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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCES

Objective

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.

Methods

IFP and subcutaneous AT samples were obtained from 11 obese women (body mass index ≥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.

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 adiponectin secretion was increased by 70%, in IFP samples 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 pathologic outcome in obese patients, the relationship between obesity and OA remains complex, since mechanical 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 α (TNFα), and IL-1β 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 groups with differences in inflammation levels and metabolic 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

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCES

Samples and laboratory methods.

We obtained IFP tissue and thigh subcutaneous AT from 11 obese women (body mass index [BMI] ≥30 kg/m2) 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 cytokines, AT explants were incubated for 3 hours in Krebs medium with 1% bovine serum albumin. Real-time quantitative 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 manufacturer'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 measured using Plateforme Technologique Phénotypage du Petit Animal et Microdosage (Hôpital Saint Antoine, Paris, France). The study was approved by the Institutional Ethics Committee of Hôpital 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

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCES

Characteristics of the patients included in this study are shown in Table 1. The 11 women were all obese (BMI ≥30 kg/m2), and all had low-grade inflammation (C-reactive protein <10 mg/liter). However, all patients had a high level of IL-6 in plasma.

Table 1. Characteristics of the 11 OA patients*
  • *

    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.

Age, years74.6 ± 1.93
BMI, kg/m231.6 ± 1.44
Glucose, mmoles/liter5.4 ± 0.60
Leptin, ng/ml4.39 ± 0.83
Adiponectin, μg/ml11.66 ± 1.49
Resistin, ng/ml2.93 ± 0.55
CRP, mg/liter6.2 ± 0.68
IL-6, pg/ml7.22 ± 1.89
sIL-6R, ng/ml22.82 ± 1.78

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 γ [PPARγ]), lipolysis (adipocyte triglyceride lipase [ATGL], lipoprotein lipase [LPL], and hormone sensitive lipase [HSL]), fatty acid reesterification (cytosolic phosphoenolpyruvate carboxykinase [PEPCK-C]), or β-oxidation (carnityl palmitoyltransferase 1 [CPT-1]) (Figure 1). Expression of genes for ATGL, LPL, HSL, FAT/CD36, and PPARγ was strikingly decreased in IFP tissue compared with subcutaneous 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.

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Figure 1. Expression pattern of genes for key enzymes involved in lipid metabolism, in subcutaneous adipose tissue (SCAT) and infrapatellar 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 μg) was analyzed using real-time quantitative polymerase chain reaction. Values were normalized to RPL13 ribosomal RNA and expressed as a percentage of the gene value obtained in subcutaneous AT (control). Values are the mean and SEM. ∗∗∗ = P ≤ 0.001 versus control. FABP-4 = fatty acid binding protein 4; PEPCK-C = cytosolic phosphoenolpyruvate carboxykinase; FAT/CD36 = fatty acid transport CD36; ATGL = adipocyte triglyceride lipase; LPL = lipoprotein lipase; HSL = hormone sensitive lipase; PPARg = peroxisome proliferator–activated receptor γ; CPT-1 = carnityl palmitoyltransferase 1.

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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 (TNFα, IL-1β, 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 subcutaneous AT, with no significant differences in the expression of the other cytokines. In addition, the rate of IL-6 release was >2-fold higher in IFP tissue than in subcutaneous AT; for the other cytokines (TNFα, 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.

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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 μg) 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; ∗∗ = 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; ∗∗ = P ≤ 0.001. TNFα = tumor necrosis factor α; MCP-1 = macrophage chemotactic protein 1.

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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 significantly different in IFP tissue compared with subcutaneous 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

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCES

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., TNFα, 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 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 involved 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 infiltration, which can be different depending on the location of the AT. Macrophages secrete proinflammatory cytokines such as IL-6, resulting in a so-called “low-grade inflammatory state” (2). In the current study, subcutaneous 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 contrast 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 metabolic 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 damage, via the local production of IL-6/sIL-6R and adiponectin. It would be of interest to know whether the same metabolic profile is found in OA patients who are not obese.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCES

We thank Dr. Maïté Corvol, Dr. Claude Forest, and Professor Robert Barouki for their participation in helpful discussions, and Nadège Brunel (Plateforme Technologique Phénotypage du Petit Animal et Microdosages, Hôpital Saint-Antoine, Paris, France) for technical assistance with cytokine measurements.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCES

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.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. AUTHOR CONTRIBUTIONS
  8. REFERENCES