Orita et al.

BMC Musculoskeletal Disorders 2011, 12:144

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RESEARCH ARTICLE Open Access

Associations between proinflammatory cytokines

in the synovial fluid and radiographic grading
and pain-related scores in 47 consecutive
patients with osteoarthritis of the knee
Sumihisa Orita1,4*, Takana Koshi2, Takeshi Mitsuka3, Masayuki Miyagi4, Gen Inoue4, Gen Arai4, Tetsuhiro Ishikawa4,
Eiji Hanaoka5, Keishi Yamashita3, Masaomi Yamashita3, Yawara Eguchi4, Tomoaki Toyone6, Kazuhisa Takahashi4 and
Seiji Ohtori4

Abstract
Background: One of the sources of knee pain in osteoarthritis (OA) is believed to be related to local chronic
inflammation of the knee joints, which involves the production of inflammatory cytokines such as tumor necrosis factor
alpha (TNFa), interleukin (IL)-6, and nerve growth factor (NGF) in the synovial membrane, and these cytokines are
believed to promote pathological OA. In the present study, correlations between proinflammatory cytokines in knee
synovial fluid and radiographic changes and functional scores and pain scores among OA patients were examined.
Methods: Synovial fluid was harvested from the knees of 47 consecutive OA patients, and the levels of TNFa, IL-6,
and NGF were measured using enzyme-linked immunosorbent assays. Osteoarthritic knees were classified using
Kellgren-Lawrence (KL) grading (1-4). The Western Ontario and McMaster University Osteoarthritis Index (WOMAC)
was used to assess self-reported physical function, pain, and stiffness.
Results: TNFa and IL-6 were detectable in knee synovial, whereas NGF was not. TNFa was not correlated with the
KL grade, whereas IL-6 had a significantly negative correlation. We observed differences in the correlations
between TNFa and IL-6 with WOMAC scores and their subscales (pain, stiffness, and physical function). TNFa
exhibited a significant correlation with the total score and its 3 subscales, whereas IL-6 exhibited a moderately
significant negative correlation only with the subscale of stiffness.
Conclusions: The present study demonstrated that the concentrations of proinflammatory cytokines are correlated
with KL grades and WOMAC scores in patients with knee OA. Although TNFa did not have a significant correlation
with the radiographic grading, it was significantly associated with the WOMAC score. IL-6 had a significant
negative correlation with the KL grading, whereas it had only a weakly significant correlation with the subscore of
stiffness. The results suggest that these cytokines play a role in the pathogenesis of synovitis in osteoarthritic knees
in different ways: TNFa is correlated with pain, whereas IL-6 is correlated with joint function.

Background membrane and subchondral bone, and OA can be

Knee osteoarthritis (OA) is a common chronic degen- recognized as an irregularity and deformity of joint
erative disease characterized by the loss of articular car- spaces in radiographic images. Its main clinical sign is
tilage components due to an imbalance between joint pain, which not only contributes to functional lim-
extracellular matrix destruction and repair [1]. The itations and reduced quality of life but is also the lead-
entire joint structure is affected, including the synovial ing cause of impaired mobility in the elderly population
[2]. Although the exact mechanism of knee pain in OA
* Correspondence: [email protected] is unclear, it is believed to be related to local chronic
1
Department of Orthopaedic Surgery, Chiba Rosai Hospital, Chiba, Japan inflammation of the knee joints, which involves the
Full list of author information is available at the end of the article

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production of inflammatory cytokines in the synovial outpatient clinics by experienced orthopedic physicians.
membrane, such as tumor necrosis factor alpha (TNFa), The samples of synovial fluid were aspirated directly
interleukin (IL)-6, and nerve growth factor (NGF), without lavage and immediately stored at -70°C until
which are generally considered to promote pathological use. Freeze-thaw cycles were avoided. Cytokine quantifi-
OA [3-5]. Proinflammatory cytokine mediators have cation was performed using a double-antibody sandwich
been reported to contribute to OA pathogenesis by enzyme-linked immunosorbent assay (ELISA) for TNFa,
increasing cartilage degradation and inducing hyperalge- IL-6 (R&D systems, Minneapolis, MN), and NGF (Bos-
sia via a number of direct and indirect actions. TNFa ter Biological Tec., Wuhan, China) without dilution
activates sensory neurons directly via its receptors and according to the manufacturers’ protocols (centrifuga-
initiates a cascade of inflammatory reactions via the pro- tion before use: for 15 min at 1,000 × g (TNFa and IL-
duction of ILs [6,7]. IL-6 is reported to have a complex 6) or for 20 min at 2,000 × g (NGF)). The detection lim-
role in OA pathogenesis by initiating inflammatory its of the assays were 0.5 pg/ml for TNFa, <0.70 pg/ml
responses such as the production of tissue inhibitors of for IL-6, and <1 pg/ml for NGF. All samples were
metalloproteinase, and this may act to limit cartilage assessed in duplicate.
damage via negative feedback [8]. NGF is reportedly
upregulated in human osteoarthritic chondrocytes and Grading of OA and pain evaluation
synovial fibroblasts, suggesting its important role in the Anteroposterior radiographs of the symptomatic knees
pathology of OA [6,9]. Another report indicated that were obtained. The X-ray beam was aimed at the lower
NGF antagonism is an important mediator of pain in pole of the patella and kept parallel to the joint surface.
OA because its antagonistic effect resulted in analgesia Radiographs were scored by experienced orthopedic sur-
in a murine OA model [10]. geons using the Kellgren-Lawrence (KL) grading scale as
Thus, investigations of the dynamic states of these follows: grade 1, doubtful narrowing of joint space and
cytokines should be conducted. Additionally, a previous possible osteophytic lipping; grade 2, definite osteo-
study indicated a strong association between the radio- phytes and possible narrowing of joint space; grade 3,
graphic images of knees with OA and with knee pain moderate multiple osteophytes, definite narrowing of
[11]. Under the hypothesis that relationships between joints space, some sclerosis, and possible deformity of
these cytokines and clinical evaluations in OA patients bone contours; and grade 4, large osteophytes, marked
are possible, the present study evaluated the association narrowing of joint space, severe sclerosis, and definite
between proinflammatory cytokines in the synovial fluid deformity of bone contours [12]. The functional status
from the knees of OA patients and radiographic severity and pain level of each patient were evaluated using the
and pain scale scores. Western Ontario McMaster University Osteoarthritis
Index (WOMAC) score [13]. The index consists of 3
Methods subscales: pain, stiffness, and physical function. A higher
Our Institutional Review Board approved the present score on the WOMAC scale represents poorer function
study. We obtained informed consent from each partici- or greater pain. The data were arranged according to
pating patient. the KL grade for each cytokine, and the correlations
between the cytokines were analyzed. Correlations
Patient selection between the cytokine concentrations and WOMAC
The present study included adult patients with knee score were also analyzed.
pain who visited our facility for clinical consultation
between August 2009 and March 2010. The present Statistical analysis
study consisted of patients with knee OA diagnosed Statistical differences between the 2 groups were deter-
using the American College of Rheumatology criteria for mined using the Mann-Whitney U test followed by Bon-
OA who had not received any prior treatment. Patients ferroni’s correction for multiple testing, and the
with clear clinical evidence of any involvement of statistical significance among the groups was determined
trauma, prior treatment, or other orthopedic diseases using the Kruskal-Wallis test. The significance of corre-
including spinal disorders causing radicular pain in the lations was determined by Spearman’s rank correlation
legs were excluded. Patients diagnosed with rheumatoid test (PASW statistics ver. 18 (SPSS Inc (IBM), Somers,
arthritis based on physical examination and laboratory NY)). A p value < 0.05 was considered significant.
data were also excluded.
Results
Synovial fluid sampling and cytokine assay Patient demographics
With the approval of patients, samples of synovial joint Table 1 shows the patient demographics. Among the 50
fluid were collected using a syringe and needle in our patients enrolled in the present study, we could not
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Table 1 Patient Demographics (CV value (%): TNFa, 5.8 ± 1.2; IL-6, 4.2 ± 0.18; NGF:
Gender Disease Total unable to be calculated).
duration The concentration of TNFa was significantly lower in
(months)**
KL grades 2 to 4 than in KL grade 1 (KL 1, 6.5 ± 2.0
Male Female pg/ml (mean ± S.E.); KL 2, 3.6 ± 0.72 pg/ml (p = 0.38
No. of Patients 22 (21) 28 (26) 50 (47) vs. KL 1); KL 3, 4.2 ± 0.48 pg/ml (p = 0.025 vs. KL 1);
Average age 69.6 ± 7.3 69.8 ± 11.3 70.0 ± 2.1 and KL 4, 3.2 ± 0.86 pg/ml (p = 0.031 vs. KL 1)) (Figure
(years)
1A). The IL-6 concentration was significantly lower in
KL grading
KL grades 3 and 4 than in KL grades 1 and 2 (KL 1,
1 6 4 4.83 ± 2.0 10
401.6 ± 33.2 pg/ml; KL 2, 292.6 ± 42.2 pg/ml; KL 3,
2 6 9 14.8 ± 8.9 15
162.9 ± 46.5 pg/ml; and KL 4, 78.6 ± 62.8 pg/ml) (p =
3 5 8 33.9 ± 18.6 13
0.032 vs. KL 1; p = 0.036 vs. KL 2) (Figure 1B). NGF
4 5 (4)* 7 (5)* 45.3 ± 21.4 12 (9)*
was not detected in any sample.
*No fluid was obtained from 3 patients (1 man and 2 women), and thus, the
numbers in parenthesis indicate the effective numbers for the present study.
**Described as mean ± SEM. Correlation between WOMAC score and cytokine
concentration
obtain any fluid from the knees of 3 patients with Figure 2 shows the correlations between the detectable
osteoarthritic knees classified as KL grade 4, and thus, cytokines and the WOMAC score. Group A shows
we analyzed the other 47 samples. Disease duration TNFa, and group B shows IL-6. TNFa exhibited a mod-
increased as the KL scores increased. erately significant positive correlation with the total
WOMAC score (A-1) and with each subscale (pain
Concentrations of the proinflammatory cytokines (A-2), stiffness (A-3), and physical function (A-4) (p <
Figure 1 shows the concentrations of proinflammatory 0.01)). IL-6 exhibited a moderately significant negative
cytokines in the synovial fluid of knee joints in relation correlation only with stiffness (B-3) (p < 0.05), whereas
to the radiographic findings for these joints. Measurable it did not exhibit any significant correlation with the
levels of TNFa and IL-6 were detected in all samples, other factors. The precise statistical values are shown in
whereas NGF was not detectable in any of the samples Table 2.

(A) TNF_ (B) IL-6

*: P < 0.05 vs. K-L 1
†: P < 0.05 vs. K-L 2

18.0 600
16.0
*: P < 0.05 vs. K-L 1 500
14.0
TNF _ (pg//mL)

mL)

12.0 400
IL-6 (pg/m

10.0
300
8.0
200
6.0
* * *
4.0 * 100 *†
†
2.0
0.0 0
1 2 3 4 1 2 3 4

K-L grade K-L g

grade

(Mean ± SEM)
Figure 1 Concentrations of proinflammatory cytokines in the synovial fluid from the knee joints. (A) The concentration of TNFa
exhibited no significant correlation with KL grading, although there was a tendency for increased TNFa concentrations at lower KL grades. (B)
The concentration of IL-6 was significantly decreased in KL grades 3 and 4 compared with those in KL grades 1 and 2. NGF was undetectable in
all patient samples.
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(A) TNF_ (B) IL-6

(A-1) (B-1)
WOMAC
80 80
Score
(WS) 60 60
Total 40 40
r= 0.69, P<0.01
20 20
r= -0.18, P=0.09
0 0
00
0.0 50
5.0 10 0
10.0 15 0
15.0 20 0
20.0 00
0.0 200.0
200 0 400 0
400.0 600 0
600.0
TNF level (pg/mL) IL-6 level (pg/mL)

(A-2) (B-2)
20 20 r= -0.23, P=0.31
15 15
WS-Pain 10 10

5 r=0.57, P<0.01 5

0 0
0.0 5.0 10.0 15.0 20.0 0.0 200.0 400.0 600.0
TNF level (pg/mL) IL-6 level (pg/mL)
(A-3) (B-3)
8 10

6 8 r= -0.48, P<0.05
WS- 6
4
Stiffness 4
2 r=0.56, P<0.01
2
0 0
0.0 5.0 10.0 15.0 20.0 0.0 200.0 400.0 600.0
TNF level (pg/mL) IL-6 level (pg/mL)
(A-4) (B-4)
80 80
r= -0.16, P=0.89
60 60
WS-
Physical 40 40
function
20 R=0.64 P<0.01 20

0 0
0.0 5.0 10.0 15.0 20.0 0.0 200.0 400.0 600.0
TNF level (pg/mL) IL-6 level (pg/mL)
Figure 2 Correlations between the detectable cytokines and the WOMAC score. Group A shows TNFa, and group B shows IL-6. TNFa
exhibited a moderately significant positive correlation with the total WOMAC score (A-1) and with the each subscale (pain (A-2), stiffness (A-3),
and physical function (A-4) (p < 0.01)). IL-6 exhibited a weakly significant negative correlation only with stiffness (B-3) (p < 0.05), whereas it
exhibited no correlation with the other subscales.
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Table 2 Statistical data of Figure 2 reported to not be regulated in the joints in late OA,
TNFa IL-6 and this is consistent with the finding of the present
r p r p study [26].
Total 0.69 0.0023 -0.18 0.09 As described in the Background section, NGF is con-
Pain 0.57 0.0069 -0.23 0.31 sidered an important factor in OA pathogenesis, and
Stiffness 0.56 0.0054 -0.48 0.039 thus, it is important to discuss why NGF was not
Physical function 0.64 0.0038 -0.16 0.89 detected in the present study. We hypothesize that,
excluding any technical errors, NGF production is insuf-
ficient for detection in the synovial fluid obtained from
Discussion the knees of OA patients. According to a previous
The present study examined whether inflammation plays a report, the mRNA expression of neurotrophins includ-
substantial role in the development of pain in OA. We ing NGF and its receptors was confirmed in the synovial
demonstrated that TNFa and IL-6 were detectable in the fluid and tissues of patients with OA, whereas NGF
synovial fluid sampled from the knees of OA patients, mRNA expression was low [27]. Furthermore, NGF is a
whereas NGF was undetectable. TNFa was not correlated basic protein, and this is disadvantageous for its exis-
with the KL grade, and IL-6 had a relatively significant tence in the relatively acidic milieu of the synovial fluid
negative correlation with KL grading. Some differences [28]. Another study reported the diagnostic usefulness
were found between TNFa and IL-6 regarding their corre- of biopsied tissue as opposed to the use of synovial fluid
lations with the WOMAC score and its subscales. TNFa [26]. Thus, in addition to examining the synovial fluid,
exhibited a moderately significant correlation with the it may be important to investigate NGF expression in
total score and its 3 subscales, whereas IL-6 exhibited a the subchondral tissue where inflammatory cytokines
weakly significant negative correlation with the subscale of are reported to be produced [29]. Moreover, to investi-
stiffness. The WOMAC scoring method used in the pre- gate any correlation between proinflammatory cytokines
sent study was translated from the English version, and including NGF, multivariable analysis among these cyto-
thus, we believe that its validity is similar as that reported kines should be performed in the future. Evaluating the
in a previous study in Asian OA patients [14]. levels of these cytokines in the synovial fluid from com-
pletely normal knees is important but also difficult for
Evidence of proinflammatory cytokines in the synovial ethical reasons. Alternatively, we can evaluate the cyto-
fluid samples kine levels in the synovial fluid from injured knees such
The results of the present study are comparable with as those with anterior cruciate ligament (ACL) injuries;
those of previous studies, which using a zymosan- however, the data may not be useful because proinflam-
induced mouse OA model, reported that TNFa is matory cytokine levels are elevated in response to any
related to synovitis and that IL-6 has a role in reducing degradation or injury in the joint. However, we can
cartilage destruction [15-18]. These studies add impor- partly infer their levels from previous studies. One study
tance to the present findings that TNFa inhibition may evaluating the cytokine levels in the knees of patients
improve the WOMAC score and that increased IL-6 with chronic ACL deficiencies reported that TNFa con-
activity in earlier phases of OA prevents cartilage centrations were lower in injured knees than in normal
destruction. Brenner et al performed a similar experi- knees, and the TNFa levels reported in that study were
ment using the synovial membranes and fluid from OA also low compared with those in the present study [30].
patients, and reported that TNFa was undetectable in Because IL-6 and TNFa levels are elevated in the early
their synovial fluid and that there were no correlations phase of knee injuries, it will be important to measure
between IL-6 levels and WOMAC pain subscores [19]. their levels in normal knees.
Regarding TNFa, some controversy exists among stu-
dies. Some previous studies reported low levels of TNFa Correlation between cytokine concentrations and KL
in the synovial fluid of OA patients [20-22], whereas grading
other studies including experiment models reported its A previous study indicated that the levels of TNFa in
detection [23-25]. The present study detected TNFa in the synovial fluid were positively correlated with Lar-
the synovial fluid. These discrepancies may be attributa- sen’s radiographic grading of bone destruction in rheu-
ble to the extremely low value of TNFa and the method matoid arthritis patients, whereas no correlation
of collecting and processing synovial fluid. However, we between the concentration of TNFa and Dahlgren’s
can suggest that TNFa is related to OA pathology and radiographic OA grade was found [31]. The report is
clinical evaluations based on the present findings, consistent with some of the results of the present study
although additional investigations with greater numbers regarding TNFa but not IL-6. The present study
of samples are needed. Additionally, TNFa was also revealed that TNFa is clearly not correlated with joint
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degeneration as assessed by KL grading, whereas IL-6 is studies. Last, we only examined the synovial fluid. It will
negatively correlated with KL grading, suggesting that be important to assess the serum levels of these cyto-
IL-6 has an important role in OA progression. Gener- kines and compare them with both the levels of cyto-
ally, members of the IL family are reported to be related kines in the synovial fluid and the grading scores.
to the severity of cartilage destruction [32,33] The pre-
sent study indicated that IL-6 production might be Conclusions
increased in the early stage of joint destruction in OA The present study demonstrated that the concentrations
patients. Other studies have reported that TNFa induces of proinflammatory cytokines can be correlated with the
IL-6 upregulation, and thus, IL-6 may still be correlated KL grades and WOMAC scores of knee OA patients.
with OA progression [34-36]. Increased IL-6 activity has TNFa did not have a significant correlation with the
been reported to be associated with increased proteogly- radiographic grading, while it did with the WOMAC
can synthesis in articular cartilage in dogs with experi- scoring. IL-6 had a significant negative correlation with
mental ACL transaction [24], and thus, IL-6 production KL grading, and only a weakly significant correlation
is highest in the early stages of joint injury. with the subscore of stiffness. These 2 cytokines were
Considering these reports, IL-6 may be related to the moderately correlated, and the results suggest that these
formal pathogenesis of OA, suggesting that active carti- cytokines play a role in the pathogenesis of synovitis in
lage destruction occurs at the greatest rate in earlier KL osteoarthritic knees in different ways. TNFa is corre-
grades. In other words, late-stage KL grading may indi- lated with pain, whereas IL-6 is correlated with joint
cate the “burnt ruins” acquired after active inflammation function. NGF was undetectable in the synovial fluid,
where IL-6 is more directly involved than TNFa. illustrating the need for differently designed experiments
in future studies.
Correlation between cytokine concentrations and the
WOMAC score
The present study demonstrated that TNFa is signifi- Acknowledgements
No funding was received for this research. None of the authors have any
cantly correlated with the WOMAC score including the conflict of interest or disclosures to report in relation to this work.
subscores. However, IL-6 was not correlated with the
WOMAC score excluding the subscore of stiffness, Author details
1
Department of Orthopaedic Surgery, Chiba Rosai Hospital, Chiba, Japan.
which indicates that IL-6 primarily affects the progress 2
Department of Orthopaedic Surgery, Seirei Sakura Citizen Hospital, Chiba,
of the degeneration of joint cartilage in OA that leads to Japan. 3Department of Orthopaedic Surgery, Social Insurance Funabashi
joint stiffness. Central Hospital, Chiba, Japan. 4Department of Orthopaedic Surgery,
Graduate School of Medicine, Chiba University, Chiba, Japan. 5Department of
Furthermore, we found a correlation only with the sub- Orthopaedic Surgery, Chiba Social Insurance Hospital, Chiba, Japan.
6
score of stiffness, which can be derived from the con- Department of Orthopaedic Surgery, Teikyo University Chiba Medical
structive degradation of the cartilage, and we found no Center, Chiba, Japan.

correlation with the subscore of pain. A previous paper Authors’ contributions

reported a negative correlation between IL-6 activity and SO designed and performed all the experiments, analyzed data, and drafted
radiographic OA scoring in dog OA models [20], and the paper. TK, TM, MM, GI, GA, TI, EH, KY, MY, and YE harvested synovial fluid
in their outpatient clinic and performed experiments. TT, KT, and SO
this coincides with the results of the present study. supervised the project and edited the manuscript. All authors contributed to
The present study has some limitations. First, we did data interpretation and have read and approved the final manuscript.
not examine the gene expression of each cytokine. Var-
Competing interests
iations in several genes that regulate inflammation have The authors declare that they have no competing interests.
been reported to be associated with the differential
expression of inflammatory mediators [37-39], some of Received: 21 November 2010 Accepted: 30 June 2011
Published: 30 June 2011
which have been associated with OA pathology [40-44].
Thus, further studies including genetic investigations are References
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Orita et al. BMC Musculoskeletal Disorders 2011, 12:144 Page 8 of 8
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Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2474/12/144/prepub

doi:10.1186/1471-2474-12-144
Cite this article as: Orita et al.: Associations between proinflammatory
cytokines in the synovial fluid and radiographic grading and pain-
related scores in 47 consecutive patients with osteoarthritis of the
knee. BMC Musculoskeletal Disorders 2011 12:144.

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