Abstract
Objective Inflammation is implicated to be involved in the pathogenesis of osteoarthritis (OA). CXCL12, also known as stromal cell-derived factor, is the unique identified natural ligand of the G-protein-coupled receptor CXCR4 and exhibits both homeostatic and proinflammatory functions. This study aims to determine whether CXCL12 levels in serum and synovial fluid (SF) of patients with knee OA are correlated with the disease severity.
Methods This study consisted of 252 patients with knee OA and 144 healthy controls. The radiological grading of OA in the knee was performed according to the Kellgren-Lawrence grading system. CXCL12 levels in serum and SF were measured by enzyme-linked immunosorbent assay.
Results Higher levels of serum CXCL12 were found in knee OA patients compared with healthy controls. The CXCL12 levels in SF of knee OA patients with KL grade 4 were significantly elevated compared with those with KL grades 2 and 3. Furthermore, knee OA patients with KL grade 3 had significantly higher SF levels of CXCL12 compared with those with KL grade 2. CXCL12 levels in SF of knee OA patients were significantly correlated with disease severity evaluated by KL grading criteria. However, there were no significant differences in the serum CXCL12 levels between patients with different KL grades.
Conclusion CXCL12 levels in SF were closely related to the radiographic severity of OA. CXCL12 levels in SF may be an alternative biomarker for the progression of OA.
Osteoarthritis (OA) is a prevalent joint disease, which causes pain, stiffness, reduced motion, swelling, crepitus, and disability.1It is characterized by the progressive destruction of articular cartilage with joint-space narrowing, osteophyte formation, subchondral sclerosis, and synovitis.2Risk factors including aging, obesity, being female, smoking, genetics, and joint injury are suggested to contribute to the structural changes of OA.3Although OA is generally considered as a noninflammatory disease, recent studies have indicated that the presence of intra-articular low-grade inflammation contributes to the development and progress of OA.4Large number of inflammation marker, such as soluble P selectin,5interleukin 6 (IL-6), tumor necrosis factor α (TNF-α),6C-reactive protein,7and YKL-40,8in the serum and synovial fluid (SF) of OA patients have been associated with the radiographic severity in patients with knee or hip OA.
Chemokines are a family of soluble peptides, which play important roles in cell movement, morphology, proliferation, and differentiation by binding to a family of 7 transmembrane G protein–coupled receptors.9Chemokines consist of 4 subfamilies including C, CC, CXC, and CX3C structures, each of which possesses different patterns of distribution and expression.10CXCL12, also called stromal cell-derived factor-1 (SDF-1), is a family member of CXC chemokines. It is a potent chemokine, which binds monogamously its receptor, called CXCR4.11Recent study showed that CXCL12 is greatly elevated in synovial fluid (SF) from patients with OA compared with healthy individuals.12Such elevation of CXCL12 concentrations in SF partially resulted from the increased synthesis of CXCL12 by synovial fibroblasts under OA condition [12]. High concentrations of CXCL12 in SF could induce chondrocyte death during the OA process.13Another study indicated that CXCL12 increased the release of matrix metalloprotease 9 (MMP-9) and MMP-13 from chondrocytes in a dose-dependent manner. The mutant CXCR4 blocked the release of MMP-9 and MMP-13 from chondrocytes by retrovirus vector.14These results suggest that CXCL12 may be involved in the mechanism of OA.
Although there were studies about the differences of CXCL12 levels between OA patients and healthy controls, the investigation on the association of CXCL12 levels with disease severity of OA has not been performed. This study aims to determine whether CXCL12 levels in serum and synovial fluid (SF) of patients with knee OA are correlated with the disease severity.
MATERIALS AND METHODS
Patients
A total of 252 patients diagnosed with knee OA according to the criteria of the American College of Rheumatology were enrolled in the present study. Patients were not included in the study if they had acute or chronic inflammatory knee disease or rheumatoid arthritis (RA), systemic or autoimmune diseases, and previous knee trauma; 144 healthy volunteers with no clinical and radiological evidence of OA were also enrolled in the present study. This study was approved by the ethics committee of our hospital, and informed consent was obtained from all subjects.
Radiographic Assessment of OA
Assessment of radiographic severity was performed using the Kellgren and Lawrence (KL) grading system.15Subjects who had radiographic knee OA of KL grade 2 or higher in at least 1 knee were defined as OA patients. 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.
Laboratory Methods
Venous blood samples were collected from all subjects after an overnight fasting. Before any treatment on OA, SF was obtained from OA patients who received the treatment of hyaluronic acid injection for the first time. The blood and SF samples were centrifuged at the speed of 3000 rpm and then stored in -80°C before measuring. The serum samples were diluted into 10 times before analysis. Then, the serum and SF samples were analyzed for CXCL12 using commercially available enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN).
Statistical Analysis
The data are presented as means ± SD or median (interquartile range). Kolmogorov-Smirnov test was performed to analyze the data normality. Unpaired t test, Mann-Whitney U test, or χ 2 test were used to evaluate significance in clinical characteristics between patients with knee OA and healthy controls. CXCL12 levels in serum and SF were compared between knee patients with different KL grades using Kruskal-Wallis test. Statistical significance of the correlation of CXCL12 levels in serum and SF with disease severity was determined using Spearman coefficient and a multinomial logistic regression analyses. As MIF levels were not normally distributed, logarithmic (log) transformed values were used for multiple linear regression analysis. All statistical analysis was performed with SPSS for windows 16.0. Statistical significance was accepted at a level of P value less than 0.05.
RESULTS
Baseline Clinical Parameters
No significant differences were found in age, sex, and body mass index (BMI) between patients with knee OA and healthy control. The baseline clinical parameters of the 2 groups are displayed in Table 1.
The CXCL12 Levels in Serum and SF
CXCL12 levels in serum and SF of knee OA patients and serum levels of CXCL12 in healthy controls are shown in Table 1. Significantly elevated levels of serum CXCL12 were observed in patients with knee OA compared with those in healthy controls (P < 0.001).
CXCL12 Levels in Knee OA Patients With Different KL Grades
The CXCL12 levels in serum and SF of knee OA patients with different KL grades are displayed in Table 2. The CXCL12 levels in SF of knee OA patients with KL grade 4 were significantly elevated compared with those with KL grades 2 and 3. Furthermore, knee OA patients with KL grade 3 had higher SF levels of CXCL12 compared with those with KL grade 2. However, no significant differences in the CXCL12 levels of serum were observed between patients with different KL grades.
Association of Clinical Parameters With KL Grades
Spearmen correlation analysis showed that the CXCL12 levels in SF were positively correlated with KL grades (r = 0.313, P < 0.001) (Fig. 1). However, there was no significant association between serum levels of CXCL12 and KL grades (r = 0.088, P = 0.165) (Fig. 2). Then, we took the characteristics, such as age, sex, BMI, serum, and SF CXCL12 levels, into a multinomial logistic regression model (Table 3). Multinomial logistic regression analysis indicated that CXCL12 levels in SF were positively correlated with KL grades (P < 0.001). No significant association was found between serum levels of CXCL12 and KL grades after multinomial logistic regression analysis (P = 0.116).
DISCUSSION
In this study, we investigated the association between the CXCL12 levels in serum and SF with the radiographic severity of OA. We found that the CXCL12 levels in SF were closely related with the radiographic severity of OA, suggesting that CXCL12 levels in SF may be a biomarker to evaluate the progression of OA. This is the first study to demonstrate that the CXCL12 levels in SF of knee OA patients are correlated with the radiographic severity of OA.
Recent studies provide evidence that inflammation is involved in the pathophysiological process of OA at both its early and end stage.16,17More than one third of the patients with OA showed mild-to-moderate synovial inflammation by histological analysis of synovial biopsies.18Chemokines are chemotactic cytokines and comprise a family of more than 50 members that are structurally related to one of the 4 subgroups of C, CC, CXC, and CX3C chemokines. Many chemokines, such as macrophage inflammatory protein (MIP-1) and RANTES (CCL5), have been found in joint fluid released by synovial tissue.19The present results indicate that knee OA patients had significantly elevated levels of serum CXCL12 compared with healthy controls. CXCL12 levels in SF were closely correlated with the radiographic severity of OA assessed by KL scores. In addition, another study showed elevated levels of CXCL12 in SF from patients with OA compared with healthy individuals. These results suggest that CXCL12 could be proposed as a biomarker for OA progression.
Matrix metalloproteases (MMPs) are a family of structurally related calcium- and zinc-dependent proteolytic enzymes, which play important roles in the degradation of many different components of extracellular matrix (ECM).20During OA and rheumatoid arthritis (RA) conditions, chondrocytes in the joint cartilage release increased amount of MMPs, which leads to cartilage destruction.21Recent evidences have implicated that CXCL12 could induce the expression or release of different MMPs. CXCL12 increased the release of MMP-9 and MMP-13 from chondrocytes in a dose-dependent manner.14,22The interaction of CXCL12 with CXCR4 in chondrocytes resulted in a specific elevation of the release of MMP-3.12In another study, CXCL12 showed an activating role on mature osteoclast by inducing bone-resorbing activity and MMP-9 enzymatic release.23These MMPs are considered as the major proteolytic enzymes involved in the ECM degradation. MMP-13 actively degrades type II collagen, the major type of collagen, in cartilage.24MMP-3 can also activate other MMPs, including collagenase 1, collagenase 3, and gelatinase.25Taken together, the release of different MMPs induced by synovial CXCL12 to maintain normal cartilage homeostasis may become excessive in the OA joint because of the significantly higher levels of CXCL12 in SF within these joints. The excessive production of different MMPs stimulated by CXCL12 may be detrimental in OA cartilage, thereby contributing to their characteristic degradation.
Recent evidence shows that the progression of joint cartilage degeneration in OA is associated with bone modification.26Increased collagen type I synthesis is one of potential mechanism responsible for bone tissue remodeling in OA.26An increased amount of collagen type I was found in OA bone tissue compared with normal bone.27In addition, CXCL12 could significantly induce the expression of collagen type I in osteoblasts from OA patients, therefore contributing to the remodeling process that occurs in the evolution of OA.28This indicates that CXCL12 may lead to joint cartilage destruction indirectly by upregulating the production of collagen type I. Furthermore, high concentrations of CXCL12 in SF were found to induce death of human chondrocytes in a necrosis-dependent manner, which suggests a novel pathological mechanism for OA.13
There are several limitations in this study. First, this is a cross-sectional study performed in a relatively small sample. Therefore, our findings should be validated by further longitudinal studies in a larger population sample. Second, we did not assess CXCL12 levels in SF from healthy controls because of ethical concerns. Third, we did not determine the association of CXCL12 levels in serum and SF with pain of knee OA patients. Further studies are needed to investigate the role of CXCL12 levels for predicting pain in knee OA patients.
In conclusion, knee OA patients had significantly elevated levels of serum CXCL12 compared with healthy controls. CXCL12 levels in SF were positively correlated with the severity of knee OA. CXCL12 levels in SF may be an alternative biomarker for assessing the progression of knee OA.