk and severity of knee OA. In addition, CXCL12 levels in plasma have the potential to serve as a marker for treatment modalities of OA but the clinical applications need further research in the future. Conflict of Interest None
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References 1. Iagnocco A, Naredo E. Osteoarthritis: research update and clinical applications. Rheumatology
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(Oxford) 2012; 51:vii2-vii5.
2. Peer MA, Lane J. The Knee Injury and Osteoarthritis Outcome Score (KOOS): a review of its
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psychometric properties in people undergoing total knee arthroplasty. J Orthop Sports Phys Ther. 2013; 43:20-28.
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3. Sandell LJ. Etiology of osteoarthritis: genetics and synovial joint development. Nat Rev Rheumatol. 2012; 8:77-89.
4. Xu Q, Sun XC, Shang XP, Jiang HS. Association of CXCL12 levels in synovial fluid with the radiographic severity of knee osteoarthritis. J Investig Med 2012; 60(6):898-901.
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5. Pantsulaia I, Kalichman L, Kobyliansky E. Association between radiographic hand osteoarthritis and RANKL, OPG and inflammatory markers. Osteoarthritis Cartilage 2010; 18:1448-1453.
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6. Saxne T, Lindell M, Ma˚nsson B, et al. Inflammation is a feature of the disease process in early knee joint osteoarthritis. Rheumatology (Oxford) 2003;42:903-904.
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7. Appelboom T, Emery P, Tant L, et al. Evaluation of technetium-99m-ciprofloxacin (Infecton) for detecting sites of inflammation in arthritis. Rheumatology (Oxford) 2003; 42:1179-1182.
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8. Iwamoto T, Okamoto H, Toyama Y, et al. Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients. FEBS J. 2008; 275:4448-4455. 9. Nanki T, Imai T, Nagasaka K, et al. Migration of CX3CR1‐positive T cells producing type 1 cytokines and cytotoxic molecules into the synovium of patients with rheumatoid arthritis. Arthritis & Rheumatism, 2002, 46(11): 2878-2883. 10. Saetan N, Honsawek S, Tanavalee A, et al. Association of plasma and synovial fluid interferon-F inducible protein-10 with radiographic severity in knee osteoarthritis. Clin Biochem. 2011; 44:1218-1222 11. Gao F, Tian J, Pan H, et al. Association of CCL13 Levels in Serum and Synovial Fluid With the Radiographic Severity of Knee Osteoarthritis. Journal of Investigative Medicine, 2015, 63(3):
ACCEPTED MANUSCRIPT 545-547. 12. Vangsness CT Jr, Burke WS, Narvy SJ, et al. Human knee synovial fluid cytokines correlated with grade of knee osteoarthritisVa pilot study. Bull NYU Hosp Jt Dis. 2011;69:122-127.
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13. Zou Y, Li Y, Lu L, et al. Correlation of fractalkine concentrations in serum and synovial fluid with the radiographic severity of knee osteoarthritis. Annals of Clinical Biochemistry 2013;
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50:571-575.
14. Aletaha D, Neogi T, SIlman A, Funovits J, Felson DT et al. 2010 rheumatoid arthritis
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classification criteria: an America college of rhuematology/European League against Rheumatism collaborative initiative. Ann Rheum Dis 2010; 69:1580–1588. 15. Kellgren JH, Lawrence JS. Radiological assessment of osteo-arthrosis. Ann Rheum Dis 1957; 16: 494–502.
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16. Veselinovic M, Barudzic N, Vuletic M, et al. Oxidative stress in rheumatoid arthritis patients: relationship to diseases activity. Molecular and cellular biochemistry, 2014, 391(1-2): 225-232.
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17. Saetan N, Honsawek S, Tanavalee A, et al. Association of plasma and synovial fluid interferon-F inducible protein-10 with radiographic severity in knee osteoarthritis. Clin Biochem.
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2011; 44:1218-1222.
18. Zhao X Y, Yang Z B, Zhang Z J, et al. CCL3 serves as a potential plasma biomarker in knee
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degeneration (osteoarthritis). Osteoarthritis Cartilage, 2015. 19. Lotz M, Blanco FJ, von Kempis J, et al. Cytokine regulation of chondrocyte functions. J Rheumatol Suppl. 1995; 43:104-108. 20. Lajeunesse D, Reboul P. Subchondral bone in osteoarthritis: a biologic link with articular cartilage leading to abnormal remodeling. Curr Opin Rheumatol. 2003; 15:628-633. 21. Pablos J L, Santiago B, Galindo M, et al. Synoviocyte-derived CXCL12 is displayed on endothelium and induces angiogenesis in rheumatoid arthritis. The Journal of Immunology, 2003, 170(4): 2147-2152.
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Table 1 Baseline characteristics of patients with knee OA and control cases
(n=244)
(n=244) 55(42-63)
NS
43.0
NS
25.4(23.9-27.5)
25.2(23.8-27.4)
NS
47.1
41.0
NS
9.43(8.16-11.55)
8.24(6.28-9.13)
<0.001
4.6(2.3-11.2)
1.6(0.8-3.4)
<0.001
88.4(38.2-168.3)
46.2(28.9-81.2)
<0.001
5.8(1.3-12.6)
1.5(0.8-2.4)
<0.001
7.7(4.5-12.7)
2.2(1.3-4.1)
<0.001
55(42-63)
Male sex (%)
43.0
BMI(kg/m2) , median(IQR) Smoking habit (%) Laboratory findings (median, IQR) Plasma levels 3
-1
Leucocyte count (×10 m L ) -1
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CRP(mg L ) -1
ASO(IU L ) -1
RF(IU L ) -1
CXCL12(ng mL ) SF levels 3
-1
WBC (×10 m L )
—
9.43(8.16-11.55)
-1
CRP(mg L )
5.2(2.9-13.2)
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ASO(IU L ) -1
RF(IU L ) -1
CXCL12(ng mL )
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KL Grades (%) Grade 1
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Grade 2 Grade 3
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Normal cases
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Age (years), median(IQR)
Patients
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Characteristics
112.4(40.3-189.5) 7.8(4.3-16.4) 9.8(5.4-16.2) — 39.8 34.8 25.4
NS, not significant；IQR, interquartile range; OA, Osteoarthritis; CXCL12, CXC chemokine ligand-12; WBC, white
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blood cells; CRP, C-reactive protein; ASO, Antistreptolysin-O; RF, Rheumatoid factor; SF, synovial fluid; KL, Kellgren–Lawrence.
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Figure legends
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Figure 1. Plasma levels of CXCL12 in patients with knee OA and controls. All data are medians and
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in-terquartile ranges (IQR). Mann–Whitney U-test. CXCL12=CXC chemokine ligand-12; OA= Osteoarthritis.
of
1-specificity
for
diagnosing
OA
based
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Figure 2. (A) Receiver operator characteristic (ROC) curve demonstrating sensitivity as a function the
combined
model
incorporating
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(CXCL12/RF/CRP/ASO) and the relative contribution of each plasma biomarker alone (initial cohort). This combined model had an area under the receiver operator characteristic curve of (AUC, 0.912; 95% CI, 0.848-0.947). (B) Receiver operator characteristic (ROC) curve demonstrating sensitivity as a function of 1-specificity for diagnosing OA active based on the
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combined model incorporating (CXCL12/RF/CRP/ASO) and the relative contribution of each plasma biomarker alone (initial cohort). This combined model had an area under the receiver
chemokine
ligand-12;
CRP=C-reactive
protein;
ASO=Antistreptolysin-O;
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CXCL12=CXC
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operator characteristic curve of (AUC, 0.902; 95% CI, 0.837-0.942). OA=Osteoarthritis;
RF=Rheumatoid factor.
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Figure 3. Levels of CXCL12 in different KL grade. (A) Plasma levels of CXCL12 in different KL grade; (B) SF levels of CXCL12 in different KL grade. All data are medians and in-terquartile ranges (IQR). Mann–Whitney U-test. CXCL12=CXC chemokine ligand-12; OA= Osteoarthritis; SF=Synovial fluid Figure 4. The correlation between plasma CXCL12 levels and SF CXCL12 levels. CXCL12=CXC chemokine ligand-12; SF=Synovial fluid
ACCEPTED MANUSCRIPT Acknowledgement This project was supported by the Funds of General Hospital of Chinese Armed Police Forces (No. WZ 2014049). The funders had no role in study design, data collection and analysis, decision to
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publish, or preparation of the manuscript. We also express our gratitude to all the patients, the
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nurses and physicians who participated in this study, and thereby made this work possible.
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S0883-5403(15)00772-X doi: 10.1016/j.arth.2015.08.018 YARTH 54670
To appear in:
Journal of Arthroplasty
Received date: Revised date: Accepted date:
2 July 2015 11 August 2015 18 August 2015
Please cite this article as: He Wei, Wang Mingxin, Wang Yang, wang Qian, Luo Bin, Plasma and synovial fluid CXCL12 levels are correlated with disease severity in patients with knee osteoarthritis, Journal of Arthroplasty (2015), doi: 10.1016/j.arth.2015.08.018
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Plasma and synovial fluid CXCL12 levels are correlated with disease severity in patients with knee osteoarthritis Wei He1, Mingxin Wang2*, Yang Wang3, Qian wang2, Bin Luo4
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1. Department of Orthopedics, the 305 Hospital of Chinese PLA, Beijing 100017, People’s Republic of China
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2.Department of Orthopedics, General Hospital of Chinese Armed Police Forces, Beijing 100039, People’s Republic of China
ai 200003, People’s Republic of China.
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3.Department of Orthopedics, Changzheng Hospital of Second Miliary Medical University, Shangh
4. Department of Orthopedics, People's Hospital of Poyang Country, Jiangxi Province, 333100, People’s Republic of China
Fax: +86- 010-57976688
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*Corresponding Author: Mingxin Wang; Email: [email protected]; Tel: +86- 010-57976688;
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Address: NO. 69, Yongding Road, Beijing 100039, People’s Republic of China
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Articles type：Original Article
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Running title：CXCL12 are correlated with OA severity
ACCEPTED MANUSCRIPT Plasma and synovial fluid CXCL12 levels are correlated with disease severity in patients with knee osteoarthritis Abstract
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Background
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The aim is to determine whether CXC chemokine ligand-12 (CXCL12) levels in plasma and synovial fluid (SF) of patients with knee osteoarthritis (OA) are correlated with the disease
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aspirating patients to determine CXCL12 levels.
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severity. In addition, we set out to investigate whether a peripheral blood test can avoid
Methods
This study consisted of 244 patients with knee OA and 244 age and sex–matched healthy controls. OA progression was classified based on Kellgren–Lawrence (KL) by evaluating x-ray changes observed in anteroposterior knee radiography. CXCL12 levels in the plasma and SF were
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measured by a quantitative sandwich ELISA.
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Results
Plasma CXCL12 levels were higher in OA patients as compared to controls (P<0.0001). There was
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a positive correlation between levels of CXCL12 and grade (P<0.0001). Base on the ROC curve, the optimal cutoff value of plasma CXCL12 levels as an indicator for screening of OA was
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estimated to be 5.5ng/ml, which yielded a sensitivity of 78.4 % and a specificity of 80.2%, with the area under the curve at 0.850 (95%CI, 0.816-0.889; P<0.0001). In multivariate analysis, there was an increased risk of active OA associated with plasma CXCL12 levels ≥10.5ng/ml (OR 6.76, 95% CI: 3.88–12.53; P<0.0001) after adjusting for possible confounders. Similarly, there was an increased risk of active OA associated with SF CXCL12 levels ≥15.0 ng/ml (OR 8.45, 95% CI: 3.23–18.22; P<0.0001) after adjusting for possible confounders. Conclusion The CXCL12 levels in the plasma and SF may serve as effective biomarkers for the severity of OA. Keywords: CXC chemokine ligand-12, knee osteoarthritis, radiographic severity, plasma, synovial fluid
ACCEPTED MANUSCRIPT Introduction Osteoarthritis (OA) is a chronic degenerative joint disease leading to pain, stiffness, reduced motion, swelling, crepitus, and disability [1]. The knee is the most clinically significant site of
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primary OA involvement [2]. It is likely that numerous etiologic factors, such as aging, obesity, being female, smoking, genetics, joint injury, inflammation, and some mechanical and metabolic
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factors, participate in the pathogenesis and are well accepted risk factors for OA [3]. Although OA is generally considered as a non-inflammatory disease, recent studies have
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indicated that Inflammation involved in the pathogenesis of OA [4]. A series of inflammatory factors in the serum and synovial fluid (SF) have been indicated to be associated with the radiographic severity in patients with knee OA [3]. Recent studies provide evidence that inflammation is involved in the pathophysiological process of OA at both its early and end stage
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[5-7].
Chemokines are small chemoattractant cytokines that play key roles in the accumulation of
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inflammatory cells at the site of inflammation [8]. Based on the conserved cysteine motifs, chemokines are classified into C, CC, CXC and CX3C chemokines [9]. Recently, chemokines such
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as CXCL10 [10], CCL13 [11], and CCL3 [12] have been shown to be involved in the mechanism of OA. Zou et al. [13] found that the CX3CL1 concentrations in serum and SF may serve as an
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effective biomarker for the severity of OA. The CXC chemokine ligand-12 (CXCL12) is a member of the CXC chemokine subfamily that is constitutively expressed in the bone marrow and other tissues [3]. CXCL12 levels in SF had been reported as an alternative biomarker for the progression of osteoarthritis (OA) [4]. However, it is difficult to obtain SF samples. Thus, this study aims to determine whether CXC chemokine ligand-12 (CXCL12) levels in plasma and synovial fluid (SF) of patients with knee Osteoarthritis (OA) are correlated with the disease severity in a cohort of Chinese patients. In addition, we also set out to investigate whether a peripheral blood test can avoid aspirating patients to determine CXCL12 levels.
ACCEPTED MANUSCRIPT Patients and methods Patients From January 2013 to May 2014, a total of 244 patients diagnosed with first ever knee OA
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according to the criteria of the American College of Rheumatology [14] were enrolled in the present study. All patients fulfilled established classification criteria and were followed up for at
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least 1 year to allow confirmation of the accuracy of the diagnosis. Patients with histories of corticosteroids, infectious or inflammatory arthritis, knee injury, aseptic osteonecrosis and
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congenital abnormality, receiving various drugs including analgesics and non-steroidal anti-inflammatory drugs (NSAIDs) were excluded from this study. The patients did not receive any treatment for knee OA before enrolling this study.
Two hundred forty-four age and sex-matched subjects with no clinical and radiological
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evidence of OA served as the control group. The control subjects were enrolled from the Health Physical Examination Center of our hospital. The present study has been approved by the ethics
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committee of xxxx. All participants or their relatives were informed of the study protocol and their written informed consent was obtained.
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Radiographic assessment of OA
Radiographic severity was assessed according to the Kellgren and Lawrence (KL) grading system
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[15](grade 1, doubtful narrowing of joint space and possible osteophytic lipping; grade 2, definite osteophytes and possible narrowing of joint space; grade 3, moderate multiple osteophytes, definite narrowing of joint space, some sclerosis, and possible deformity of bone contour; grade 4, large osteophytes, marked narrowing of joint space, severe sclerosis, and definite deformity of bone contour). The radiographs were taken with the patients standing. The grading of radiographs was performed by an experienced observer who was blinded to the source of subjects. OA patients were defined as having radiographic knee OA of KL grade ≥2 in at least one knee. In our study, patients with grade 4 were considered as active OA. Subjects who had KL grades of 0 for both knees were defined as healthy controls. The grading scale used for analysis was the one found higher upon comparison between both knees.
ACCEPTED MANUSCRIPT Laboratory test Fasting venous blood was collected at 8:00 AM after overnight fasting from all participants in vacutainer tubes and quickly centrifuged to avoid glycolysis. Plasma samples were kept at
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−80°C until assay. Before any treatment on OA, SF was obtained from OA patients. CXCL12 levels of patients and normal controls were blindly assessed simultaneously with a quantitative
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sandwich ELISA kit (Quantikine; R&D Systems, Minneapolis, Minn., USA). The inter-assay and intra-assay coefficients of variation for CXCL12 were shown to be: 3.5-5.5% and 4.2%-6.4%. In
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addition, the other biomarkers, such as white blood cells (WBC), C-reactive protein (CRP), Antistreptolysin-O(ASO) and Rheumatoid factor(RF) were also tested using standard detection methods. Statistical Analysis
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The results were expressed as percentages for categorical variables and as medians (interquartile ranges，IQRs) for continuous variables. The Mann-Whitney U test and chi-square test were used
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to compare the two groups. Correlations among laboratory parameters were analyzed by using
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Spearman’s rank correlation test. Associations between the levels of CXCL12 and OA and radiographic severity were assessed by using ordered logistic regression models with
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multivariate adjustment for possible confounders, for instance, age, gender, BMI, smoking habit, levels of WBC, CRP, ASO and RF. The results were expressed as adjusted OR (odds ratios), with the corresponding 95% confidence interval (CI). Receiver operating characteristic (ROC) curve was used to evaluate the accuracy of CXCL12 in diagnosing OA and radiographic severity. The area under the curve (AUC) was calculated as a measure of the accuracy of the test. All calculations were performed using SPSS for Windows, version 19.0 (SPSS Inc., Chicago, IL, USA). Differences were considered significant at P < 0.05 Results Baseline Clinical Parameters In the study population, 105 (43.0%) were male and the median age was 55 years (IQR, 42–63). 97 patients were defined as Grade 2, 85 were as Grade 3 and 62 were as Grade 4. The baseline
ACCEPTED MANUSCRIPT clinical parameters of patients with knee OA and control subjects are presented in Table 1. The CXCL12 Levels and knee OA risk The results indicated that the plasma CXCL12 levels were significantly higher in OA patients as
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compared to normal controls [7.7(IQR, 4.5-12.4) ng/mL vs. 2.2(IQR, 1.3-4.1)ng/mL; Z=15.136, P<0.0001; Figure 1]. Based on the ROC curve, the optimal cutoff value of plasma CXCL12 levels as
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an indicator for screening of OA was estimated to be 5.5ng/ml, which yielded a sensitivity of 78.4 % and a specificity of 80.2%, with the area under the curve at 0.850 (95%CI, 0.816-0.889;
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P<0.0001; Figure 2A). With an AUC of 0.850, CXCL12 showed a significantly greater discriminatory ability as compared with CPR (AUC, 0.730; 95% CI, 0.685–0.774; P<0.01), ASO (AUC, 0.597; 95% CI, 0.546–0.678; P< 0.001) and RF (AUC, 0.753; 95% CI, 0.707–0.802; P<0.001). Interestingly, the combination model (CXCL12/CRP/ASO/RF) had a higher discriminatory accuracy (AUC, 0.912; 95%
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CI, 0.848-0.947) than those markers alone.
The CXCL12 Levels and radiographic severity
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Plasma CXCL12 levels increased with increasing severity of OA as defined by the KL grades. There was a positive correlation between levels of CXCL12 and KL grade(r=0.461, P<0.0001). Knee OA
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patients with KL grade 4 had higher CXCL12 levels in serum than did those with KL grades 3 and 2[13.9(IQR, 10.4-16.2) ng/mL vs. 7.5(IQR, 4.2-10.0)ng/mL vs. 6.1(IQR, 4.0-8.5)ng/mL; P<0.0001,
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respectively; Figure 3A]. However, no differences in the CXCL12 serum levels were observed between patients with KL grades 2 and 3 (P= 0.144; figure 3A). Similarly, Knee OA patients with KL grade 4 had higher CXCL12 levels in SF than did those with KL grades 3 and 2[21.9(IQR, 16.2-25.3) ng/mL vs. 12.0(IQR, 6.6-16.0)ng/mL vs. 11.7(IQR, 6.0-15.2)ng/mL; P<0.0001, respectively; Figure 3B]. Again, no differences in the CXCL12 levels of SF were observed between patients with KL grades 2 and 3 (P= 0.123; Figure 3B). In addition, there was a correlation between levels of CXCL12 in plasma and in SF(r=0.344, P<0.0001; Figure 4). We also found that there was a positive correlation between plasma levels of CXCL12 levels and CRP(r=0.244, P<0.001), age(r=0.168, P=0.015). Similarly correlation was found between SF levels of CXCL12 levels and CRP(r=0.268, P<0.001), age (r=0.176, P=0.012). Statistical analysis here also revealed no influence of sex, BMI,
ACCEPTED MANUSCRIPT ASO, RF and WBC on CXCL12 in OA patients (P>0.05, respectively). Based on the ROC curve, the optimal cutoff value of plasma CXCL12 levels as an indicator for screening of active OA was estimated to be 10.5 ng/ml, which yielded a sensitivity of 78.4 % and
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a specificity of 80.2%, with the area under the curve at 0.839 (95%CI, 0.707-0.913; P<0.0001; Figure 2B). With an AUC of 0.839, CXCL12 showed a significantly greater discriminatory ability as
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compared with CPR (AUC, 0.722; 95% CI, 0.676–0.771; P<0.01), ASO (AUC, 0.603; 95% CI, 0.547–0.685; P<0.001) and RF (AUC, 0.744; 95% CI, 0.712–0.807; P<0.01). Interestingly, the
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combination model (CXCL12/CRP/ASO/RF) had a higher discriminatory accuracy (AUC, 0.902; 95% CI, 0.837-0.942) than those markers alone. Further, in our study, we found that an increased risk of active OA was associated with plasma CXCL12 levels ≥10.5ng/ml (unadjusted OR 12.32, 95% CI: 5.22–27.15). In multivariate analysis, there was an increased risk of active OA associated with
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plasma CXCL12 levels ≥10.5ng/ml (OR 6.76, 95% CI: 3.88–12.53; P<0.0001) after adjusting for possible confounders.
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Similarly, based on the ROC curve, the optimal cutoff value of SF CXCL12 levels as an indicator for screening of active OA was estimated to be 15.0 ng/ml, which yielded a sensitivity of
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81.3 % and a specificity of 78.4%, with the area under the curve at 0.855 (95%CI, 0.721-0.919; P<0.0001). With an AUC of 0.855, CXCL12 showed a significantly greater discriminatory ability as
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compared with CPR (AUC, 0.754; 95% CI, 0.695–0.802; P<0.01), ASO (AUC, 0.643; 95% CI, 0.589–0.732; P<0.001) and RF (AUC, 0.766; 95% CI, 0.710–0.813; P<0.01). Interestingly, the combination model (CXCL12/CRP/ASO/RF) had a higher discriminatory accuracy (AUC, 0.915; 95% CI, 0.845-0.99) than those markers alone. In multivariate analysis, there was an increased risk of active OA associated with SF CXCL12 levels ≥15.0 ng/ml (OR 8.45, 95% CI: 3.23–18.22; P<0.0001) after adjusting for possible confounders. Discussion Recently, chemokines have been shown to be involved in the mechanism of OA [11]. In ours study, we determined whether CXCL12 levels in plasma and synovial fluid (SF) of patients with knee OA are correlated with the disease severity. We firstly found that, in our cohort, elevated plasma
ACCEPTED MANUSCRIPT levels of CXCL12 was a diagnose marker of knee OA. Elevated plasma and SF levels of CXCL12 may be useful in evaluating the radiographic severity of OA, which were better than traditional biomarkers. Similarly, in another study, stronger response in samples with higher diseases activity
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suggested that CXCL12 may be useful in evaluating the progression of RA as well as in elucidating the mechanisms of disease pathogenesis [16]. In addition, spearman correlation analysis showed
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that CXCL12 levels in plasma and SF were correlated with KL grades in patients with OA (P<0.0001).
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Consistent with our findings, chemokines such as CXCL10 [17], CCL13 [11], and macrophage inflammatory protein 1> (CCL3) [18] were all indicated to be associated with the severity of OA. Xu et al. [4] demonstrate d that the CXCL12 levels in SF of knee OA patients are correlated with the radiographic severity of OA. In our study, the plasma and SF sample were collected. We found
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that there was a correlation between levels of CXCL12 in plasma and in SF. We suggested that plasma levels of CXCL12 had a better prospect for clinical application than SF levels. In addition,
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in our study, plasma and SF levels of ASO, CRP and RF were tested, and we were able to control for those multiple variables.
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Inflammatory factors have been shown to be associated with pathogenesis of OA [5]. CXCL12 may contribute to the development and progression of OA indirectly by enhancing
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inflammation response. In our study, we found that there a positive trend between plasma levels of CXCL12 levels and CRP (P<0.001). However, in multivariate analysis, there was an increased the risk of active OA associated with plasma CXCL12 levels ≥10.5ng/ml (OR: 6.76) after adjusting for possible confounders, such as, CRP, RF and ASO, suggesting the different underlying mechanism of CXCL12 production and consumption from the acute reactive protein. Whether higher circulating CXCL12 level is an accelerator or only be a marker of OA remains uncertain. It is important to discuss whether CXCL12 in patients with OA have pathological roles or just was as indicator. In addition to inflammation, some other roles should be considered. Firstly, recent evidences have implicated that CXCL12 could induce the expression or release of different MMPs. During OA conditions, chondrocytes in the joint cartilage release increased
ACCEPTED MANUSCRIPT amount of MMPs, which leads to cartilage destruction [19]. Second, CXCL12 may lead to joint cartilage destruction indirectly by upregulating the production of collagen type I [20]. One study indicated that synoviocyte-derived CXCL12 accumulates and it was immobilized on heparan
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sulfate molecules of endothelial cells, where it can promote angiogenesis and inflammatory cell infiltration [21].
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This study has several potential limitations. First, the sample size was not large enough to reach definitive conclusions. Further studies with great numbers are warranted. Second, our
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study is of cross-sectional design, and the causative relation must be confirmed by future longitudinal studies. Third, we only tested the levels of CXCL12 at admission. Without serial measurement of the circulating CXCl12 levels, this study yielded no data regarding when and how long biomarkers were elevated in these patients. Fourth, we did not test other chemokines and
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pre-inflammation markers, thus we did not obtain the correlation between those markers and OA. Last, we did not assess the differences of CXCL12 levels in SF between knee OA patients and
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control subjects because of ethical concerns. Conclusions
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In conclusion, CXCL12 levels in plasma and SF were positively correlated with the severity of knee OA. CXCL12 levels in plasma may serve as a new biomarker in addition of the traditional methods
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for assessing the risk and severity of knee OA. In addition, CXCL12 levels in plasma have the potential to serve as a marker for treatment modalities of OA but the clinical applications need further research in the future. Conflict of Interest None
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References 1. Iagnocco A, Naredo E. Osteoarthritis: research update and clinical applications. Rheumatology
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(Oxford) 2012; 51:vii2-vii5.
2. Peer MA, Lane J. The Knee Injury and Osteoarthritis Outcome Score (KOOS): a review of its
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psychometric properties in people undergoing total knee arthroplasty. J Orthop Sports Phys Ther. 2013; 43:20-28.
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3. Sandell LJ. Etiology of osteoarthritis: genetics and synovial joint development. Nat Rev Rheumatol. 2012; 8:77-89.
4. Xu Q, Sun XC, Shang XP, Jiang HS. Association of CXCL12 levels in synovial fluid with the radiographic severity of knee osteoarthritis. J Investig Med 2012; 60(6):898-901.
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5. Pantsulaia I, Kalichman L, Kobyliansky E. Association between radiographic hand osteoarthritis and RANKL, OPG and inflammatory markers. Osteoarthritis Cartilage 2010; 18:1448-1453.
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6. Saxne T, Lindell M, Ma˚nsson B, et al. Inflammation is a feature of the disease process in early knee joint osteoarthritis. Rheumatology (Oxford) 2003;42:903-904.
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7. Appelboom T, Emery P, Tant L, et al. Evaluation of technetium-99m-ciprofloxacin (Infecton) for detecting sites of inflammation in arthritis. Rheumatology (Oxford) 2003; 42:1179-1182.
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8. Iwamoto T, Okamoto H, Toyama Y, et al. Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients. FEBS J. 2008; 275:4448-4455. 9. Nanki T, Imai T, Nagasaka K, et al. Migration of CX3CR1‐positive T cells producing type 1 cytokines and cytotoxic molecules into the synovium of patients with rheumatoid arthritis. Arthritis & Rheumatism, 2002, 46(11): 2878-2883. 10. Saetan N, Honsawek S, Tanavalee A, et al. Association of plasma and synovial fluid interferon-F inducible protein-10 with radiographic severity in knee osteoarthritis. Clin Biochem. 2011; 44:1218-1222 11. Gao F, Tian J, Pan H, et al. Association of CCL13 Levels in Serum and Synovial Fluid With the Radiographic Severity of Knee Osteoarthritis. Journal of Investigative Medicine, 2015, 63(3):
ACCEPTED MANUSCRIPT 545-547. 12. Vangsness CT Jr, Burke WS, Narvy SJ, et al. Human knee synovial fluid cytokines correlated with grade of knee osteoarthritisVa pilot study. Bull NYU Hosp Jt Dis. 2011;69:122-127.
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13. Zou Y, Li Y, Lu L, et al. Correlation of fractalkine concentrations in serum and synovial fluid with the radiographic severity of knee osteoarthritis. Annals of Clinical Biochemistry 2013;
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14. Aletaha D, Neogi T, SIlman A, Funovits J, Felson DT et al. 2010 rheumatoid arthritis
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classification criteria: an America college of rhuematology/European League against Rheumatism collaborative initiative. Ann Rheum Dis 2010; 69:1580–1588. 15. Kellgren JH, Lawrence JS. Radiological assessment of osteo-arthrosis. Ann Rheum Dis 1957; 16: 494–502.
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16. Veselinovic M, Barudzic N, Vuletic M, et al. Oxidative stress in rheumatoid arthritis patients: relationship to diseases activity. Molecular and cellular biochemistry, 2014, 391(1-2): 225-232.
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17. Saetan N, Honsawek S, Tanavalee A, et al. Association of plasma and synovial fluid interferon-F inducible protein-10 with radiographic severity in knee osteoarthritis. Clin Biochem.
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18. Zhao X Y, Yang Z B, Zhang Z J, et al. CCL3 serves as a potential plasma biomarker in knee
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degeneration (osteoarthritis). Osteoarthritis Cartilage, 2015. 19. Lotz M, Blanco FJ, von Kempis J, et al. Cytokine regulation of chondrocyte functions. J Rheumatol Suppl. 1995; 43:104-108. 20. Lajeunesse D, Reboul P. Subchondral bone in osteoarthritis: a biologic link with articular cartilage leading to abnormal remodeling. Curr Opin Rheumatol. 2003; 15:628-633. 21. Pablos J L, Santiago B, Galindo M, et al. Synoviocyte-derived CXCL12 is displayed on endothelium and induces angiogenesis in rheumatoid arthritis. The Journal of Immunology, 2003, 170(4): 2147-2152.
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Table 1 Baseline characteristics of patients with knee OA and control cases
(n=244)
(n=244) 55(42-63)
NS
43.0
NS
25.4(23.9-27.5)
25.2(23.8-27.4)
NS
47.1
41.0
NS
9.43(8.16-11.55)
8.24(6.28-9.13)
<0.001
4.6(2.3-11.2)
1.6(0.8-3.4)
<0.001
88.4(38.2-168.3)
46.2(28.9-81.2)
<0.001
5.8(1.3-12.6)
1.5(0.8-2.4)
<0.001
7.7(4.5-12.7)
2.2(1.3-4.1)
<0.001
55(42-63)
Male sex (%)
43.0
BMI(kg/m2) , median(IQR) Smoking habit (%) Laboratory findings (median, IQR) Plasma levels 3
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Leucocyte count (×10 m L ) -1
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CRP(mg L ) -1
ASO(IU L ) -1
RF(IU L ) -1
CXCL12(ng mL ) SF levels 3
-1
WBC (×10 m L )
—
9.43(8.16-11.55)
-1
CRP(mg L )
5.2(2.9-13.2)
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ASO(IU L ) -1
RF(IU L ) -1
CXCL12(ng mL )
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KL Grades (%) Grade 1
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Grade 2 Grade 3
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Normal cases
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Age (years), median(IQR)
Patients
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Characteristics
112.4(40.3-189.5) 7.8(4.3-16.4) 9.8(5.4-16.2) — 39.8 34.8 25.4
NS, not significant；IQR, interquartile range; OA, Osteoarthritis; CXCL12, CXC chemokine ligand-12; WBC, white
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blood cells; CRP, C-reactive protein; ASO, Antistreptolysin-O; RF, Rheumatoid factor; SF, synovial fluid; KL, Kellgren–Lawrence.
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Figure legends
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Figure 1. Plasma levels of CXCL12 in patients with knee OA and controls. All data are medians and
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in-terquartile ranges (IQR). Mann–Whitney U-test. CXCL12=CXC chemokine ligand-12; OA= Osteoarthritis.
of
1-specificity
for
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OA
based
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Figure 2. (A) Receiver operator characteristic (ROC) curve demonstrating sensitivity as a function the
combined
model
incorporating
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(CXCL12/RF/CRP/ASO) and the relative contribution of each plasma biomarker alone (initial cohort). This combined model had an area under the receiver operator characteristic curve of (AUC, 0.912; 95% CI, 0.848-0.947). (B) Receiver operator characteristic (ROC) curve demonstrating sensitivity as a function of 1-specificity for diagnosing OA active based on the
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combined model incorporating (CXCL12/RF/CRP/ASO) and the relative contribution of each plasma biomarker alone (initial cohort). This combined model had an area under the receiver
chemokine
ligand-12;
CRP=C-reactive
protein;
ASO=Antistreptolysin-O;
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CXCL12=CXC
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operator characteristic curve of (AUC, 0.902; 95% CI, 0.837-0.942). OA=Osteoarthritis;
RF=Rheumatoid factor.
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Figure 3. Levels of CXCL12 in different KL grade. (A) Plasma levels of CXCL12 in different KL grade; (B) SF levels of CXCL12 in different KL grade. All data are medians and in-terquartile ranges (IQR). Mann–Whitney U-test. CXCL12=CXC chemokine ligand-12; OA= Osteoarthritis; SF=Synovial fluid Figure 4. The correlation between plasma CXCL12 levels and SF CXCL12 levels. CXCL12=CXC chemokine ligand-12; SF=Synovial fluid
ACCEPTED MANUSCRIPT Acknowledgement This project was supported by the Funds of General Hospital of Chinese Armed Police Forces (No. WZ 2014049). The funders had no role in study design, data collection and analysis, decision to
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publish, or preparation of the manuscript. We also express our gratitude to all the patients, the
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nurses and physicians who participated in this study, and thereby made this work possible.
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