- Email: [email protected]
Association of MIF in serum and synovial fluid with severity of knee osteoarthritis
Clinical Biochemistry 45 (2012) 737–739
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Association of MIF in serum and synovial fluid with severity of knee osteoarthritis Minghui Liu a, Chunhe Hu b,⁎ a b
Department of Trauma, Union Medicine Centre, Tianjin, PR China Department of Hand Surgery, Hebei Medical University third Hospital, Shijiazhuang, PR China
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 7 March 2012 Accepted 8 March 2012 Available online 16 March 2012 Keywords: Macrophage migration inhibitory factor Serum Synovial fluid Severity Osteoarthritis
a b s t r a c t Objective: Recent evidences suggest that inflammation contributes to the development and progression of osteoarthritis (OA). This study aims to determine macrophage migration inhibitory factor (MIF) levels in serum and synovial fluid (SF) of patients with knee OA and to analyze the association of MIF levels with the radiographic severity of OA. Design and methods: 224 patients with knee OA and 186 healthy controls were enrolled in this study. Results: Higher levels of serum MIF were found in knee OA patients compared with healthy controls. Knee OA patients with Kellgren and Lawrence (KL) grade 4 showed significantly elevated MIF levels in serum and SF compared with those with KL grade 2 and 3. MIF levels in serum and SF of knee OA patients were significantly related to disease severity evaluated by KL grading criteria. Conclusion: MIF levels in serum and SF were closely related to the radiographic severity of OA. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Knee osteoarthritis (OA), a common chronic degenerative disease, is characterized by the loss of articular cartilage components due to an imbalance between extracellular matrix destruction and repair [1]. OA leads to functional limitations, reduced quality of life, and even disability [2]. The diagnosis of OA is generally based on clinical and radiographic changes, which reflect disease severity by grading the joint destruction [3]. Nowadays, biochemical markers are studied as a promising indicator for OA. Recent evidences have demonstrated that radiographic grading of OA is correlated with some biochemical markers in synovial fluid (SF) such as P selectin [4], soluble lectinlike oxidized low-density lipoprotein receptor-1 [5], as well as interleukin (IL)-8 and CCL5 [6]. Risk factors such as aging, obesity, being female, smoking, genetics, and joint injury are considered to be associated with OA [7]. However, the etiology and pathogenesis of OA remain poorly understood. In addition, inflammation has been implicated in the pathogenesis of OA [8]. Recent in-vivo and in-vitro studies have demonstrated that chondrocytes can produce and/or respond to a number of inflammatory cytokines and chemokines present in joint tissues and fluids in OA [9]. Macrophage migration inhibitory factor (MIF) was originally identified as a protein derived from T activated lymphocytes [10]. MIF is a proinflammatory cytokine produced by macrophages in response ⁎ Corresponding author at: Department of Hand Surgery, Hebei Medical University third Hospital, 139 Ziqiang Road, 050017, Shijiazhuang, Hebei, PR China. Fax: + 86 0311 88602006. E-mail address: [email protected] (C. Hu).
to a variety of inflammatory stimuli [11]. MIF induces the release of proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), interferon-γ, IL-1β, IL-6, IL-8, nitric oxide, and cyclo-oxygenase 2 [12]. In synovial fibroblasts from rheumatoid arthritis patients, MIF also upregulates the expression of matrix metalloproteinases (MMP)-1, MMP-2, and MMP-3 which play important roles in synovial invasion [13]. In addition, MIF indirectly induces joint destruction by promoting angiogenesis in synovial fibroblasts from rheumatoid arthritis patients [14]. Therefore, MIF may be involved in the mechanism of OA, and MIF levels in the serum and SF may be related to the severity of knee OA. In the present study, we aim to determine the association of MIF concentrations in serum and SF with the radiographic disease severity in patients with knee OA in order to assess its role in OA pathophysiology.
Materials and methods Subjects This study consists of 224 patients diagnosed with knee OA according to the criteria of the American College of Rheumatology. Patients with previous knee surgery, meniscectomy in both knee compartments, osteochondritis dissecans, fracture in or adjacent to the knee, septic arthritis, osteonecrosis, any ligament injury, or radiographic signs of knee OA were excluded. The control group included 186 healthy check-up examinees who matched to the cases by age, gender, and body mass index (BMI). They had no clinical and radiological evidence of OA. This study was approved by the ethics
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2012.03.012
738
M. Liu, C. Hu / Clinical Biochemistry 45 (2012) 737–739
Results
Table 1 The characteristics between patients with knee OA and healthy controls. Characteristics
Knee OA patients (n = 224)
Healthy controls (n = 186)
P value
Age (years) Gender (male/female) BMI (kg/m2) CRP (mg/L) Diabetes (%) Cardiovascular diseases (%) Asthma (%) MIF in serum (ng/mL) MIF in SF (ng/mL)
59.60 ± 7.69 94/130 24.12 ± 3.76 2.88 ± 0.86 24 (10.71%) 13 (5.80%) 8 (3.57%) 15.49 (12.46–19.00) 3.72 (2.88–4.37)
60.39 ± 7.53 71/115 24.41 ± 3.49 1.48 ± 0.46 18 (9.68%) 8 (4.30%) 2 (1.08%)
0.297 0.436 0.427 b 0.001 0.730 0.492 0.103 b 0.001
6.06 (4.97–7.30)
Baseline clinical parameters The baseline clinical parameters of patients with knee OA and healthy controls are shown in Table 1. Knee OA patients showed significantly higher levels of CRP compared with healthy controls (P b 0.001). There were no significant differences in age, gender, and BMI, as well as the percentage of diabetes, cardiovascular diseases, and asthma between the two groups. The MIF levels in serum and SF
committee of our hospital, and informed consent was obtained from all participants. Disease severity assessment was performed using the Kellgren and Lawrence (KL) grading system [15]. OA patients were defined as having radiographic knee OA of KL grade ≥ 2 in at least one knee. Controls were defined as having no radiographic knee OA as indicated by KL grades of 0 for both knees. The grading of the worst affected knee in each patient was used for data analysis.
Laboratory methods Venous blood samples were obtained from all participants after a 12-h overnight fasting. SF was taken from OA patients who received the treatment of hyaluronic acid injection for the first time. Quantitative determination of MIF concentration in serum and SF was performed using commercial enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA). C-reactive protein (CRP) was tested using an auto biochemistry instrument (Hitachi 7170, Tokyo, Japan).
MIF levels in serum and SF of knee OA patients and serum levels of MIF in healthy controls are presented in Table 1. Patients with knee OA had significantly elevated levels of serum MIF compared with those in healthy controls (P b 0.001). MIF levels in knee OA patients with different KL grades The MIF levels in serum and SF of knee OA patients with KL grade 4 were significantly elevated compared with those with KL grade 2 and 3. Furthermore, knee OA patients with KL grade 3 had significantly higher MIF levels in serum and SF compared with those with KL grade 2. The MIF levels in serum and SF of knee OA patients with different KL grades are shown in Table 2. Association of clinical parameters with KL grades Spearman correlation analysis showed that the MIF levels in serum and SF were strongly related to KL grades (r = 0.509, P b 0.001 and r = 0.548, P b 0.001 respectively). Multinomial logistic regression analysis indicated that MIF levels in serum and SF were both positively associated with KL grades (both P b 0.001). Discussion
Statistical analysis The data are presented as means ± SD, median (interquartile range), or frequencies. Kolmogorov–Smirnov test was performed to analyze the data normality. Comparison of the characteristics between patients with knee OA and healthy controls were performed by an unpaired t test, Mann–Whitney U test, or Chi-square test as indicated. Characteristics were compared between knee patients with different KL grades using Kruskal–Wallis test. Correlations of MIF levels in serum and SF with disease severity were calculated using Spearman correlation analysis. A multinomial logistic regression analysis was performed to determine the association of various factors with the severity of OA. As MIF levels were not normally distributed, logarithmic (log) transformed values were used for multiple linear regression analysis. All analyses were performed using the Statistical Package for the Social Sciences program (SPSS for Windows, version 16; Chicago, IL). Statistical significance was accepted at a level of P-value less than 0.05.
Inflammation has been implicated to be involved in the mechanism of OA. Synovial inflammation contributes to an imbalance between the catabolic and anabolic activities of the chondrocyte in remodeling extracellular matrix of the cartilage, and then the presence and progression of cartilage damage [16]. Inflammatory molecules such as interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, and IL-18 have been shown to be potential biomarkers for OA [17]. The current results indicate that serum levels of MIF were significantly elevated in patients with knee OA compared with healthy controls. Furthermore, MIF concentrations were strongly related to KL grades. This is consistent with other studies which demonstrated that OA patients had significantly higher MIF levels in serum and SF than normal controls [12,18]. These results suggest that MIF, a proinflammatory cytokine produced by macrophages, play an important role in the pathogenesis of OA. MIF levels could serve a new biomarker for predicting the presence and progression of OA. A recent study showed that serum levels of MIF in patients with rheumatoid arthritis were also significantly higher compared with healthy controls [19,20].
Table 2 The MIF levels of serum and SF in knee OA patients with different KL grades. MIF (ng/mL)
Grade 2 (n = 72)
Grade 3 (n = 88)
Grade 4 (n = 64)
P value
Serum SF
13.34 (10.97–16.40)a 3.14 (2.55–3.79)a
14.02 (11.28–16.97)b 3.42 (2.81–4.23)b
19.72 (16.87–23.22)b,a 5.18 (3.92–6.26)b,a
b0.001 b0.001
a b
P b 0.01 vs KL grade 3. P b 0.01 vs KL grade 2.
M. Liu, C. Hu / Clinical Biochemistry 45 (2012) 737–739
In addition, synovial MIF immunostaining was found to be related strongly to disease activity as measured by CRP concentration [21]. The mechanisms of OA and rheumatoid arthritis were both associated with inflammation. Therefore, MIF seems to contribute to the presence of OA and rheumatoid arthritis as an inflammatory factor. Degradation of extracellular matrix components is a typical pathological process of OA [22]. The main proteinases responsible for the degradation of extracellular matrix components are matrix metalloproteinases (MMP) [23]. MMP is a large group of zinc-dependent extracellular endopeptidase proteinases involved in the cleavage of extracellular matrix macromolecules [24]. Recent studies have shown that MIF could upregulate the expression of different MMP. MIF induces MMP-2 expression of synovial fibroblast from rheumatoid arthritis patients in a timedependent and concentration-dependent manner [25]. Furthermore, MMP-2 protein levels were significantly decreased in the knee joint of MIF gene-deficient mice compared with wild-type ones [25]. In another study, the expression levels of MMP-1 and MMP-3 mRNA were significantly elevated after stimulation by MIF in synovial fibroblasts from patients with rheumatoid arthritis [13]. Furthermore, MIF was found to up-regulate MMP-9 and -13 in rat osteoblasts [26]. These results indicate that MIF could induce extracellular matrix degradation and then the deterioration of cartilage tissue by promoting the expression of MMP which can digest extracellular matrix. MIF induces the production of inflammatory molecules such as TNF-α, IL-1β, and IL-8 in synovial fibroblasts of patients with rheumatoid arthritis [12,27]. These inflammatory factors have been shown to be associated with pathogenesis of OA. MIF may contribute to the development and progression of OA indirectly by promoting the release of different inflammatory cytokines. On the other hand, MIF was demonstrated to be induced by interferon-γ, CD40 ligand, IL-15, IL-1β, TNF-α, and transforming growth factor-β in synovial fibroblasts of patients with rheumatoid arthritis [28]. Therefore, MIF is hypothesized to interact with inflammatory cytokines and then amply the inflammatory response in chondrocytes, at last lead to the damage of cartilage. There are several limitations in this study. First, this is a crosssectional study performed in a relatively small sample. Therefore, the results of this study need to be confirmed in prospective longitudinal studies with a larger population. Second, we did not assess MIF levels in SF from healthy controls due to ethical concerns. In conclusion, MIF levels in serum and SF were strongly related to the severity of knee OA. MIF levels in serum and SF may serve as a new biomarker in addition of the traditional methods for assessing the risk and severity of knee OA.
References [1] Todhunter PG, Kincaid SA, Todhunter RJ, Kammermann JR, Johnstone B, Baird AN, et al. Immunohistochemical analysis of an equine model of synovitis-induced arthritis. Am J Vet Res 1996;57:1080–93. [2] Guccione AA, Felson DT, Anderson JJ, Anthony JM, Zhang Y, Wilson PW, et al. The effects of specific medical conditions on the functional limitations of elders in the Framingham Study. Am J Public Health 1994;84:351–8. [3] Melikoglu MA, Yildirim K, Senel K. Relationship between radiographic grading of osteoarthritis and serum beta-2 microglobulin. Ir J Med Sci 2009;178:151–4. [4] Cheng T, Li FF, Zhao S, Peng XC, Zhang XL. Soluble P selectin in synovial fluid level is correlated with the radiographic severity of knee osteoarthritis. Clin Chim Acta 2010;411:1529–31.
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[5] Yang CZ, Zhang YY, Zheng M. Soluble lectin-like oxidized low-density lipoprotein receptor-1 levels in synovial fluid are correlated with disease severity of knee osteoarthritis. Clin Biochem 2011;44:1094–6. [6] Pierzchala AW, Kusz DJ, Hajduk G. CXCL8 and CCL5 expression in synovial fluid and blood serum in patients with osteoarthritis of the knee. Arch Immunol Ther Exp (Warsz) 2011;59:151–5. [7] Zhang Y, Jordan JM. Epidemiology of osteoarthritis. Clin Geriatr Med 2010;26: 355–69. [8] Bonnet CS, Walsh DA. Osteoarthritis, angiogenesis and inflammation. Rheumatology (Oxford) 2005;44:7–16. [9] Pantsulaia I, Kalichman L, Kobyliansky E. Association between radiographic hand osteoarthritis and RANKL, OPG and inflammatory markers. Osteoarthritis Cartilage 2010;18:1448–53. [10] Bloom BR, Bennett B. Mechanism of a reaction in vitro associated with delayedtype hypersensitivity. Science 1966;153:80–2. [11] Calandra T, Bernhagen J, Mitchell RA, Bucala R. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med 1994;179:1895–902. [12] Leech M, Metz C, Hall P, Hutchinson P, Gianis K, Smith M, et al. Macrophage migration inhibitory factor in rheumatoid arthritis: evidence of proinflammatory function and regulation by glucocorticoids. Arthritis Rheum 1999;42: 1601–8. [13] Onodera S, Kaneda K, Mizue Y, Koyama Y, Fujinaga M, Nishihira J. Macrophage migration inhibitory factor up-regulates expression of matrix metalloproteinases in synovial fibroblasts of rheumatoid arthritis. J Biol Chem 2000;275:444–50. [14] Kim HR, Park MK, Cho ML, Yoon CH, Lee SH, Park SH, et al. Macrophage migration inhibitory factor upregulates angiogenic factors and correlates with clinical measures in rheumatoid arthritis. J Rheumatol 2007;34:927–36. [15] Kellgren JH, Lawrence JS. Radiological assessment of osteo-arthrosis. Ann Rheum Dis 1957;16:494–502. [16] Loeser RF. Molecular mechanisms of cartilage destruction: mechanics, inflammatory mediators, and aging collide. Arthritis Rheum 2006;54:1357–60. [17] Patra D, Sandell LJ. Recent advances in biomarkers in osteoarthritis. Curr Opin Rheumatol 2011;23:465–70. [18] Onodera S, Tanji H, Suzuki K, Kaneda K, Mizue Y, Sagawa A, et al. High expression of macrophage migration inhibitory factor in the synovial tissues of rheumatoid joints. Cytokine 1999;11:163–7. [19] Wakabayashi K, Otsuka K, Sato M, Takahashi R, Odai T, Isozaki T, et al. Elevated serum levels of macrophage migration inhibitory factor and their significant correlation with rheumatoid vasculitis disease activity. Mod Rheumatol 2012;22: 59–65. [20] Llamas-Covarrubias MA, Valle Y, Navarro-Hernández RE, Guzmán-Guzmán IP, Ramírez-Dueñas MG, Rangel-Villalobos H, et al. Serum levels of macrophage migration inhibitory factor are associated with rheumatoid arthritis course. Rheumatol Int May 24 2011 [Electronic publication ahead of print]. [21] Morand EF, Leech M, Weedon H, Metz C, Bucala R, Smith MD. Macrophage migration inhibitory factor in rheumatoid arthritis: clinical correlations. Rheumatology (Oxford) 2002;41:558–62. [22] van den Berg WB. Osteoarthritis year 2010 in review: pathomechanisms. Osteoarthritis Cartilage 2011;19:338–41. [23] Murphy G, Nagase H. Reappraising metalloproteinases in rheumatoid arthritis and osteoarthritis: destruction or repair? Nat Clin Pract Rheumatol 2008;4: 128–35. [24] Cawston TE, Wilson AJ. Understanding the role of tissue degrading enzymes and their inhibitors in development and disease. Best Pract Res Clin Rheumatol 2006;20:983–1002. [25] Pakozdi A, Amin MA, Haas CS, Martinez RJ, Haines III GK, Santos LL, et al. Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis. Arthritis Res Ther 2006;8:R132. [26] Onodera S, Nishihira J, Iwabuchi K, Koyama Y, Yoshida K, Tanaka S, et al. Macrophage migration inhibitory factor up-regulates matrix metalloproteinase-9 and -13 in rat osteoblasts. Relevance to intracellular signaling pathways. J Biol Chem 2002;277:7865–74. [27] Onodera S, Nishihira J, Koyama Y, Majima T, Aoki Y, Ichiyama H, et al. Macrophage migration inhibitory factor up-regulates the expression of interleukin-8 messenger RNA in synovial fibroblasts of rheumatoid arthritis patients: common transcriptional regulatory mechanism between interleukin-8 and interleukin-1beta. Arthritis Rheum 2004;50:1437–47. [28] Kim HR, Park MK, Cho ML, Kim KW, Oh HJ, Park JS, et al. Induction of macrophage migration inhibitory factor in ConA-stimulated rheumatoid arthritis synovial fibroblasts through the P38 map kinase-dependent signaling pathway. Korean J Intern Med 2010;25:317–26.
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Association of MIF in serum and synovial fluid with severity of knee osteoarthritis Minghui Liu a, Chunhe Hu b,⁎ a b
Department of Trauma, Union Medicine Centre, Tianjin, PR China Department of Hand Surgery, Hebei Medical University third Hospital, Shijiazhuang, PR China
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 7 March 2012 Accepted 8 March 2012 Available online 16 March 2012 Keywords: Macrophage migration inhibitory factor Serum Synovial fluid Severity Osteoarthritis
a b s t r a c t Objective: Recent evidences suggest that inflammation contributes to the development and progression of osteoarthritis (OA). This study aims to determine macrophage migration inhibitory factor (MIF) levels in serum and synovial fluid (SF) of patients with knee OA and to analyze the association of MIF levels with the radiographic severity of OA. Design and methods: 224 patients with knee OA and 186 healthy controls were enrolled in this study. Results: Higher levels of serum MIF were found in knee OA patients compared with healthy controls. Knee OA patients with Kellgren and Lawrence (KL) grade 4 showed significantly elevated MIF levels in serum and SF compared with those with KL grade 2 and 3. MIF levels in serum and SF of knee OA patients were significantly related to disease severity evaluated by KL grading criteria. Conclusion: MIF levels in serum and SF were closely related to the radiographic severity of OA. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Knee osteoarthritis (OA), a common chronic degenerative disease, is characterized by the loss of articular cartilage components due to an imbalance between extracellular matrix destruction and repair [1]. OA leads to functional limitations, reduced quality of life, and even disability [2]. The diagnosis of OA is generally based on clinical and radiographic changes, which reflect disease severity by grading the joint destruction [3]. Nowadays, biochemical markers are studied as a promising indicator for OA. Recent evidences have demonstrated that radiographic grading of OA is correlated with some biochemical markers in synovial fluid (SF) such as P selectin [4], soluble lectinlike oxidized low-density lipoprotein receptor-1 [5], as well as interleukin (IL)-8 and CCL5 [6]. Risk factors such as aging, obesity, being female, smoking, genetics, and joint injury are considered to be associated with OA [7]. However, the etiology and pathogenesis of OA remain poorly understood. In addition, inflammation has been implicated in the pathogenesis of OA [8]. Recent in-vivo and in-vitro studies have demonstrated that chondrocytes can produce and/or respond to a number of inflammatory cytokines and chemokines present in joint tissues and fluids in OA [9]. Macrophage migration inhibitory factor (MIF) was originally identified as a protein derived from T activated lymphocytes [10]. MIF is a proinflammatory cytokine produced by macrophages in response ⁎ Corresponding author at: Department of Hand Surgery, Hebei Medical University third Hospital, 139 Ziqiang Road, 050017, Shijiazhuang, Hebei, PR China. Fax: + 86 0311 88602006. E-mail address: [email protected] (C. Hu).
to a variety of inflammatory stimuli [11]. MIF induces the release of proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), interferon-γ, IL-1β, IL-6, IL-8, nitric oxide, and cyclo-oxygenase 2 [12]. In synovial fibroblasts from rheumatoid arthritis patients, MIF also upregulates the expression of matrix metalloproteinases (MMP)-1, MMP-2, and MMP-3 which play important roles in synovial invasion [13]. In addition, MIF indirectly induces joint destruction by promoting angiogenesis in synovial fibroblasts from rheumatoid arthritis patients [14]. Therefore, MIF may be involved in the mechanism of OA, and MIF levels in the serum and SF may be related to the severity of knee OA. In the present study, we aim to determine the association of MIF concentrations in serum and SF with the radiographic disease severity in patients with knee OA in order to assess its role in OA pathophysiology.
Materials and methods Subjects This study consists of 224 patients diagnosed with knee OA according to the criteria of the American College of Rheumatology. Patients with previous knee surgery, meniscectomy in both knee compartments, osteochondritis dissecans, fracture in or adjacent to the knee, septic arthritis, osteonecrosis, any ligament injury, or radiographic signs of knee OA were excluded. The control group included 186 healthy check-up examinees who matched to the cases by age, gender, and body mass index (BMI). They had no clinical and radiological evidence of OA. This study was approved by the ethics
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2012.03.012
738
M. Liu, C. Hu / Clinical Biochemistry 45 (2012) 737–739
Results
Table 1 The characteristics between patients with knee OA and healthy controls. Characteristics
Knee OA patients (n = 224)
Healthy controls (n = 186)
P value
Age (years) Gender (male/female) BMI (kg/m2) CRP (mg/L) Diabetes (%) Cardiovascular diseases (%) Asthma (%) MIF in serum (ng/mL) MIF in SF (ng/mL)
59.60 ± 7.69 94/130 24.12 ± 3.76 2.88 ± 0.86 24 (10.71%) 13 (5.80%) 8 (3.57%) 15.49 (12.46–19.00) 3.72 (2.88–4.37)
60.39 ± 7.53 71/115 24.41 ± 3.49 1.48 ± 0.46 18 (9.68%) 8 (4.30%) 2 (1.08%)
0.297 0.436 0.427 b 0.001 0.730 0.492 0.103 b 0.001
6.06 (4.97–7.30)
Baseline clinical parameters The baseline clinical parameters of patients with knee OA and healthy controls are shown in Table 1. Knee OA patients showed significantly higher levels of CRP compared with healthy controls (P b 0.001). There were no significant differences in age, gender, and BMI, as well as the percentage of diabetes, cardiovascular diseases, and asthma between the two groups. The MIF levels in serum and SF
committee of our hospital, and informed consent was obtained from all participants. Disease severity assessment was performed using the Kellgren and Lawrence (KL) grading system [15]. OA patients were defined as having radiographic knee OA of KL grade ≥ 2 in at least one knee. Controls were defined as having no radiographic knee OA as indicated by KL grades of 0 for both knees. The grading of the worst affected knee in each patient was used for data analysis.
Laboratory methods Venous blood samples were obtained from all participants after a 12-h overnight fasting. SF was taken from OA patients who received the treatment of hyaluronic acid injection for the first time. Quantitative determination of MIF concentration in serum and SF was performed using commercial enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA). C-reactive protein (CRP) was tested using an auto biochemistry instrument (Hitachi 7170, Tokyo, Japan).
MIF levels in serum and SF of knee OA patients and serum levels of MIF in healthy controls are presented in Table 1. Patients with knee OA had significantly elevated levels of serum MIF compared with those in healthy controls (P b 0.001). MIF levels in knee OA patients with different KL grades The MIF levels in serum and SF of knee OA patients with KL grade 4 were significantly elevated compared with those with KL grade 2 and 3. Furthermore, knee OA patients with KL grade 3 had significantly higher MIF levels in serum and SF compared with those with KL grade 2. The MIF levels in serum and SF of knee OA patients with different KL grades are shown in Table 2. Association of clinical parameters with KL grades Spearman correlation analysis showed that the MIF levels in serum and SF were strongly related to KL grades (r = 0.509, P b 0.001 and r = 0.548, P b 0.001 respectively). Multinomial logistic regression analysis indicated that MIF levels in serum and SF were both positively associated with KL grades (both P b 0.001). Discussion
Statistical analysis The data are presented as means ± SD, median (interquartile range), or frequencies. Kolmogorov–Smirnov test was performed to analyze the data normality. Comparison of the characteristics between patients with knee OA and healthy controls were performed by an unpaired t test, Mann–Whitney U test, or Chi-square test as indicated. Characteristics were compared between knee patients with different KL grades using Kruskal–Wallis test. Correlations of MIF levels in serum and SF with disease severity were calculated using Spearman correlation analysis. A multinomial logistic regression analysis was performed to determine the association of various factors with the severity of OA. As MIF levels were not normally distributed, logarithmic (log) transformed values were used for multiple linear regression analysis. All analyses were performed using the Statistical Package for the Social Sciences program (SPSS for Windows, version 16; Chicago, IL). Statistical significance was accepted at a level of P-value less than 0.05.
Inflammation has been implicated to be involved in the mechanism of OA. Synovial inflammation contributes to an imbalance between the catabolic and anabolic activities of the chondrocyte in remodeling extracellular matrix of the cartilage, and then the presence and progression of cartilage damage [16]. Inflammatory molecules such as interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, and IL-18 have been shown to be potential biomarkers for OA [17]. The current results indicate that serum levels of MIF were significantly elevated in patients with knee OA compared with healthy controls. Furthermore, MIF concentrations were strongly related to KL grades. This is consistent with other studies which demonstrated that OA patients had significantly higher MIF levels in serum and SF than normal controls [12,18]. These results suggest that MIF, a proinflammatory cytokine produced by macrophages, play an important role in the pathogenesis of OA. MIF levels could serve a new biomarker for predicting the presence and progression of OA. A recent study showed that serum levels of MIF in patients with rheumatoid arthritis were also significantly higher compared with healthy controls [19,20].
Table 2 The MIF levels of serum and SF in knee OA patients with different KL grades. MIF (ng/mL)
Grade 2 (n = 72)
Grade 3 (n = 88)
Grade 4 (n = 64)
P value
Serum SF
13.34 (10.97–16.40)a 3.14 (2.55–3.79)a
14.02 (11.28–16.97)b 3.42 (2.81–4.23)b
19.72 (16.87–23.22)b,a 5.18 (3.92–6.26)b,a
b0.001 b0.001
a b
P b 0.01 vs KL grade 3. P b 0.01 vs KL grade 2.
M. Liu, C. Hu / Clinical Biochemistry 45 (2012) 737–739
In addition, synovial MIF immunostaining was found to be related strongly to disease activity as measured by CRP concentration [21]. The mechanisms of OA and rheumatoid arthritis were both associated with inflammation. Therefore, MIF seems to contribute to the presence of OA and rheumatoid arthritis as an inflammatory factor. Degradation of extracellular matrix components is a typical pathological process of OA [22]. The main proteinases responsible for the degradation of extracellular matrix components are matrix metalloproteinases (MMP) [23]. MMP is a large group of zinc-dependent extracellular endopeptidase proteinases involved in the cleavage of extracellular matrix macromolecules [24]. Recent studies have shown that MIF could upregulate the expression of different MMP. MIF induces MMP-2 expression of synovial fibroblast from rheumatoid arthritis patients in a timedependent and concentration-dependent manner [25]. Furthermore, MMP-2 protein levels were significantly decreased in the knee joint of MIF gene-deficient mice compared with wild-type ones [25]. In another study, the expression levels of MMP-1 and MMP-3 mRNA were significantly elevated after stimulation by MIF in synovial fibroblasts from patients with rheumatoid arthritis [13]. Furthermore, MIF was found to up-regulate MMP-9 and -13 in rat osteoblasts [26]. These results indicate that MIF could induce extracellular matrix degradation and then the deterioration of cartilage tissue by promoting the expression of MMP which can digest extracellular matrix. MIF induces the production of inflammatory molecules such as TNF-α, IL-1β, and IL-8 in synovial fibroblasts of patients with rheumatoid arthritis [12,27]. These inflammatory factors have been shown to be associated with pathogenesis of OA. MIF may contribute to the development and progression of OA indirectly by promoting the release of different inflammatory cytokines. On the other hand, MIF was demonstrated to be induced by interferon-γ, CD40 ligand, IL-15, IL-1β, TNF-α, and transforming growth factor-β in synovial fibroblasts of patients with rheumatoid arthritis [28]. Therefore, MIF is hypothesized to interact with inflammatory cytokines and then amply the inflammatory response in chondrocytes, at last lead to the damage of cartilage. There are several limitations in this study. First, this is a crosssectional study performed in a relatively small sample. Therefore, the results of this study need to be confirmed in prospective longitudinal studies with a larger population. Second, we did not assess MIF levels in SF from healthy controls due to ethical concerns. In conclusion, MIF levels in serum and SF were strongly related to the severity of knee OA. MIF levels in serum and SF may serve as a new biomarker in addition of the traditional methods for assessing the risk and severity of knee OA.
References [1] Todhunter PG, Kincaid SA, Todhunter RJ, Kammermann JR, Johnstone B, Baird AN, et al. Immunohistochemical analysis of an equine model of synovitis-induced arthritis. Am J Vet Res 1996;57:1080–93. [2] Guccione AA, Felson DT, Anderson JJ, Anthony JM, Zhang Y, Wilson PW, et al. The effects of specific medical conditions on the functional limitations of elders in the Framingham Study. Am J Public Health 1994;84:351–8. [3] Melikoglu MA, Yildirim K, Senel K. Relationship between radiographic grading of osteoarthritis and serum beta-2 microglobulin. Ir J Med Sci 2009;178:151–4. [4] Cheng T, Li FF, Zhao S, Peng XC, Zhang XL. Soluble P selectin in synovial fluid level is correlated with the radiographic severity of knee osteoarthritis. Clin Chim Acta 2010;411:1529–31.
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