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RESEARCH ARTICLE Open Access
Plasma proteins present in osteoarthritic synovial
fluid can stimulate cytokine production via Toll-
like receptor 4
Dong Hyun Sohn
1,2†
, Jeremy Sokolove
1,2†
, Orr Sharpe
1,2†
, Jennifer C Erhart
3,4
, Piyanka E Chandra
1,2
,
Lauren J Lahey
1,2
, Tamsin M Lindstrom
1,2
, Inyong Hwang
1,2
, Katherine A Boyer
3,4
, Thomas P Andriacchi
3,4
and
William H Robinson
1,2*
Abstract
Introduction: Osteoarthritis (OA) is a degenerative disease characterized by cartilage breakdown in the synovial
joints. The presence of low-grade inflammation in OA joints is receiving increasing attention, with synovitis shown
to be present even in the early stages of the disease. How the synovial inflammation arises is unclear, but proteins
in the synovial fluid of affected joints could conceivably contribute. We therefore surveyed the proteins present in
OA synovial fluid and assessed their immunostimulatory properties.
Methods: We used mass spectrometry to survey the proteins present in the synovial fluid of patients with knee
OA. We used a multiplex bead-based immunoassay to measure levels of inflammatory cytokines in serum and
synovial fluid from patients with knee OA and from patients with rheumatoid arthritis (RA), as well as in sera from
healthy individuals. Significant differences in cytokine levels between groups were determined by significance
analysis of microarrays, and relations were determined by unsupervised hierarchic clustering. To assess the
immunostimulatory properties of a subset of the identified proteins, we tested the proteins’ability to induce the
production of inflammatory cytokines by macrophages. For proteins found to be stimulatory, the macrophage
stimulation assays were repeated by using Toll-like receptor 4 (TLR4)-deficient macrophages.
Results: We identified 108 proteins in OA synovial fluid, including plasma proteins, serine protease inhibitors,
proteins indicative of cartilage turnover, and proteins involved in inflammation and immunity. Multiplex cytokine
analysis revealed that levels of several inflammatory cytokines were significantly higher in OA sera than in normal
sera, and levels of inflammatory cytokines in synovial fluid and serum were, as expected, higher in RA samples than
in OA samples. As much as 36% of the proteins identified in OA synovial fluid were plasma proteins. Testing a
subset of these plasma proteins in macrophage stimulation assays, we found that Gc-globulin, a
1
-microglobulin,
and a
2
-macroglobulin can signal via TLR4 to induce macrophage production of inflammatory cytokines implicated
in OA.
Conclusions: Our findings suggest that plasma proteins present in OA synovial fluid, whether through exudation
from plasma or production by synovial tissues, could contribute to low-grade inflammation in OA by functioning
as so-called damage-associated molecular patterns in the synovial joint.
* Correspondence: wrobins@stanford.edu
†Contributed equally
1
GRECC, VA Palo Alto Health Care System, 3801 Miranda Ave., Palo Alto, CA
94304, USA
Full list of author information is available at the end of the article
Sohn et al.Arthritis Research & Therapy 2012, 14:R7
http://arthritis-research.com/content/14/1/R7
© 2012 Robinson et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attri bution License (http://creativecommons.org /licenses/by/2.0), which permits unrestricte d use, distribution, and
reproductio n in any medium, provided the original work is properly cited.
Introduction
Osteoarthritis (OA) is a degenerative disease of the
joints that is characterized by destruction of articular
cartilage, inflammation of the synovial membrane (syno-
vitis), and remodeling of periarticular bone. Which of
these pathogenic processes occurs first is unknown. One
proposed scenario is that cartilage breakdown (due to
injury or mechanical stress) releases components of the
damaged extracellular matrix (ECM) into synovial fluid,
and that these ECM components elicit the local produc-
tion of inflammatory molecules by binding to receptors
on resident synovial cells or infiltrating inflammatory
cells [1,2]. The inflammatory molecules produced may
in turn stimulate production of cartilage-degrading
enzymes and recruit inflammatory cells to the affected
joint [3,4], thus establishing a vicious cycle of cartilage
destruction and inflammation that perpetuates and pro-
motes the OA pathology. Therefore, OA has been
described as a chronic wound in which molecules in
synovial fluid function as damage-associated molecular
patterns (DAMPs; that is, endogenous molecules pro-
duced during injury that signal through inflammatory
toll-like receptors (TLRs) to effect tissue remodeling)
[2,5,6]. Although the identities of the endogenous mole-
cules that mediate synovial inflammation have yet to be
confirmed in OA patients or animal models, a continu-
ous supply of DAMPs could perpetuate the early
response to injury and thereby damage the joint.
Besides ECM components, many other molecules may
act as DAMPs [2]. One such molecule is fibrinogen,
which stimulates macrophage production of chemokines
in a TLR4-dependent manner [7-9]. Fibrinogen is present
at abnormally high levels in OA synovial fluid [10], and
the amount of fibrin (the thrombin-cleaved form of fibri-
nogen [11]) deposited in the synovial membrane corre-
lates with the severity of OA [12]. Although classically a
plasma protein, fibrinogen exudes from the vasculature
at sites of inflammation, such as the inflamed OA joint,
owing to the retraction of inflamed endothelial cells [11].
Fibrinogen is not the only protein to extravasate at sites
of inflammation, however, and several other plasma pro-
teins have been detected in OA synovial fluid [10,13].
The extravascular function of most of these plasma pro-
teins is unclear. It is possible that, like fibrinogen, some
of these plasma proteins could have an immunoregula-
tory role at sites of inflammation or tissue damage.
Inflammation is present even in the early stages of OA
[14,15], and clinical signs of synovitis correlate with
radiographic progression of knee OA [16]. Insight into
the cause of synovial inflammation is therefore impor-
tant in understanding the pathogenesis of OA. Here we
used proteomic techniques to survey the proteins pre-
sent in OA synovial fluid and to evaluate levels of
inflammatory cytokines in OA serum and synovial fluid.
We then determined whether a subset of the identified
proteins could promote inflammation by functioning as
immunostimulatory DAMPs.
Material and methods
Synovial fluid and serum samples
Serum and synovial fluid samples were obtained from
patients with knee OA, patients with rheumatoid arthri-
tis (RA), or healthy individuals under protocols
approved by the Stanford University Institutional Review
Board and with the patients’informed consent. Synovial
fluid aspiration was performed by a board-certified
rheumatologist by fine-needle arthrotomy, and the syno-
vial fluid samples obtained were free from obvious con-
tamination with blood or debris. OA serum and synovial
fluid samples were obtained from patients diagnosed
with knee OA (of Kellgren-Lawrence score 2 to 4 [17])
according to the 1985 criteria of the American Rheuma-
tism Association [18]. For mass spectrometric analysis,
OA synovial fluid samples were from five Caucasian
men aged 50 to 75 years who met the 1985 OA criteria
[18]; exclusion criteria included radiographic evidence of
chondrocalcinosis or evidence of crystals under polariz-
ing microscopy. Demographics and clinical characteris-
tics of these five individuals are shown in Table 1.
Synovial fluids from the other OA patients and from the
RA patients were provided as de-identified remnant
clinical samples, and patient demographics were there-
fore unavailable for these samples. All RA patients met
the 1987 American Rheumatism Association criteria for
RA [19] and had RA of less than 6 months’duration;
exclusion criteria included concurrent infectious or crys-
tal arthritis. Samples of “normal”serum were obtained
from healthy individuals who had no joint pain and no
radiographic evidence of knee arthritis [20]. OA and
normal sera were matched by age, sex, and BMI. Serum
and synovial fluid samples were not matched but were
derived from patients with the characteristics described
earlier. All samples were aliquoted and stored at -80°C.
Table 1 Clinical and demographic characteristics of OA
patients whose synovial fluid was analyzed with mass
spectrometry
Subject
a
Age (years) K-L score SF cell count (cells/mm
3
)
1 43 3 1,090
2 70 3 400
3 68 2 850
4 72 4 Not measured
5 74 4 Not measured
K-L, Kellgren-Lawrence OA score; SF, synovial fluid.
a
All subjects were male
and Caucasian with symptomatic pain in the aspirated knee.
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Mass spectrometric analysis
Synovial fluid proteins were separated by 1D or 2D
polyacrylamide gel electrophoresis (PAGE), trypsinized,
and identified by liquid chromatography tandem mass
spectrometry (LCMS), as follows. Fifty microliters of fro-
zen synovial fluid was diluted to a final volume of 1 ml
in phosphate buffered saline (PBS) containing Halt pro-
tease and phosphatase inhibitor (Thermo Fisher Scienti-
fic), and then depleted of the highly abundant proteins
albumin and immunoglobulin G (IgG) by using the Pro-
teoPrep Immunoaffinity Albumin & IgG Depletion Kit
(Sigma-Aldrich) according to the manufacturer’s
instructions. In brief, synovial fluids were twice passed
over spin columns prepacked with a mixture of two
beaded mediums containing recombinantly expressed,
small, single-chain antibody ligands. The flow-through
fractions containing synovial fluid depleted of albumin
and IgG were diluted 1:1 with Laemmli Sample Buffer
(BioRad) and then subjected to 1D-PAGE or 2D-PAGE
analysis. Because a small number of proteins other than
albumin and IgG may bind to the medium in the spin
columns, the bound proteins were eluted with Laemmli
sample buffer and also subjected to PAGE analysis. For
1D-PAGE analysis, proteins were boiled for 10 minutes
and separated on Precast Criterion XCT gels (4% to
12% linear gradient, BioRad). After electrophoresis, the
gels were stained for 1 hour with Gelcode blue (Pierce)
and destained overnight. For 2D-PAGE analysis, meth-
ods were as previously described [21]. In brief, 100 μgof
synovial fluid proteins was dissolved in 150 μl of isoelec-
tric focusing (IEF) buffer (ReadyPrep Sequential Extrac-
tion Kit Reagent 3, BioRad). For the first-dimension
electrophoresis, 150 μl(ata1μg/μl concentration) of
sample solution was applied to an 11-cm Ready-Strip
Immobilized pH Gradient (IPG) strip, pH 3 to 10
(BioRad). The IPG strips were soaked in the sample
solution for 1 hour, to allow uptake of the proteins, and
then actively rehydrated in the Protean IEF cell (BioRad)
for 12 hours at 50 V. IEF was performed for 1 hour at
each of 100, 200, 500, and 1,000 V, and then for 10
hours at 8,000 V. For second-dimension electrophoresis,
IPG strips were equilibrated for 20 minutes in 50 mM
Tris-HCl, pH 8.8, containing 6 Murea, 1%SDS, 30% gly-
cerol, and 65 mMdithiothreitol (DTT), and then re-
equilibrated for 20 minutes in the same buffer contain-
ing 260 mMiodacetamide in place of DTT. Precast Cri-
terion XCT gels (4% to 12% linear gradient, BioRad)
were used for the second-dimension electrophoresis, as
was done for the 1D-PAGE. After electrophoresis, the
gels were stained for 1 hour with Gelcode blue (Pierce)
and destained overnight.
The stained protein bands and spots (from the 1D-
PAGE and 2D-PAGE, respectively) were cut out of the
gels, immersed in 10 mMammonium bicarbonate
containing 10 mMDTT and 100 mMiodoacetamide,
treated with 100% acetonitrile, and then digested over-
night at 37°C with 0.1 mg trypsin (Sigma-Aldrich) in 10
mMammonium acetate containing 10% acetonitrile.
The trypsinized proteins were identified with LCMS by
using the Agilent 1100 LC system and the Agilent XCT
Ultra Ion Trap (Agilent Technologies, Santa Clara, CA)
as previously described [22]. We scanned the LCMS
data against the SwissProt database by using the Spec-
trumMill software (Agilent). We required the detection
of at least two peptides for identification of a protein,
and a significance level of P≤0.05 for identification of
each peptide. The significance level of peptide identifica-
tion takes into account the number of ionization forms
of the fragmented peptide that match with a particular
protein in the SwissProt database (with penalties for
ionization forms not identified), as well as the total
intensity of each ionization form [23].
Multiplex cytokine analysis
Multiplex analysis of cytokines and chemokines in
human serum and synovial fluid samples was performed
by using both the 27-plex and the 21-plex Bio-Plex Pro
Human Cytokine Assay (BioRad) run on the Luminex
200 platform, as recommended by the manufacturers.
Performing the Bio-Plex assay with the kit reagents, we
found that several commercial reagents designed to block
the confounding effect of heterophilic antibodies, includ-
ingonesweusedpreviouslywithothercytokineassay
kits [24], did not significantly affect the readout of the
Bio-Plex assay; we therefore did not use such blocking
reagents with the Bio-Plex assay. Data processing was
performed by using Bio-Plex Manager 5.0, and analyte
concentrations (in picograms per milliliter) were interpo-
lated from standard curves. Statistical differences in cyto-
kine levels were calculated with significance analysis of
microarrays (SAM [25]), and the SAM-generated results
with a false discovery rate (FDR) of less than 10% were
selected. To identify relations and to display our results
most effectively, we normalized the analyte concentra-
tions as follows: all values less than 1 were designated as
1, and the mean concentration of each analyte in the
“normal serum”samples was calculated; the analyte value
in the sample was then divided by the mean analyte value
in normal serum, and finally, a log-base-2 transformation
was applied. Results were subjected to unsupervised hier-
archic clustering by using Cluster 3.0, which arranges the
SAM-generated results according to similarities in cyto-
kine levels, and the clustering results were displayed by
using Java Treeview (Version 1.1.3).
Macrophage stimulation assays
To generate mouse macrophages, we differentiated
bone-marrow cells isolated from wild-type C57BL/6
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mice and from B6.B10ScN-Tlr4
lps-del
mice (Jackson
Laboratory) according to standard procedures [26]. In
brief, the femur and tibia were flushed with a-minimal
essential medium (MEM; Invitrogen) by using a 1-ml
syringeanda25-gaugeneedle.Theresultingcellsus-
pension was lysed with ACK Lysing Buffer (Invitrogen)
for removal of erythrocytes. Cell clumps were removed
by filtering through a 70-μm cell strainer (BD). The
remaining cells in the suspension were cultured on 100-
mm culture dishes in a-MEM supplemented with 10%
fetal bovine serum (FBS), 100 units/ml of penicillin, 100
μg/ml of streptomycin, and 2 mMglutamine (Invitro-
gen)for16to24hoursin5%CO
2
at 37°C. Nonadher-
ent cells were collected, plated on 100-mm dishes, and
differentiated into bone-marrow-derived macrophages
(BMMs) for 6 days in the presence of 30 ng/ml of
macrophage colony-stimulating factor (PeproTech). To
generate human monocyte-derived macrophages
(MDMs), we collected peripheral blood mononuclear
cells (PBMCs) by performing density-gradient centrifu-
gation of LRS chamber content (Stanford Blood Center)
over Ficoll (Invitrogen), purified human monocytes by
negative selection by using a monocyte isolation kit
(Miltenyi Biotec), and differentiated the monocytes into
macrophages by culturing them for 7 days in RPMI con-
taining 10% FBS and 30 ng/ml of human M-CSF.
For stimulation assays, mouse BMMs were plated in
96-well plates at 1 × 10
5
cells/well, and human macro-
phages at 7 × 10
4
cells/well. Cells were incubated for 24
hours with lipopolysaccharide (LPS; Sigma-Aldrich),
peptidoglycan (InvivoGen), a
1
-microglobulin (Cell
Sciences), a2-macroglobulin (EMD Chemicals), a1-acid
glycoprotein (EMD Chemicals), Gc-globulin (also known
as vitamin D-binding protein; Abcam), haptoglobin
(Sigma-Aldrich), or human serum albumin (Sigma-
Aldrich). We measured levels of interleukin-1b(IL-1b),
interleukin-6 (IL-6), and vascular endothelial growth fac-
tor (VEGF) in the culture supernatants with Luminex
analysis, by using a 27-plex Bio-Plex Pro Human Cyto-
kine Assay kit (BioRad) according to the manufacturer’s
instructions. We measured TNF levels with enzyme-
linked immunosorbent assay (ELISA; PeproTech). For
the TNF ELISA, the limits of detection were 16 to 2,000
pg/ml for mouse TNF, and 23 to 1,500 pg/ml for
human TNF. For the Luminex assay, the limits of detec-
tion were 3.2 to 3,261 pg/ml for IL-1b, 2.3 to 18,880 pg/
ml for IL-6, and 5.5 to 56,237 pg/ml for VEGF. To
exclude a contribution of endotoxin contamination, we
included 10 μg/ml of polymyxin B (Sigma-Aldrich) in
some of the stimulation assays. As an additional control
for endotoxin contamination, we tested whether prein-
cubating the plasma proteins with proteinase K and b-
mercaptoethanol at 55°C for 4 hours (and then at 100°C
for 10 minutes to inactivate the proteinase K) abrogated
their ability to induce the production of cytokines (the
plasma proteins, but not any contaminating endotoxin,
would be denatured under these conditions).
Statistical analysis
One-way ANOVA and unpaired ttest (Graph-Pad Soft-
ware) were used to analyze differences in levels of cyto-
kines. Pvalues less than 0.05 were considered
significant.
Results and Discussion
We first used mass spectrometry to survey the proteins
present in the synovial fluid of patients with knee OA.
Synovial fluid proteins from five OA patients were sepa-
rated by 1D- or 2D-PAGE and then identified by LCMS.
Analysis of all five samples identified a total of 111
unique proteins; three of these were keratin proteins,
skin proteins most likely obtained as a result of the
cutaneous puncture performed during aspiration of the
synovial joints. Eliminating these keratins left 108
unique proteins (Tables 2 and 3), most of which were
detected in all synovial fluid samples analyzed. Of these,
44 were identified in a previous proteomic survey of
highly abundant proteins in OA synovial fluid [10]
(Table 2). Thus, we confirmed the presence of serine
protease inhibitors (for example, antithrombin III, a
1
-
antitrypsin, a
1
-antichymotrypsin, kininogen 1) and of
proteins important in regulating proteases that degrade
cartilage ECM. We also confirmed the presence of pro-
teins involved in cartilage (for example, fibronectin)
and/or collagen (for example, gelsolin and collagen a
1
,
a
2
,anda
3
chains) metabolism, and of proteins involved
in inflammation or immunity (for example, fibrinogen,
AGP 1, complement factors, immunoglobulins, cyto-
kines) (Table 2), findings consistent with the inflamma-
tion, ECM degradation, and immune-cell infiltration
that characterize OA. Among the 64 proteins that we
newly identified (Table 3) were histone-related proteins,
macrophage-related proteins, proinflammatory receptors,
and proteins related to the proinflammatory transcrip-
tion factor nuclear factor kappa B (Table 4), presumably
reflecting the turnover of resident synovial cells or infil-
trating inflammatory cells.
Our mass-spectrometric findings revealed the presence
of many molecules associated with inflammation.
Although cytokines are also classically associated with
inflammation, PAGE-based mass spectrometry is not
well suited to the detection of small proteins such as
cytokines. We therefore used a multiplex immunoassay
to measure levels of inflammatory cytokines and chemo-
kines in synovial fluid samples from 12 patients with
knee OA and 14 patients with RA, as well as in serum
samplesfrom24patientswithkneeOA,23patients
with RA, and 35 healthy individuals. Samples from
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Table 2 Proteins identified in OA synovial fluids in this study and in a previous proteomic study [10]
Protein name
a
Accession no.
b
Score
c
% Coverage
d
Number of peptides
e
Transferrin P02787 877.49 65 57
a
2
-Macroglobulin P01023 569.94 34 57
Serum albumin P02768 514.49 60 34
Complement C3 P01024 426.92 27 34
Apolipoprotein A-I P02647 405.32 67 26
a
1
-Antitrypsin (a
1
protease inhibitor) P01009 388.37 66 27
Apolipoprotein A-IV P06727 370.16 60 27
Haptoglobin P00738 331.09 49 22
Hemopexin (b-1B-glycoprotein) P02790 273.36 40 20
Gc-globulin (vitamin D-binding protein precursor) P02774 269.18 43 21
a
1
B-glycoprotein P04217 215.74 47 16
Complement factor B (C3/C5 convertase) P00751 163.54 20 12
a
1
-Antichymotrypsin P01011 144.7 27 10
Antithrombin-III P01008 142.4 32 10
Ceruloplasmin (EC 1.16.3.1) (Ferroxidase) P00450 132.65 12 9
Transthyretin P02766 129.52 69 8
Plasma protease C1 inhibitor P05155 111.87 17 8
Ig mu chain C region P01871 98.51 16 7
Actin, cytoplasmic 2 (g-actin) P63261 89.13 33 8
Fibrinogen bchain P02675 88.28 18 7
a
2
-HS-glycoprotein P02765 85.26 22 7
Complement factor H (H factor 1) P08603 80.47 8 8
a
1
-Acid glycoprotein 1 (Orosomucoid-1) P02763 68.98 23 5
Complement C5 P01031 55.88 8 7
Prothrombin (coagulation factor II) P00734 52.57 9 4
Plasma retinol-binding protein P02753 50.79 20 4
Apolipoprotein E P02649 49.32 21 4
Afamin P43652 48.67 7 4
Fibrinogen achain P02671 48.63 6 4
Gelsolin P06396 47.03 4 3
Complement C4 P01028 39.34 8 6
Vitronectin P04004 38.1 6 3
Apolipoprotein A-II P02652 36.26 22 3
Hemoglobin bsubunit (Hemoglobin bchain) P68871 31.88 17 2
b
2
-Glycoprotein I (apolipoprotein H) P02749 30.33 6 2
Inter-a-trypsin inhibitor heavy chain H4 Q14624 28.43 5 3
Fibronectin P02751 23.84 2 2
Clusterin P10909 21.17 12 2
Complement component C8 gchain P07360 20.93 10 2
Histidine-rich glycoprotein P04196 20.11 5 2
Fibrinogen gchain P02679 19.9 7 2
Kininogen-1 P01042 16.02 4 2
Desmoplakin P15924 11.59 1 2
a
1
-Microglobulin/bikunin precursor protein precursor P02760 11.13 9 2
a
Shown in bold are the proteins classified as plasma proteins (as assessed by www.HPRD.org).
b
UniProtKB/Swiss-Prot database.
c
An aggregate score of the quality
of the peptide spectra obtained, the total number of peptides identified, and percentage coverage of the protein.
d
The number of amino acids identified as a
percentage of the total number of amino acids in the corresponding protein.
e
The number of peptides identified that correspond to the protein indicated.
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Table 3 Proteins newly identified in OA synovial fluids
Protein name
a
Accession
no.
b
Score
c
%
Coverage
d
No. of
peptides
e
Zinc-a
2
-glycoprotein P25311 140.54 35 10
Ig a1 chain C region P01876 128.82 37 9
Ig g-1 chain C region P01857 115.3 35 9
Ig chain C region P01834 87.06 85 6
Calgranulin A (MRP-8) P05109 67.87 33 5
Collagen a
1
(I) chain P02452 40.14 4 4
Complement component C9 P02748 36.76 9 4
Serum paraoxonase/arylesterase 1 P27169 34.77 13 3
Ig chain V-III region SIE P01620 33.65 38 3
Ig heavy-chain V-III region BRO P01766 28.94 24 2
NF-B-repressing factor O15226 23.76 8 3
A-kinase anchor protein-like protein 8 Q9ULX6 23.65 11 3
Structural maintenance of chromosomes 4-like 1 protein Q9NTJ3 23.43 1 2
Bromodomain adjacent to zinc-finger domain 2B (hWALp4) Q9UIF8 22.6 3 3
Histone deacetylase 5 Q9UQL6 22.4 4 3
Voltage-dependent R-type calcium channel a-1E subunit Q15878 22.29 1 2
Inhibitor of nuclear factor B kinase bsubunit O14920 22.07 8 3
bplatelet-derived growth factor receptor precursor (EC 2.7.1.112) (CD140b antigen) P09619 20.93 5 2
Leucine-rich a
2
-glycoprotein P02750 20.55 5 2
Mitochondrial 28S ribosomal protein S29 P51398 19.76 6 2
Glucose-6-phosphate 1-dehydrogenase P11413 19.58 14 3
Collagen a
2
(I) chain P08123 18.98 7 3
Histone H4 P62805 18.73 16 2
Macrophage inflammatory protein 2-bP19876 18.72 28 2
Collagen a
3
(IV) chain Q01955 18.15 5 2
Histone-lysine N-methyltransferase, H3 lysine-9 specific 4 Q15047 17.82 4 2
Toll-like receptor 6 Q9Y2C9 17.38 2 2
Nuclear factor NF-B p100 subunit Q00653 17.22 7 2
Possible global transcription activator SNF2L1 (SWI/SNF-related matrix-associated actin-dependent
regulator of chromatin subfamily A member 1)
P28370 16.37 5 2
RRP5 protein homologue Q14690 16.35 3 2
Interleukin-20 receptor achain Q9UHF4 16.22 11 2
NF-B inhibitor-like protein 1 Q9UBC1 16.13 14 2
Platelet endothelial cell-adhesion molecule P16284 15.87 10 2
Fibroblast growth factor receptor 2 P21802 15.4 2 2
Interleukin-1 receptor-associated kinase 1 P51617 14.92 11 2
Cadherin EGF LAG seven-pass G-type receptor 1 Q9NYQ6 14.84 1 2
Collagen a
2
(V) chain P05997 14.78 5 2
Zinc-finger A20 domain-containing protein 1 Q6GQQ9 14.61 6 2
Low-density lipoprotein receptor-related protein 1 (apolipoprotein E receptor) Q07954 14.54 1 2
Microtubule-actin crosslinking factor 1, isoforms 1/2/3/5 Q9UPN3 14.53 1 2
Zinc-finger DHHC domain-containing protein 13 Q8IUH4 14.48 11 2
Death-associated protein kinase 1 P53355 14.41 3 2
Collagen a
1
(V) chain P20908 13.78 4 2
Integrin a-V (Vitronectin receptor asubunit) P06756 13.52 3 2
Misshapen-like kinase 1(MAPK/ERK kinase kinase kinase 6) Q8N4C8 13.36 4 2
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patients with RA, a classic inflammatory arthritis, were
used as a comparator. Figure 1 shows a heatmap of the
relative levels of cytokines in the five groups of samples.
Compared with cytokine levels in normal sera, cytokine
levels in OA sera were generally slightly higher, and
thoseinRAseraweremuchhigher(Figure1).SAM
analysis revealed that levels of several inflammatory
cytokines (for example, IL-1band IL-6), chemokines
(for example, IP-10 (also known as CXCL10), MCP-1,
IL-8, MIG, and MIP-1b), and growth factors (for exam-
ple, VEGF and SCGF-b) were significantly higher in OA
sera than in normal sera (FDR < 10%; Figure 2), consis-
tent with previous reports of the association of OA with
such inflammatory mediators [27]. Interestingly, we also
found OA-associated elevations in levels of IL-9 and
cutaneous T-cell attracting chemokine (CTACK), sup-
porting the concept that T cells play a role in OA [28].
As expected, cytokine levels were significantly higher in
RA sera than in OA sera (Figure 3; FDR < 10%).
Unlike RA, OA is considered a disorder that is
restricted to the joints. Indeed, levels of multiple cyto-
kines were much higher in OA synovial fluids than in
OA sera (Figure 1 and Table 5); levels of TNF were neg-
ligible in OA sera but substantial in OA synovial fluid
(Figure 1 and Table 5). Our findingssuggestthatthe
abnormally high levels of cytokines in OA sera largely
reflect overproduction of these cytokines in the joint,
consistent with the finding that levels of high-sensitivity
C-reactive protein in the serum of OA patients correlate
withthedegreeofinflammatoryinfiltrateinthe
Table 4 Classes of proteins newly identified in OA synovial fluids
Histone-related proteins NF-B-related proteins Inflammatory
receptors
Macrophage-related proteins
Histone deacetylase 5 NF--B-repressing factor IL-12-receptor b
2
Macrophage inflammatory
protein 2-b
Histone H4 Inhibitor of nuclear factor B kinase b
subunit
IL-18-receptor 1 Macrophage receptor MARCO
Histone-lysine N- methyltransferase, H3 lysine-9
specific 4
Nuclear factor B p100 subunit IL-20-receptor a
chain
Histone acetyltransferase MYST3 NF-B inhibitor-like protein 1 Toll-like receptor 6
Integrin-aV
Table 3 Proteins newly identified in OA synovial fluids (Continued)
Cadherin EGF LAG seven-pass G-type receptor Q9NYQ7 13.26 1 3
Regulator of G-protein signaling 14 O43566 13.21 8 2
Collagen a
1
(VII) chain Q02388 13.19 1 2
Interleukin-12 receptor b
2
chain Q99665 13.1 5 2
Interleukin-1b(IL-1b) P01584 12.94 20 2
Platelet-derived growth factor B chain P01127 12.91 18 2
Collagen a
1
(II) chain P02458 12.65 6 2
Pappalysin-1 Q13219 12.58 2 2
Complement C1q tumor necrosis factor-related protein 5 Q9BXJ0 11.97 23 2
Interleukin-18 receptor 1 precursor (IL-1 receptor-related protein) Q13478 11.8 4 2
A kinase anchor protein 10, mitochondrial (PRKA10) O43572 11.36 8 2
Neutrophil collagenase P22894 11.34 9 2
Protein-arginine deiminase type I Q9ULC6 11.34 5 2
TNF receptor-associated factor 5 (RING finger protein 84) O00463 10.92 8 2
Macrophage receptor MARCO Q9UEW3 10.89 8 2
Collagen a
3
(V) chain P25940 10.32 1 2
Histone acetyltransferase MYST3 Q92794 10.14 2 2
Leukocyte immunoglobulin-like receptor subfamily B member 4 Q8NHJ6 9.43 11 2
Mast/stem cell growth-factor receptor P10721 9.3 5 2
a
Shown in bold are the proteins classified as plasma proteins (as assessed by www.HPRD.org).
b
UniProtKB/Swiss-Prot database.
c
An aggregate score of the quality
of the peptide spectra obtained, the total number of peptides identified, and percentage coverage of the protein.
d
The number of amino acids identified as a
percentage of the total number of amino acids in the corresponding protein.
e
The number of peptides identified that correspond to the protein indicated.
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patients’joints [29]. Thus, OA is associated with low-
grade inflammation that may originate in the joints.
Interestingly, 39 (36%) of the proteins we identified in
OA synovial fluid are classically considered plasma pro-
teins (Tables 2 and 3). Indeed, plasma proteins form a
large proportion of the proteins enriched in OA synovial
fluid relative to healthy synovial fluid [10]. What might
these plasma proteins be doing in the OA joint? Like cer-
tain products of ECM breakdown [2,5,6], the plasma pro-
tein fibrinogen can function as a DAMP and has been
proposed to contribute to the pathogenesis of inflamma-
tory arthritis [7-9]. We therefore examined whether
other plasma proteins in OA synovial fluid can function
as immunostimulatory DAMPs that could contribute to
the low-grade inflammation associated with OA.
Key players in OA-associated inflammation are the
macrophages [27,30,31]. The cell infiltrate in human OA
joints consists mainly of macrophages, and mice
depleted of macrophages are relatively resistant to col-
lagenase-induced OA [30]. Macrophages from OA joints
produce a number of growth factors, such as VEGF, and
inflammatory cytokines, such as the major OA-asso-
ciated cytokines IL-1band TNF [30]. We detected
VEGF, IL-1b, and TNF in OA synovial fluid in our cyto-
kine screen (Figure 1 and Table 5) and found that levels
of VEGF and IL-1bare significantly higher in OA sera
than in normal sera (Figure 2). VEGF may promote OA
pathology by inducing angiogenesis (and thereby osteo-
phyte formation) and by inducing matrix metallopro-
tease production (and thereby cartilage degradation)
Normal1
Normal10
Normal11
Normal12
Normal13
Normal15
Normal16
Normal17
Normal18
Normal19
Normal2
Normal20
Normal21
Normal22
Normal23
Normal24
Normal25
Normal26
Normal27
Normal28
Normal29
Normal3
Normal30
Normal31
Normal32
Normal33
Normal34
Normal35
Normal36
Normal4
Normal5
Normal6
Normal7
Normal8
Normal9
102a
116a
19a
30a
47a
48a
57a
65a
82a
91a
92a
94a
97a
9a
104a
108a
111a
115a
117a
43a
46a
49a
53a
75a
36_OA
66_OA
26_OA
15_OA
384_OA
291_OA
320_OA
220_OA
221_OA
7_OA
214_OA
8_OA
001-32A
003-33A
005-31A
004-31A
006-31A
003-29A
002-29A
004-29A
002-28A
007-31A
001-31A
003-32A
004-32A
002-31A
001-34A
003-34A
009-31A
002-34A
003-12A
006-20A
005-33A
004-20A
004-21A
80_RA
40_RA
24_RA
13_RA
44_RA
17_RA
38_RA
99_RA
14_RA
48_RA
12_RA
37_RA
6_RA
18_RA
MIG
IP-10
IFN-α2
IL-7
IL-12(p70)
IL-10
IL-13
β-NGF
IL-18
IL-2Rα
SCF
IL-3
IL-12p40
M-CSF
HGF
SCGF-β
MCP-1
IL-8
IL-1ra
VEGF
IL-6
IL-16
IL-1α
TNF-β
LIF
SDF-1α
TNF-α
IL-9
IFN-γ
IL-5
IL-4
MCP-3
IL-1β
G-CSF
GM-CSF
IL-2
IL-15
IL-17
FGF basic
CTACK
GROα
MIP-1β
MIP-1α
Normal sera OA sera OA SF RA sera RA SF
0.5 x normal
0.125 x normal
0.25 x normal
Normal mean
2 x normal
4 x normal
8 x normal
Figure 1 Inflammatory cytokines are associated with osteoarthritis. Relative cytokine levels in serum and synovial fluid (SF) samples from
patients with osteoarthritis (OA) or rheumatoid arthritis (RA) and in serum samples from healthy individuals (normal sera). Cytokine levels were
measured with a multiplex bead-based immunoassay. Samples from individual patients are listed above the heatmap, and the individual
cytokines are listed to the right of the heatmap. IL, interleukin; IFN, interferon; MIG, monokine induced by IFN-g; IP-10, interferon gamma-induced
protein 10; IL-1ra, interleukin-1 receptor antagonist; VEGF, vascular endothelial growth factor; GM-CSF, granulocyte macrophage colony-
stimulating factor; FGF, fibroblast growth factor; MCP, monocyte chemotactic protein; IL-2Ra, interleukin-2 receptor achain; HGF, hepatocyte
growth factor; GROa, growth-regulated oncogene a; MIP-1, macrophage inflammatory protein; b-NGF, bnerve growth factor; SCF, stem cell
factor; M-CSF, macrophage colony-stimulating factor; SCGF-b, stem cell growth factor b; LIF, leukemia inhibitory factor; SDF-1a, stromal cell-
derived factor 1a; G-CSF, granulocyte colony-stimulating factor; CTACK, cutaneous T-cell attracting chemokine.
Sohn et al.Arthritis Research & Therapy 2012, 14:R7
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[32]. The cytokines produced by macrophages amplify
the inflammation in the joints by inducing synovial cells
to produce further cytokines and chemokines, as well as
matrix metalloproteases [30]. Moreover, macrophages
express many of the receptors that mediate DAMP sig-
naling, and they can thus trigger an inflammatory cas-
cade in response to DAMPs present in OA synovial
fluid [7-9].
We therefore assessed whether a subset of the identi-
fied plasma proteins could induce macrophages to pro-
duce TNF, a key cytokine that is thought to drive the
inflammatory cascade in OA [27]. We tested a
1
-micro-
globulin (a
1
m), a
1
-acid glycoprotein 1 (AGP 1; also
known as orosomucoid 1), a
2
-macroglobulin (a
2
m), Gc-
globulin (also known as vitamin D-binding protein),
albumin, and haptoglobin, all of them plasma proteins
detected in our survey of synovial fluid proteins (Table
2) and shown to be enriched in OA synovial fluid [10].
With mouse macrophages, we found that a
1
m, a
2
m,
and Gc-globulin, at concentrations similar to those mea-
sured in synovial fluid [33-35], each dose-dependently
stimulated the production of TNF, whereas AGP 1,
albumin, and haptoglobin did not (Figure 4a). The
plasma proteins ceruloplasmin, complement component
C3, complement component C4, b
2
-glycoprotein (also
known as apolipoprotein H) also did not stimulate TNF
production (data not shown).
We next examined the effect of a
1
m, a
2
m, and Gc-
globulin on cytokine production in human macrophages.
Because the endotoxin LPS is a common contaminant
and is itself an agonist of TLR4, we tested the stimula-
tory properties of the plasma proteins in the presence of
polymyxin B, a compound that neutralizes LPS. In the
presence of polymyxin B, a
1
m-, a
2
m-, and Gc-globulin-
induced TNF production was not significantly reduced,
whereas LPS-induced TNF production was abrogated
(Figure 4b). Additionally, pretreatment with proteinase
K significantly abrogated TNF production induced by
the plasma proteins but not TNF production induced by
LPS (Figure 5). Although we cannot exclude the possibi-
lity that a small component of the observed stimulation
is due to endotoxin, this result confirms that the plasma
proteins are themselves immunostimulatory. Gc-globu-
lin, a
1
m, and a
2
mwerealsoabletoinducethe
Normal25
Normal18
Normal34
Normal28
Normal15
Normal20
Normal33
Normal5
Normal6
Normal26
Normal36
Normal35
Normal29
Normal30
Normal27
Normal13
Normal19
Normal21
Normal24
Normal17
Normal8
Normal3
9a
57a
Normal12
Normal7
Normal16
Normal32
Normal23
92a
75a
97a
111a
19a
Normal2
Normal9
Normal4
53a
43a
Normal31
30a
Normal10
115a
Normal22
Normal11
104a
Normal1
117a
116a
108a
94a
46a
49a
102a
91a
47a
82a
65a
48a
IL-9
GM-CSF
IL-1ra
GROα
VEGF
SCGF-β
MCP-1
IL-7
IL-1β
IL-10
IL-8
IL-6
G-CSF
MIP-1β
HGF
M-CSF
CTACK
MIG
IP-10
0.5 x normal
0.125 x normal
0.25 x normal
Normal mean
2 x normal
4 x normal
8 x normal
Mostly normal sera Mostl
y
OA sera
Figure 2 Levels of inflammatory cytokines are higher in OA compared with healthy sera. Cytokines whose levels differ significantly
between sera from individuals with osteoarthritis (OA) and sera from age-matched healthy individuals (FDR < 10%). Significance analysis of
microarrays (SAM) was used to identify statistically significant differences, and the SAM-generated results were subjected to unsupervised
hierarchic clustering. Cytokine levels were measured with a multiplex bead-based immunoassay. Samples from individual patients are listed
above the heatmap, and the individual cytokines are listed to the right of the heatmap. IL, interleukin; MIG, monokine induced by IFN-g; IP-10,
interferon gamma-induced protein 10; IL-1ra, interleukin-1 receptor antagonist; VEGF, vascular endothelial growth factor; GM-CSF, granulocyte
macrophage colony-stimulating factor; MCP, monocyte chemotactic protein; HGF, hepatocyte growth factor; GROa, growth-regulated oncogene
a; MIP-1b, macrophage inflammatory protein 1b; M-CSF, macrophage colony-stimulating factor; SCGF-b, stem cell growth factor b; G-CSF,
granulocyte colony-stimulating factor; CTACK, cutaneous T-cell attracting chemokine.
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65a
30a
9a
92a
111a
75a
57a
43a
82a
48a
108a
104a
94a
91a
47a
117a
116a
53a
19a
115a
97a
46a
003-29A
102a
002-34A
002-31A
004-31A
001-31A
001-34A
001-32A
007-31A
49a
003-33A
004-29A
002-29A
005-33A
006-31A
009-31A
005-31A
004-20A
003-12A
003-32A
004-21A
003-34A
002-28A
006-20A
004-32A
FGF
TNF-α
IL-2Rα
IL-3
IL-12p40
IL-16
HGF
M-CSF
MCP-3
G-CSF
IL-1β
IL-6
IL-9
IL-15
IL-2
IL-10
IL-12(p70)
SCGF-β
IL-1ra
MCP-1
IL-8
MIP-1α
0.5 x normal
0.125 x normal
0.25 x normal
Normal mean
2 x normal
4 x normal
8 x normal
Mostl
y
OA sera Mostl
y
RA sera
Figure 3 Levels of inflammatory cytokines are higher in RA compared with OA sera. Cytokines whose levels differ significantly between
sera from individuals with osteoarthritis (OA) and sera from individuals with rheumatoid arthritis (RA) (FDR < 10%). Significance analysis of
microarrays (SAMs) was used to identify statistically significant differences, and the SAM-generated results were subjected to unsupervised
hierarchic clustering. Cytokine levels were measured with a multiplex bead-based immunoassay. Samples from individual patients are listed
above the heatmap, and the individual cytokines are listed to the right of the heatmap. IL, interleukin; IL-1ra, interleukin-1 receptor antagonist;
FGF, fibroblast growth factor; MCP, monocyte chemotactic protein; HGF, hepatocyte growth factor; MIP-1, macrophage inflammatory protein; M-
CSF, macrophage colony-stimulating factor; SCGF-b, stem cell growth factor b; G-CSF, granulocyte colony-stimulating factor.
Table 5 Absolute and relative cytokine levels in healthy and OA serum, and in OA synovial fluid
Cytokine Normal serum levels
(pg/ml)
a
OA serum levels
(pg/ml)
b
OA SF levels
(pg/ml)
c
Ratio of OA serum levels to normal
serum levels
Ratio of OA SF levels to OA
serum levels
IL-6 3.02
(2.7-4.4)
5.13
(4.4-5.8)
975.39
(454.0-2,689.5)
1.7 190.1
IL-1b1.22
(1.0-1.3)
1.58
(1.4-1.8)
1.14
(1.0-1.7)
1.3 0.7
TNF 0.00
(0.0-0.0)
0.00
(0.0-0.0)
2.92
(0.0-13.3)
--
VEGF 20.53
(11.7-65.6)
78.22
(31.3-124.6)
496.31
(245.0-577.8)
3.8 6.3
MCP-1 3.59
(0.0-11.9)
18.53
(13.0-28.0)
107.67
(84.8-191.1)
5.2 5.8
IP-10 537.00
(376.1-750.4)
795.55
(684.7-1,029.7)
2,105.40
(923.2-4,913.3)
1.5 2.6
MIG 244.51
(173.5-374.6)
420.13
(308.4-568.6)
1,047.14
(389.8-1,925.3)
1.7 2.5
OA, osteoarthritis; SF, synovial fluid; IL, interleukin ; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; MCP-1, macrophage chemo tactic protein
1; IP-10, interferon g-induced protein 10; MIG, monokine-induced by interferon g.
a
The median (IQR); n= 34 healthy individuals.
b
The median (IQR); n=23
individuals with OA.
c
The median (IQR); n= 10 individuals with OA.
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production of several other inflammatory cytokines that
were upregulated in OA serum and synovial fluid (Fig-
ures 1 and 2): IL-1b, IL-6, and VEGF (Figure 4c). Thus,
Gc-globulin, a
1
m, and a
2
m can each induce the produc-
tion of TNF, IL-1b, IL-6, and VEGF, all molecules impli-
cated in the pathogenesis of OA [27,30,31].
But how do these plasma proteins stimulate cytokine
production? To determine whether these immunostimu-
latory plasma proteins signal through TLR4, we exam-
ined whether Gc-globulin, a
1
m, and a
2
mcouldalso
induce TNF production in TLR4-deficient macrophages.
TLR4 deficiency inhibited Gc-globulin-, a
1
m-, and a
2
m-
induced TNF production (Figure 6). Confirming that the
defect in inflammatory signaling in the Tlr4
lps-del
macro-
phages was specific to the TLR4 pathway, theTLR2-spe-
cificagonistpeptidoglycanwasabletoinduceTNF
production in these cells-in fact, to a greater degree
than in wild-type cells (possibly because of compensa-
tory mechanisms operating within the TLR family) (Fig-
ure 6). Thus, Gc-globulin-, a
1
m-, and a
2
m-induced
production of TNF is dependent on TLR4.
Interest in the putative immunomodulatory effects of
a
1
m, a
2
m, and Gc-globulin is increasing, with both
proinflammatory and antiinflammatory properties sug-
gested for each of them [36-38].
For example, a
1
m has been shown to bind to the sur-
face of various inflammatory cells and to either stimu-
late or inhibit the activation of human lymphocytes [38].
The immunoregulatory role of a
1
m in health and dis-
ease is likely to be context dependent. Gc-globulin, how-
ever, appears to be primarily proinflammatory: it
enhances the neutrophil- and monocyte-chemotactic
activity of the anaphylatoxin C5a [36] and, in its sialic-
acid-free form, activates macrophages [39]. Here, we
No Polymyxin B
Polymyxin B
Unstim. Gc-glob. LPS
1
m
2
m
Unstim. Gc-glob. LPS
1
m
2
m
Unstim. Gc-glob. LPS
1
m
2
m
IL-1 (pg/ml)
0
20
40
60
80
100
0
2000
4000
6000
8000
IL-6 (pg/ml)
500
1500
1000
0
VEGF (pg/ml)
*
NS
** *
No Polymyxin B
Polymyxin B
No Polymyxin B
Polymyxin B
C
100
10
1
100
10
1
100
10
1
100
10
1
100
10
1
100
10
1
0.0001
0.001
0.01
TNF (pg/ml)
0
200
400
600
800
***
***
*** ***
a
Unstim. Gc-glob. LPS PGN
1
m
2
m
TNF (pg/ml)
0
500
1000
1500
2000
**
NS
No Polymyxin
B
Polymyxin B
NS
NS
NS
b
SPL.bolg-cG.mitsnU
1
m
2
m
AGP 1 Album. Haptogl.
NS NS
NS
NS
NS
NS
NS
NS
Figure 4 Plasma proteins detected in osteoarthritic synovial fluid are immunostimulatory. Mouse bone-marrow-derived macrophages
(BMMs) and human monocyte-derived macrophages (MDMs) were stimulated for 24 hours with plasma proteins detected in osteoarthritic
synovial fluid (Table 2), after which cytokine levels in the supernatants were measured with ELISA or Luminex immunoassay. (a) Levels of TNF
produced by mouse BMMs stimulated with the indicated concentrations (in μg/ml) of Gc-globulin, a
1
-microglobulin (a
1
m), a
2
-macroglobulin
(a
2
m), a
1
-acid glycoprotein 1 (AGP 1), albumin, or haptoglobin. Lipopolysaccharide (LPS) was used as a positive control. (b) Levels of TNF
produced by human MDMs stimulated with 50 μg/ml of a
1
m, a
2
m, or Gc-globulin in the presence or absence of 10 μg/ml of polymyxin B, an
inhibitor of LPS. LPS (1 ng/ml) was used as a positive control for the efficacy of polymyxin B, and peptidoglycan (PGN; 5 μg/ml), as a negative
control. (c) Levels of interleukin-1b(IL-1b), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF) produced by human MDMs
stimulated with 50 μg/ml of Gc-globulin, a
1
m, or a
2
m. LPS (1 ng/ml) was used as a positive control. Results are representative of experiments
performed at least twice. In (a), data are shown as the mean ± SEM of duplicates. In (b) and (c), data are shown as the mean ± SEM of
triplicates. *P< 0.05; **P< 0.01, ***P< 0.001; NS, not significant.
Sohn et al.Arthritis Research & Therapy 2012, 14:R7
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uncover an additional mechanism by which these
plasma proteins could promote inflammation. We spec-
ulate that exudation into extravascular spaces at sites of
tissue damage and inflammation may render these
plasma proteins inflammatory by bringing them into
contact with TLR-expressing macrophages. Our finding
that certain plasma proteins present in OA synovial
fluid can induce macrophage production of inflamma-
tory cytokines supports the model of local production of
inflammatory mediators in the joints in OA.
Conclusions
We identified 108 proteins in OA synovial fluid and
showed that OA is associated with low-grade inflamma-
tion. We found that plasma proteins form a large propor-
tion of the proteins present in OA synovial fluid and that
certain of these plasma proteins can signal through TLR4
to induce the production of an array of inflammatory
cytokines, including those upregulated in OA. Our find-
ings suggest that plasma proteins present in OA synovial
fluid, whether through exudation from the plasma or
production by synovial tissues, could contribute to low-
grade inflammation in OA by functioning as DAMPs.
Abbreviations
α
1
m: α
1
-microglobulin; AGP 1: α
1
-acid glycoprotein 1; α
2
m: α
2
-macroglobulin;
BMM: bone-marrow-derived macrophage; DAMP: damage-associated
molecular pattern; ECM: extracellular matrix; IL-1β: interleukin-1β; IL-6:
interleukin-6; IP-10: interferon gamma-induced protein 10; LCMS:
chromatography tandem mass spectrometry; LPS: lipopolysaccharide; MCP-1:
macrophage chemotactic protein-1; MDM: monocyte-derived macrophage;
MEM: minimal essential medium; MIG: monokine induced by interferon-γ;
MIP-1: macrophage inflammatory protein-1; OA: osteoarthritis; PAGE:
polyacrylamide gel electrophoresis; RA: rheumatoid arthritis; TLR: Toll-like
receptor; TNF: tumor necrosis factor; VEGF: vascular endothelial growth
factor.
Acknowledgements
This study was funded by VA RR&D Merit Review Award and N01-HV-00242
NHLBI Proteomics Center funding to WHR.
Author details
1
GRECC, VA Palo Alto Health Care System, 3801 Miranda Ave., Palo Alto, CA
94304, USA.
2
Division of Immunology and Rheumatology, Department of
Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
3
Bone and Joint Center, VA Palo Alto Health Care System, 3801 Miranda
Ave., Palo Alto, CA 94304, USA.
4
Department of Mechanical Engineering,
Stanford University, Stanford, CA 94305, USA.
Authors’contributions
JS and WHR conceived the studies. OS performed the mass spectrometric
analysis of synovial fluid. PEC, LJL, and JCE performed the multiplex cytokine
analysis. JCE, KAB, and TPA collected and provided OA sera. DHS performed
the in vitro macrophage stimulation assays. JS, WHR, DHS, OS, TML, and IH
analyzed the resulting datasets. TML and JS wrote and edited the
manuscript with the input of WHR, OS, and DHS. All authors read and
approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 16 May 2011 Revised: 4 November 2011
Accepted: 8 January 2012 Published: 8 January 2012
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No treatment
Proteinase K
Unstim. Gc-
g
lob. LPS
α
1
mα
2
m
TNF
(
pg
/
ml
)
500
**
NS
0
1000
1500
2000
2
5
00
**
*
Figure 5 Induction of TNF production by plasma proteins is
not due to endotoxin contamination. RAW264.7 macrophages
were stimulated for 24 hours with 50 μg/ml of Gc-globulin, a
1
-
microglobulin (a
1
m), or a
2
-macroglobulin (a
2
m) that had been
incubated with proteinase K (20 μg/ml) at 55°C for 4 hours in the
presence of b-mercaptoethanol and then heated to 100°C for 10
minutes. TNF levels in the supernatants were determined with
ELISA. Lipopolysaccharide (LPS; 1 ng/ml) was used as a positive
control. Data are shown as the mean ± SEM of duplicates from one
of two representative experiments. *P< 0.05; **P< 0.01.
Unstim. Gc-glob. α
1
mα
2
mLPS PGN
wt
Tlr4
lps-del
TNF (pg/ml)
0
500
1000
1500
2000
** ** ** *
Figure 6 Plasma proteins detected in osteoarthritic synovial
fluid stimulate macrophage TNF production via TLR4. Levels of
TNF produced by wild-type (wt) or TLR4-deficient (Tlr4
lps-del
) mouse
bone-marrow-derived macrophages stimulated for 24 hours with 50
μg/ml of Gc-globulin, a
1
-microglobulin (a
1
m), or a
2
-macroglobulin
(a
2
m), after which TNF levels in the supernatants were determined
with ELISA. Lipopolysaccharide (LPS; 1 ng/ml) was used as a positive
control for TLR4-dependent TNF production, and peptidoglycan
(PGN; 5 μg/ml) as a positive control for TLR4-independent TNF
production. Data are shown as the mean ± SEM of triplicates from
one of three representative experiments. *P< 0.05; **P< 0.01.
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doi:10.1186/ar3555
Cite this article as: Sohn et al.: Plasma proteins present in osteoarthritic
synovial fluid can stimulate cytokine production via Toll-like receptor 4.
Arthritis Research & Therapy 2012 14:R7.
Sohn et al.Arthritis Research & Therapy 2012, 14:R7
http://arthritis-research.com/content/14/1/R7
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