The Infrapatellar Fat Pad in Knee Osteoarthritis An Important Source of Interleukin-6 and Its Soluble Receptor

ArticleinArthritis & Rheumatology 60(11):3374-7 · November 2009with44 Reads
Impact Factor: 7.76 · DOI: 10.1002/art.24881 · Source: PubMed
Abstract

Obesity is a potent risk factor in knee osteoarthritis (OA). It has been suggested that adipokines, secreted by adipose tissue (AT) and largely found in the synovial fluid of OA patients, derive in part from the infrapatellar fat pad (IFP), also known as Hoffa's fat pad. The goal of this study was to characterize IFP tissue in obese OA patients and to compare its features with thigh subcutaneous AT to determine whether the IFP contributes to local inflammation in knee OA via production of specific cytokines. IFP and subcutaneous AT samples were obtained from 11 obese women (body mass index > or =30 kg/m2) with knee femorotibial OA. Gene expression was measured by real-time quantitative polymerase chain reaction. Cytokine concentrations in plasma and in conditioned media of cultured AT explants were determined by enzyme-linked immunosorbent assay or by Luminex xMAP technology. In IFP tissue versus subcutaneous AT, there was a decrease in the expression of genes for key enzymes implicated in adipocyte lipid metabolism, whereas the expression levels of genes for AT markers remained similar. A 2-fold increase in the expression of the gene for interleukin-6 (IL-6), a 2-fold increase in the release of IL-6, and a 3.6-fold increase in the release of soluble IL-6 receptor (sIL-6R) were observed in IFP samples, compared with subcutaneous AT, but the rates of secretion of other cytokines in IFP samples were similar to the rates in subcutaneous AT. In addition, leptin secretion was decreased by 40%, whereas adiponectin secretion was increased by 70%, in IFP samples versus subcutaneous AT. Our results indicate that the IFP cytokine profile typically found in OA patients could play a role in paracrine inflammation via the local production of IL-6/sIL-6R and that such a profile might contribute to damage in adjacent cartilage.

Full-text

Available from: Xavier Chevalier, May 26, 2015
ARTHRITIS & RHEUMATISM
Vol. 60, No. 11, November 2009, pp 3374–3377
DOI 10.1002/art.24881
© 2009, American College of Rheumatology
The Infrapatellar Fat Pad in Knee Osteoarthritis
An Important Source of Interleukin-6 and Its Soluble Receptor
Emilie Distel,
1
Thomas Cadoudal,
1
Sylvie Durant,
1
Alexandre Poignard,
2
Xavier Chevalier,
2
and Chantal Benelli
1
Objective. Obesity is a potent risk factor in knee
osteoarthritis (OA). It has been suggested that adipo-
kines, secreted by adipose tissue (AT) and largely found
in the synovial fluid of OA patients, derive in part from
the infrapatellar fat pad (IFP), also known as Hoffa’s
fat pad. The goal of this study was to characterize IFP
tissue in obese OA patients and to compare its features
with thigh subcutaneous AT to determine whether the
IFP contributes to local inflammation in knee OA via
production of specific cytokines.
Methods. IFP and subcutaneous AT samples were
obtained from 11 obese women (body mass index >30
kg/m
2
) with knee femorotibial OA. Gene expression was
measured by real-time quantitative polymerase chain
reaction. Cytokine concentrations in plasma and in
conditioned media of cultured AT explants were deter-
mined by enzyme-linked immunosorbent assay or by
Luminex xMAP technology.
Results. In IFP tissue versus subcutaneous AT,
there was a decrease in the expression of genes for key
enzymes implicated in adipocyte lipid metabolism,
whereas the expression levels of genes for AT markers
remained similar. A 2-fold increase in the expression of
the gene for interleukin-6 (IL-6), a 2-fold increase in the
release of IL-6, and a 3.6-fold increase in the release of
soluble IL-6 receptor (sIL-6R) were observed in IFP
samples, compared with subcutaneous AT, but the rates
of secretion of other cytokines in IFP samples were
similar to the rates in subcutaneous AT. In addition,
leptin secretion was decreased by 40%, whereas adi-
ponectin secretion was increased by 70%, in IFP sam-
ples versus subcutaneous AT.
Conclusion. Our results indicate that the IFP
cytokine profile typically found in OA patients could
play a role in paracrine inflammation via the local
production of IL-6/sIL-6R and that such a profile might
contribute to damage in adjacent cartilage.
Obesity is a potent risk factor in the development
and progression of knee osteoarthritis (OA) (1). While
mechanical stress obviously contributes to this patho-
logic outcome in obese patients, the relationship be-
tween obesity and OA remains complex, since mechan-
ical stress alone cannot explain the link between obesity
and pathology in non–weight-bearing joints, such as
hand OA (1). Thus, other pathophysiologic functions of
the adipose tissue (AT) in obese patients could also be
involved. It has been well-established that AT releases
cytokines that may act in an autocrine, paracrine, or
endocrine manner (2). More specifically, among the
many biologically active proteins secreted by AT,
interleukin-6 (IL-6), tumor necrosis factor
(TNF
),
and IL-1
are known to be involved in inflammation (3).
Adipokines are largely found in the synovial fluid
of patients with OA and rheumatoid arthritis (4). It has
been suggested that these adipokines might originate in
the infrapatellar fat pad (IFP), also known as Hoffa’s fat
pad. Hoffa’s fat pad is located between the lower surface
of the patella and the trochlear surface of the femur, in
an intracapsular but extrasynovial location, and was
initially thought to have a mainly biomechanical function
(5). The presence of several cytokines in the IFP has
been noted in a few previous studies (6,7). However,
these studies were performed in heterogeneous patient
1
Emilie Distel, Thomas Cadoudal, PhD, Sylvie Durant, PhD,
Chantal Benelli, PhD: INSERM UMR-S 747, Universite´ Paris Des-
cartes, Paris, France;
2
Alexandre Poignard, MD, Xavier Chevalier,
MD, PhD: Hoˆpital Henri Mondor, Cre´teil, France.
Ms Distel and Dr. Cadoudal contributed equally to this work.
Drs. Chevalier and Benelli contributed equally to this work.
Address correspondence and reprint requests to Chantal
Benelli, PhD, INSERM UMR-S 747, Centre Universitaire des Saints-
Pe`res, 45 Rue des Saints-Pe`res, F-75006 Paris, France. E-mail: chantal.
benelli@parisdescartes.fr.
Submitted for publication April 1, 2009; accepted in revised
form July 11, 2009.
3374
Page 1
groups with differences in inflammation levels and met-
abolic status. Furthermore, these studies provided no
evidence that this particular AT was different from other
“classic” ATs, such as subcutaneous AT, although it has
been well-established that AT has specific characteristics
according to its localization.
The aim of the present study was to characterize
IFP tissue from obese OA patients and to compare its
features with samples of thigh subcutaneous AT taken
from the same individuals, in order to determine
whether the IFP contributes to local inflammation in
knee OA via the production of specific cytokines.
PATIENTS AND METHODS
Samples and laboratory methods. We obtained IFP
tissue and thigh subcutaneous AT from 11 obese women (body
mass index [BMI] 30 kg/m
2
) with knee femorotibial OA
(grade IV, according to the Kellgren/Lawrence scale [8]) who
were undergoing total knee replacement surgery. None of the
patients had known metabolic or malignant diseases, and none
were taking medications known to alter adipocyte metabolism.
Both IFP tissue and thigh subcutaneous AT were carefully
dissected in order to obtain AT explants. In IFP samples,
special care was taken to remove the synovial membrane. For
experiments with messenger RNA (mRNA), AT explants were
frozen in liquid nitrogen, whereas, for experiments with cyto-
kines, AT explants were incubated for 3 hours in Krebs
medium with 1% bovine serum albumin. Real-time quantita-
tive polymerase chain reaction was used to analyze mRNA.
Cytokine concentrations in plasma and in conditioned media
of cultured AT explants were determined by enzyme-linked
immunosorbent assay (ELISA), according to the manufactur-
er’s protocols for the Human Adiponectin/Acrp30 Quantikine
ELISA kit and the Human sIL-6R Quantikine ELISA kit
(R&D Systems, Abingdon, UK); other cytokines were mea-
sured using Plateforme Technologique Phe´notypage du Petit
Animal et Microdosage (Hoˆpital Saint Antoine, Paris, France).
The study was approved by the Institutional Ethics Committee
of Hoˆpital Henri Mondor.
Statistical analysis. Pairwise comparisons were made
using the nonparametric Mann-Whitney U test, and analyses
were performed using StatView A (SAS Institute, Cary, NC).
Results are presented as the mean SEM. P values less than
0.05 were considered significant.
RESULTS
Characteristics of the patients included in this
study are shown in Table 1. The 11 women were all
obese (BMI 30 kg/m
2
), and all had low-grade inflam
-
mation (C-reactive protein 10 mg/liter). However, all
patients had a high level of IL-6 in plasma.
First, in both IFP tissue and subcutaneous AT, we
determined expression levels of genes for key enzymes
that are involved, in one of the following ways, in
adipocyte lipid metabolism: lipid uptake (fatty acid
transport CD36 [FAT/CD36]), intracellular fatty acid
trafficking (fatty acid binding protein 4/aP2 [FABP-4/
aP2]), nuclear receptor (peroxisome proliferator–
activated receptor
[PPAR
]), lipolysis (adipocyte tri-
glyceride lipase [ATGL], lipoprotein lipase [LPL], and
hormone sensitive lipase [HSL]), fatty acid reesterifica-
Figure 1. Expression pattern of genes for key enzymes involved in
lipid metabolism, in subcutaneous adipose tissue (SCAT) and infrapa-
tellar fat pad (IFP) explants. Total RNA was extracted from 300 mg of
subcutaneous AT and IFP explants from 11 obese patients with
osteoarthritis. Complementary DNA (1.25
g) was analyzed using
real-time quantitative polymerase chain reaction. Values were normal-
ized to RPL13 ribosomal RNA and expressed as a percentage of the
gene value obtained in subcutaneous AT (control). Values are the
mean and SEM. ⴱⴱⴱ P 0.001 versus control. FABP-4 fatty acid
binding protein 4; PEPCK-C cytosolic phosphoenolpyruvate car-
boxykinase; FAT/CD36 fatty acid transport CD36; ATGL
adipocyte triglyceride lipase; LPL lipoprotein lipase; HSL hor-
mone sensitive lipase; PPARg peroxisome proliferator–activated
receptor
; CPT-1 carnityl palmitoyltransferase 1.
Table 1. Characteristics of the 11 OA patients*
Age, years 74.6 1.93
BMI, kg/m
2
31.6 1.44
Glucose, mmoles/liter 5.4 0.60
Leptin, ng/ml 4.39 0.83
Adiponectin,
g/ml 11.66 1.49
Resistin, ng/ml 2.93 0.55
CRP, mg/liter 6.2 0.68
IL-6, pg/ml 7.22 1.89
sIL-6R, ng/ml 22.82 1.78
* Values are the mean SEM. OA osteoarthritis; BMI body
mass index; CRP C-reactive protein; IL-6 interleukin-6; sIL-6R
soluble IL-6 receptor.
INFRAPATELLAR FAT PAD AND IL-6/sIL-6R IN KNEE OA 3375
Page 2
tion (cytosolic phosphoenolpyruvate carboxykinase
[PEPCK-C]), or
-oxidation (carnityl palmitoyltrans-
ferase 1 [CPT-1]) (Figure 1). Expression of genes for
ATGL, LPL, HSL, FAT/CD36, and PPAR
was strik-
ingly decreased in IFP tissue compared with subcutane-
ous AT, whereas differences in expression of genes for
CPT-1, FABP-4/aP2, and PEPCK-C, also considered to
be AT markers, were not significant.
We then investigated the patterns of expression
and secretion of various cytokines in both IFP tissue and
subcutaneous AT. Expression of the genes for major
proinflammatory cytokines (TNF
, IL-1
, IL-6, IL-8,
and macrophage chemotactic protein 1 [MCP-1]) was
observed in both IFP and subcutaneous AT explants
(Figure 2A). A nearly 2-fold increase in IL-6 gene
expression was observed in IFP samples versus subcuta-
neous AT, with no significant differences in the expres-
sion of the other cytokines. In addition, the rate of IL-6
release was 2-fold higher in IFP tissue than in subcu-
taneous AT; for the other cytokines (TNF
, IL-8, and
MCP-1), the differences in the rates of release were not
significant (Figure 2B). Concomitantly, the release of
soluble IL-6 receptor (sIL-6R) in IFP samples was
3.6-fold higher than in subcutaneous AT.
Obesity has also been linked to a modulation of
AT secretion of adipokines, such as adiponectin, leptin,
and resistin (2). Notably, these adipokines are known to
be present in the synovial fluid of OA patients (4).
Expression of the gene for adiponectin was not signifi-
cantly different in IFP tissue compared with subcutane-
ous AT; however, there was a significant decrease in
expression of the gene for leptin (Figure 2A). While
resistin secretion was undetectable in both IFP and
subcutaneous AT explants, leptin secretion in IFP was
decreased by 40% of that observed in subcutaneous AT,
and adiponectin secretion in IFP was increased by 70%
of that observed in subcutaneous AT (Figure 2B).
DISCUSSION
Our results clearly demonstrate that IFP tissue
from obese patients, compared with subcutaneous AT
from the same individuals, exhibits an original pattern of
expression and secretion of cytokines. Rates of IL-6
expression and secretion were increased in IFP tissue
compared with rates in subcutaneous AT, whereas the
rates of expression and secretion of other cytokines
known to be involved in the pathogenesis of OA (e.g.,
TNF
, IL-8, etc.) remained similar between IFP tissue
and subcutaneous AT. It is now accepted that cytokines
can act through endocrine, paracrine, or autocrine
mechanisms. Long-term high levels of IL-6 within AT
Figure 2. Expression pattern and secretion of adipokines and proinflammatory cytokines, in subcutaneous adipose tissue (SCAT) and infrapatellar
fat pad (IFP) explants. A, Adipokine and cytokine gene expression. Total RNA was extracted from 300 mg of subcutaneous AT and IFP explants
from 11 obese patients with osteoarthritis. Complementary DNA (1.25
g) was analyzed using real-time quantitative polymerase chain reaction.
Values were normalized to RPL13 ribosomal RNA and are expressed as the mean and SEM percentage of the gene value obtained in subcutaneous
AT (control). P 0.01 versus control; ⴱⴱ P 0.001 versus control. B, Adipokine and cytokine secretion. Subcutaneous AT explants (300 mg)
were incubated for 3 hours in Krebs medium containing 1% bovine serum albumin. Cytokine levels were measured in incubation medium. Leptin
and interleukin-6 (IL-6) levels were determined using Luminex technology; adiponectin and soluble IL-6 receptor (IL-6sR) levels were determined
using enzyme-linked immunosorbent assay. Values are the mean and SEM. P 0.01; ⴱⴱ P 0.001. TNF
tumor necrosis factor
;
MCP-1 macrophage chemotactic protein 1.
3376 DISTEL ET AL
Page 3
are concomitant with altered expression of lipolytic
genes or proteins (7). The high concentration of IL-6
that we observed in IFP samples might explain the
reduction in the expression of genes for proteins in-
volved in adipocyte lipid metabolism (2).
In addition to its autocrine effect, IL-6 from the
IFP could have paracrine effects on articular cartilage
mediated by sIL-6R, which is also increased in the IFP.
In fact, to be fully active, IL-6 must bind to its receptor,
which can be either membrane-bound or soluble. As
chondrocytes express low levels of the IL-6 membrane-
bound receptor, the presence of sIL-6R is required to
obtain the full effect of IL-6 (9).
Obesity is associated with AT macrophage infil-
tration, which can be different depending on the location
of the AT. Macrophages secrete proinflammatory cyto-
kines such as IL-6, resulting in a so-called “low-grade
inflammatory state” (2). In the current study, subcuta-
neous AT and IFP tissue similarly express macrophage
markers (CD14, CD68; data not shown), suggesting that
IL-6 overexpression and production by the IFP in knee
OA are probably not related to a general phenotype in
obesity but rather to a specific characteristic of the IFP.
The overproduction of IL-6/sIL-6R in the IFP is
associated with a decrease in local leptin secretion and
an increase in adiponectin secretion in IFP tissue. The
results of previous studies have suggested that, in con-
trast to its protective role against obesity, adiponectin in
skeletal joints might be proinflammatory and involved in
matrix degradation (10). Moreover, it has been shown
that adiponectin may strongly induce IL-6 secretion in a
cultured chondrogenic cell line (10), amplifying the
inflammatory profile.
The decrease of locally produced leptin by the
IFP is unexpected in light of the increase of circulating
leptin and IL-6 previously observed in obese patients
(11) and the proposed proinflammatory role of leptin in
the regulation of cartilage metabolism (12,13). Further
research will be needed to clarify this finding.
In conclusion, our results show a different meta-
bolic pattern in IFP tissue than in subcutaneous AT and
strongly suggest that the specific cytokine profile found
in the IFP tissue of obese OA patients may contribute to
paracrine inflammation and progressive cartilage dam-
age, via the local production of IL-6/sIL-6R and adi-
ponectin. It would be of interest to know whether the
same metabolic profile is found in OA patients who are
not obese.
ACKNOWLEDGMENTS
We thank Dr. Maı¨t e´ Corvol, Dr. Claude Forest, and
Professor Robert Barouki for their participation in helpful
discussions, and Nade`ge Brunel (Plateforme Technologique
Phe´notypage du Petit Animal et Microdosages, Hoˆpital Saint-
Antoine, Paris, France) for technical assistance with cytokine
measurements.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Benelli had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Chevalier, Benelli.
Acquisition of data. Distel, Cadoudal, Durant, Poignard, Chevalier.
Analysis and interpretation of data. Distel, Cadoudal, Durant, Benelli.
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INFRAPATELLAR FAT PAD AND IL-6/sIL-6R IN KNEE OA 3377
Page 4
    • "For instance, leptin and chemerin, positively correlated with the severity of osteoarthritis91011. Actually, the local production by joint tissues has been postulated as an important source of these adipokines as well as other inflammatory mediators [9,121314. Therefore, the alteration of their secretion pattern during OA could impact cartilage and synovium homeostasis. In fact, IPFPs and synovial fibroblasts, exposed to pro-inflammatory cytokines such as IL-1β, released large amounts of pro-inflammatory mediators, including prostaglandinE 2 , TNF-α or IL-6, and adipokines such as leptin [15]. "
    [Show abstract] [Hide abstract] ABSTRACT: Emerging data suggest that several metabolic factors, released mainly by white adipose tissue (WAT) and joint tissues, and collectively named adipokines, might have a role in the pathophysiology of OA. Recently, novel adipokines such as SERPINE2, WISP2, GPNMB and ITIH5 have been identified in WAT. The main goal of this study was to analyse the expression of these novel adipokines in synovium, infrapatellar fat pad and chondrocytes and to compare the expression of these molecules in healthy and OA tissues. Synovial tissues, infrapatellar fat pad and chondrocytes were obtained from 36 OA patients (age 52-85; mean BMI 28.9) who underwent total knee replacement surgery. Healthy synovial tissues and infrapatellar fat pad were obtained from 15 traumatic knee patients (age 23-53; mean BMI 23.5). mRNA and protein expression were determined by qRT-PCR and western blot analysis respectively. All the novel adipokines, matter of our study, are expressed in OA synovium, infrapatellar fat pad and chondrocytes. Moreover, we detected a differential expression of SERPINE2 and ITIH5 in OA synovial tissues as compared to healthy samples. Finally, we also observed an increased expression of WISP2 in OA infrapatellar fat pad in comparison to healthy controls. In this study we demonstrated for the first time the expression of four novel adipokines in different joint tissues and how these molecules are differentially expressed in healthy and OA joint tissues.
    Full-text · Article · Apr 2015 · PLoS ONE
    • "When the subcutaneous fat tissue (SCFT) and fat pad tissue (FPT)-derived cells were compared, IPFP adipocytes showed a twofold increase in IL-6 gene expression and in IL-6 release. Interestingly, leptin secretion was 40 % lower and adiponectin was increased by 70 % in IPFP cells compared with the subcutaneous adipose tissue [73] . Moreover, it was demonstrated that the culture media from OA patients' IPFP adipocytes induced MMP13 and MMP1 expression in articular chondrocytes and that the leptin level positively correlated with the expression of both MMPs and cartilage collagen de- struction [20]. "
    [Show abstract] [Hide abstract] ABSTRACT: Osteoarthritis (OA) is one of the most common causes of musculoskeletal disability in the world. Traditionally, it has been thought that obesity contributes to the development and progression of OA by increased mechanical load of the joint structures. Nevertheless, studies have shown that adipose tissue-derived cytokines (adipocytokines) are a possible link between obesity and OA. Furthermore, according to recent findings, not only articular cartilage may be the main target of these cytokines but also the synovial membrane, subchondral bone and infrapatellar fat pad may be encompassed in the process of degradation. This review presents the most recent reports on the contribution of adipocytokines to the knee joint cartilage degradation, osteophyte formation, infrapatellar fat pad alterations and synovitis.
    Full-text · Article · Feb 2015 · International Orthopaedics
    • "Indeed, genes contributing to an inflammatory joint state were in general not highly differentially expressed between IFP and SAT among those with endstage OA, In other words, SAT gene expression was more similar to that of IFP among those with later stage disease. In the only other previous work comparing IFP to SAT fat samples, Distal et al. [21] studied differences in gene expression between IFP and SAT among individuals with endstage knee OA. Consistent with our findings, the authors reported that the majority of cytokines demonstrated similar expressions between the two adipose tissues, with only small fold increases in IL-6 and IL-6 receptor (2 to 3 fold) in IFP tissue and decreased expression of genes related to lipid metabolism in SAT. "
    Preview · Article · Jan 2015
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