4-1BB, a member of the TNF receptor superfamily, has a role in various inflammatory pathologies through its interaction with 4-1BB ligand. We previously demonstrated that it participates in initiating and promoting obesity-induced adipose inflammation in a rodent model.
In this study, we examined whether 4-1BB is related to obesity-induced adipose inflammation and metabolic parameters in humans.
A total of 50 subjects, 25 obese (body mass index (BMI)⩾25 kg m−2) and 25 lean (BMI<23 kg m−2) participated in the study. The levels of 4-1BB transcripts and soluble 4-1BB protein (s4-1BB) in subcutaneous adipose tissue were measured by quantitative real-time PCR and enzyme-linked immunosorbent assay, respectively. Inflammatory and metabolic parameters were measured by enzymatic analysis and immunoassay.
Obese subjects had higher levels of both 4-1BB transcripts and s4-1BB protein in subcutaneous adipose tissue than lean controls, and the levels were correlated with BMI and the expression of inflammatory markers, as well as with serum metabolic parameters. Moreover, s4-1BB was released from human adipocytes, and elicited chemotactic responses from human monocytes/T cells as well as enhancing their inflammatory activity, indicating that it may promote human adipose inflammation.
Our data demonstrate that elevated levels of 4-1BB transcripts and s4-1BB in adipose tissue are closely associated with obesity-induced inflammation and metabolic dysregulation. They suggest that both 4-1BB transcripts and s4-1BB could serve as novel biomarkers and/or therapeutic targets for obesity-induced inflammation and metabolic syndrome in humans.
Obesity is a chronic low-grade inflammatory state,1 and obesity-induced inflammation is considered to be responsible for the development of metabolic complications such as insulin resistance, type 2 diabetes, dyslipidemia and atherosclerosis.2, 3, 4 Adipose tissue is a major site of obesity-induced inflammation, which is characterized by the accumulation of infiltrated immune cells (T cells, macrophages) and elevated levels of inflammatory cytokines.5, 6 Studies have shown that cell–cell interactions have a crucial role in triggering the production of adipose inflammatory cytokines/chemokines (for example, tumor necrosis factor alpha, TNF-α; interleukin-6, IL-6; monocyte chemoattractant protein-1, MCP-1),7, 8, 9 which cause metabolic dysfunction.10, 11 Several inflammatory receptor–ligand (for example, CD40/CD40L and HVEM/LIGHT) interactions deliver inflammatory signals to adipocytes–macrophages and thus have been implicated in adipose inflammation.8, 12, 13 Moreover, it is likely that soluble forms of the inflammatory receptors or ligands (for example, sTNFR2, sCD40L and sLIGHT) are generated by proteolytic cleavage or alternative splicing14 and are elevated in the plasma of obese humans and associated with obesity-induced inflammation, insulin resistance and hyperglycemia,15, 16 as well as cardiovascular disease.17 Hence, the inflammatory receptor–ligand molecules are considered to be attractive targets for modulating obesity-induced adipose inflammation.
4-1BB (CD137, TNFRSF9) belongs to the TNF receptor superfamily and provides a costimulatory signal by binding to its ligand 4-1BBL (CD137L). 4-1BB signals have been implicated in inflammatory responses mediated by cells of innate immunity such as T cells, natural killer and natural killer T cells.18, 19, 20 4-1BBL is expressed mainly on antigen presenting cells, such as macrophages and dendritic cells, and these cells can be activated by 4-1BBL engagement to secrete cytokines and chemokines.21, 22 The 4-1BB/4-1BBL interaction influences many inflammatory conditions,23, 24, 25 and increased levels of 4-1BB and its soluble form are associated with several inflammatory diseases.26, 27, 28, 29, 30 Interestingly, 4-1BB is likely to have a role in the development of inflammatory metabolic diseases such as atherosclerosis, insulin resistance and obesity.20 For example, it is expressed in human atherosclerotic plaques and in patients with acute coronary syndrome,27, 31, 32 and 4-1BB deficiency reduces atherosclerosis in hyperlipidemic mice.23 Moreover, we previously demonstrated that 4-1BB participated in initiating and promoting obesity-induced adipose inflammation in contact cocultured murine adipocytes/macrophages,9, 20 and ablation of 4-1BB reduced adipose inflammation, ameliorated insulin resistance and improved insulin signaling in mice fed a high-fat diet.20 However, it is not known whether 4-1BB is involved in obesity-induced adipose inflammation and its metabolic complications in humans.
In this study, we demonstrate that both 4-1BB transcripts and soluble 4-1BB (s4-1BB) levels in subcutaneous adipose tissue are higher in obese subjects than in lean controls, and that their levels are positively correlated with the levels of adipose inflammatory markers and metabolic parameters. These findings suggest that 4-1BB may be useful as a novel biomarker and/or therapeutic target for obesity-induced adipose inflammation and metabolic syndrome in obese individuals.
Subjects and methods
Patients undergoing cholecystectomy without evidence of infection at Ulsan University Hospital were enrolled in this study. Obesity was estimated from body mass index (BMI⩾25 kg m−2) based on the criteria of the World Health Organization (WHO) Expert Consultation for Asian populations.33 Fifty subjects participated in the study; twenty-five subjects (fifteen women and ten men) had BMI⩾25 kg m−2, and twenty-five (twenty women and five men) had BMI<23 kg m−2, which is not associated with any significant illness. Excluded from this study were individuals with severe inflammatory or infectious diseases, cancer, secondary causes of obesity, pregnant or lactating women, subjects with evidence of severe hepatic or renal disease and subjects on medication, including antihypertensive drugs, oral hypoglycemic agents, insulin and hypolipidemic agents. All individuals were subjected to a medical evaluation by a physician, including a full medical history and physical examination. The study was approved by the Ulsan University Hospital Institutional Review Board and was conducted according to the principles of the Helsinki Declaration.
Anthropometric and biochemical analyses
BMI (kg m−2) was calculated from the participant’s weight and height. Systolic and diastolic blood pressures (BPs) were measured with a mercury sphygmomanometer on the right arm after at least 5 min rest. Fasting blood samples were taken into BD SST II Plus blood collection tube (BD, Franklin Lakes, NJ, USA) and centrifuged with Eppendorf centrifuge 5810R (Eppendorf AG, Hamburg, Germany, 3500 r.p.m., 10 min). Serum samples were stored at −70 °C in multiple aliquots. Serum triglyceride (TG) was measured by enzyme immunoassay (glycerol phosphate oxidase without serum blank), total cholesterol (TC) was measured by enzyme immunoassay (NECP 9), and high-density lipoprotein (HDL) cholesterol concentration was measured by direct enzymatic assay, low-density lipoprotein (LDL) cholesterol was calculated from the Friedwald formula: LDL-C=TC−(HDL-C+TG/5).34 Serum glucose level was measured by the hexokinase (UV) method. Insulin was measured by electrochemiluminescence immunoassay. Glycated hemoglobin (A1C) was measured by high performance liquid chromatography (HPLC) and C-reactive protein (CRP) was measured by tubidimetric immunoassay. The estimate of insulin resistance by homeostatic model assessment for insulin resistance (HOMA-IR) score was calculated with the formula: fasting serum insulin (μU ml−1)xfasting blood glucose (mmol l−1)/22.5.35
Subcutaneous adipose tissue was obtained from patients undergoing surgery. It was promptly washed with DMEM (Dulbecco’s Modified Eagle Medium, Gibco BRL, Grand Island, NY, USA) and visible blood vessels were removed. Finely cut tissue pieces were digested with type II collagenase (1 mg ml−1, Sigma, St Louis, MO, USA), and, after removing erythrocytes and filtration, total cell numbers were determined microscopically; the cells were grown in DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) for 48 h at 37 °C in 5% CO2. Differentiation of primary preadipocytes was induced by incubation for 3 days in DMEM/F12 (1:1) supplemented with 10 μg ml−1 insulin (Sigma-Aldrich, St Louis, MO, USA), 0.25 μM dexamethasone (Sigma-Aldrich), 0.5 mM isobutylmethylxanthine (Sigma-Aldrich) and 10 μM troglitazone (Cayman, Ann Arbor, MI, USA). Mature adipocytes were maintained in this culture medium, which was replaced every 2 days. Jurkat T cells and THP-1 monocytes were obtained from the Korean Cell Line Bank (Seoul, South Korea) and maintained in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum. THP-1 monocytes were incubated at a density of 5 × 105 cells ml−1 with 10−7 M phorbol myristate acetate (Sigma) for 48 h to induce their differentiation into macrophages. Human primary adipocytes and Jurkat T cells were incubated with palmitic acid 500 μM and lipopolysaccharide 100 ng ml−1 for 24 h; or Jurkat T cells and THP-1-derived macrophages were incubated with s4-1BB (Recombinant human 4-1BB receptor; Peprotech, Rocky Hill, NJ, USA) in 24-well plates in the presence or absence of neutralizing monoclonal antibody against 4-1BBL (TKS-1; e-Bioscience, San Diego, CA, USA) for 12 h. Rat immunoglobulin G (Sigma-Aldrich) was used as control. Free fatty acids (FFAs, palmitic acid mixture, Sigma-Aldrich) were dissolved in ethanol containing bovine serum albumin (50 μM) and conjugated with bovine serum albumin at a 10:1 molar ratio before use.
Human adipose tissue explant culture
Human adipose tissue was immediately transported to the laboratory and handled under aseptic conditions. The tissue was washed in phosphate-buffered saline supplemented with 0.4% Fungizone (Gibco) and cut with scissors into small pieces (20–30 mg). These were incubated in phosphate-buffered saline plus 0.5% bovine serum albumin for 30 min to remove diffusible factors and blood cells, then centrifuged for 30 s at 400 g to remove erythrocytes and pieces of tissues containing insufficient adipocytes to float. The explants were separated from the medium plus sedimented cells and resuspended in fresh DMEM in 100 mm2 dishes. They (100 mg ml−1) were then incubated for 3 days in suspension culture at 37 °C in 5% CO2.
Measurement of s4-1BB and inflammatory cytokines
Human adipose tissue (0.1 g) was homogenized in 1 ml 100 mmol l−1 Tris-HCl and 250 mmol l−1 sucrose buffer, pH 7.4, supplemented with protein inhibitors. Lipids were removed by centrifugation at 10 000g for 10 min. s4-1BB in the homogenate was measured with an enzyme-linked immunosorbent assay using a human 4-1BB/TNFRSF9 set (R&D System, Minneaoplis, MN, USA).
Levels of MCP-1, TNF-α, IL-6 and adiponectin in homogenates were measured with human MCP-1, TNF-α, IL-6 and adiponectin kits (R&D System). Values for cytokine levels were derived from standard curves using the curve-fitting program SOFTmax (Molecular Devices, Sunnyvale, CA, USA).
Quantitative real-time PCR
Total RNA extracted from human subcutaneous adipose tissue was reverse transcribed into complementary DNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Real-time PCR of complementary DNA was performed in duplicate with a SYBR premix Ex Taq kit (Takara Bio Inc., Foster, CA, USA) using a Thermal Cycler Dice (Takara Bio Inc., Otsu, Japan). All reactions were performed by the same procedure: initial denaturation at 95 °C for 10 s, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. Results were analyzed with real-time system TP800 software and all values for genes of interest were normalized to values for a housekeeping gene (18S). Human primer sequences were used: 4-1BB, forward 5′-IndexTermACTGGTGCCATTTCAGGAACAA-3′, reverse 5′-IndexTermACACCATGTGTCCAAAGCCAAG-3′; 4-1BBL, forward 5′-IndexTermGGGGGCCTGAGCTACAAAGA-3′, reverse 5′-IndexTermGGCAGGTCCACGGTCAAAG-3′; MCP-1, forward 5′-IndexTermCTTCTGTGCCTGCTGCTCATA-3′, reverse 5′-IndexTermCTTTGGGACACTTGCTGCTG-3′; TNF-α, forward 5′-IndexTermGTGACAAGCCTGTAGCCCATGTT-3′, reverse 5′-IndexTermTTATCTCTCAGCTCCACGCCATT-3′; IL-6, forward 5′-IndexTermAAGCCAGAGCTGTGCAGATGAGTA-3′, reverse 5′-IndexTermTGTCCTGCAGCCACTGGTTC-3′; adiponectin, forward 5′-IndexTermCTGGCTATGCTCACAGTCTCACATC-3′, reverse 5′-IndexTermCTCTGTGCCTCTGGTTCCACAA-3′; F4/80, forward 5′-IndexTermCAGTGTTAATGCCGAAGTCTCAA-3′, reverse 5′-IndexTermGACACCTGCCACAGGTCCAA-3′; CD247, forward 5′-IndexTermAGAACCCTCAGGAAGGCCTGTA-3′, reverse 5′-IndexTermCGCCTTTCATCCCAATCTCAC-3′; 18S, forward 5′-IndexTermTTTGCGAGTACTCAACACCAACATC-3′, reverse 5′-IndexTermGAGCATATCTTCGGCCCACAC-3′.
Culture supernatants were incubated with anti-4-1BB antibody (1 μg; Ancell Corp., Bayport, MN, USA) for 1 h at 4 °C. Recombinant protein G agarose (Invitrogen, Carlsbad, CA, USA) was continuously added to the antibody-incubated supernatants. Immunoprecipitation was carried out overnight at 4 °C. Precipitates were collected by centrifugation at 14 000 g for 30 s and washed three times with lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 0.1 mM EDTA, 50 mM NaF, 10 mM Na4P2O7, 1 mM MgCl2, 0.5% deoxycholate, 1% IGEPAL). Immunoprecipitates were dissolved in 2 × SDS sample buffer and analyzed by western blot analysis with anti-4-1BB antibody (Ancell) and secondary antibody (mouse IgG-heavy and light chain antibody; Bethyl Laboratories, Montgomery, TX).
Cell migration was assessed in a multi-well microchemotaxis chamber (Neuroprobe, Gaithersburg, MD, USA). Briefly, THP-1 monocytes or Jurkat T cells were suspended in RPMI 1640 medium at 2 × 106 cells ml−1 in the presence or absence of TKS-1, and a 60 μl aliquot was placed in the upper well of a 96-well chamber separated from a lower chamber containing s4-1BB (Peprotech) by a 5-μm polycarbonate filter. After incubation for 4 h at 37 °C, nonmigrated cells were removed by scraping, and the cells that had migrated across the filter were fixed and stained with Diff-Quick (International Reagent Corp., Kobe, Japan). Stained cells were counted and counts averaged in five randomly chosen fields ( × 200) examined with an Axio-Star Plus microscope (Carl Zeiss, Gottingen, Germany); images were captured with Axiovision AC software (Carl Zeiss). Cell migration induced by the medium served as control and was designated as 100% migration in each experiment. Results are expressed as mean±s.e.m. of quadruplicate samples.
For statistical analysis we used the SPSS/PC+statistical software package (SPSS 18.0, IBM, Seoul, South Korea). Differences in anthropometric and biochemical values were analyzed by the Mann–Whitney U-test. For correlation analysis, Pearson’s correlation test was used. Anthropometric and laboratory data are expressed as median and range. A P-value below 0.05 (two-tailed) was considered to be statistically significant.
General characteristics of obese and control subjects
The general characteristics and laboratory parameters of the obese and lean subjects (controls) are shown in Table 1. Compared with the lean subjects, the obese subjects had higher BMI (P<0.0001), higher serum levels of atherogenic factors (for example, increased systolic (P<0.0001) and diastolic (P=0.004) BP, triglyceride (P<0.0001), TC (P=0.05)) and decreased HDL-cholesterol (HDL-C) (P=0.01); they also had higher levels of insulin resistance markers (such as increased insulin level (P=0.03), A1C% (P=0.03) and HOMA-IR index (P=0.03)), and of inflammatory markers (for example, increased CRP (P=0.003)) (Table 1). There were no significant differences between obese and lean subjects in fasting glucose or LDL-cholesterol (Table 1).
Increased levels of 4-1BB transcripts in human obese adipose tissue
We have shown previously that 4-1BB transcripts are upregulated in the adipose tissue of obese mice fed a high-fat diet, and this is accompanied by increased inflammatory cytokines.9, 20 To examine the association of 4-1BB with human adipose inflammation, we first compared 4-1BB transcripts in the adipose tissue of obese subjects and lean subjects. As shown in Figure 1a, there was a higher level of 4-1BB transcripts in the adipose tissue of obese subjects (two-fold increase, P=0.012) (Figure 1a), and the level was positively correlated with BMI (R=0.417, P=0.003) (Figure 1b). Moreover, inflammatory cytokines were elevated in the obese adipose tissue, and levels of 4-1BB transcripts were positively correlated with levels of inflammatory cytokines (MCP-1 (R=0.342, P=0.015), TNF-α (R=0.611, P<0.0001), IL-6 (R=0.527, P<0.0001)) and negatively correlated with adiponectin levels (R=−0.416, P=0.002) (Figure 1c).
Elevated levels of s4-1BB in human obese adipose tissue
Levels of the s4-1BB are associated with certain inflammatory diseases.26, 27, 28, 29, 30 The upregulation of 4-1BB transcript in human obese adipose tissue led us to inquire whether s4-1BB is elevated in the inflamed adipose tissue of the obese subjects. We found that this was the case (6.9±4.3 vs 15.7±20.6 pg mg−1 protein, P=0.012) (Figure 2a), and s4-1BB levels were also positively correlated with BMI (R=0.356, P=0.011) (Figure 2b) and with 4-1BB mRNA levels (R=0.422, P=0.002) (Figure 2c). Levels of s4-1BB were also positively associated with levels of the inflammatory cytokines MCP-1 (R=0.298, P=0.035) (Figure 2d) and IL-6 (R=0.300, P=0.035) (Figure 2e), as well as with serum levels of CRP (R=0.823, P<0.0001) (Figure 2f). Although we found that the levels of 4-1BB transcripts were negatively correlated with adiponectin transcript levels (Figure 1c), there was no correlation between s4-1BB levels and levels of adiponectin protein in human adipose tissue (data not shown). Moreover, to assess the ability of s4-1BB to predict the obesity-related inflammation and metabolic syndrome, we divided obese subjects into two groups, those with and those without metabolic syndrome, which is defined as the presence of three or more of the five features such as abdominal obesity, hyperglycemia, hypertension, hypertriglyceridemia and low HDL-C,36, 37 and observed that the s4-1BB levels in adipose tissue were significantly elevated in the former than in the latter (3.95±0.32 vs 8.33±1.65 vs 26.43±7.58 pg mg−1 protein) (Figure 2a). These indicate that the adipose tissue s4-1BB may be useful to predict the obesity-related inflammation and metabolic syndrome.
Release of s4-1BB by human adipocytes and T cells
To see whether adipose tissue secretes s4-1BB, we cultured human adipose tissue explants and measured s4-1BB in the cultured medium by ELSIA. Adipose tissue explants from obese subjects secreted considerably higher amounts of s4-1BB than those from lean subjects (4.06±0.37 vs 16.18±0.23 pg mg−1 protein, P=0.001) (Figure 3a). Next, to see whether adipocytes were responsible for the s4-1BB release, we prepared human stromal vascular fraction-derived adipocytes and measured the level of s4-1BB in the culture medium by immunoprecipitation-western blotting. Levels of 4-1BB transcripts in adipocytes, and s4-1BB release from the adipocytes, increased during adipogenesis (Figure 3b) and rose further in response to treatment with obesity-related factors such as FFAs and lipopolysaccharide (Figure 3c). Because activated T cells, which are known to release s4-1BB,38 increase in obese adipose tissue,5, 6 we tested whether obesity factors could enhance the release of s4-1BB from human Jurkat T cells. We found that this was indeed the case (Figure 3d). These data suggest that adipocytes and activated T cells are the source of s4-1BB in inflamed human adipose tissue.
S4-1BB-induced cell migration and activation
It is well known that obesity-induced adipose inflammation results from increased infiltration of immune cells (macrophages, T cells) into the adipose tissue. In agreement with this, we confirmed that the expression of markers for macrophages (F4/80) and T cells (CD247) was significantly increased in the adipose tissue of the obese subjects (Figure 4a). Given that soluble receptors exert their inflammatory effects through their ligands,39, 40, 41 we wished to test whether adipose tissue-derived s4-1BB directly participates in adipose inflammation; we therefore measured the effect of s4-1BB on the infiltration of immune cells into adipose tissue. We confirmed that human monocytic THP-1 cells and Jurkat T cells strongly expressed 4-1BBL (data not shown), and found that the s4-1BB elicited chemotactic responses from these cells (Figure 4b). These effects were completely blocked by the treatment with neutralizing anti-4-1BBL antibody (TKS-1 Ab). We subsequently demonstrated elevated levels of TNF-α transcript in s4-1BB-activated THP-1 macrophages as well as Jurkat T cells. The inflammatory effect of s4-1BB was completely inhibited in the presence of TKS-1, which blocks the 4-1BBL receptor on these cells (Figures 4c and d). These findings strongly suggest that s4-1BB contributes to adipose inflammatory responses (for example, chemotaxis and proinflammatory cytokine release) by interacting with 4-1BBL on human adipose macrophages and/or T cells.
Correlation between s4-1BB levels and metabolic complications
Next we investigated the association between levels of s4-1BB in adipose tissue and obesity-related metabolic complications such as atherosclerosis and insulin resistance. Our data showed that levels of s4-1BB were positively correlated with atherosclerotic parameters such as systolic BP (R=0.319, P=0.023), diastolic BP (R=0.290, P=0.041) and TG levels (R=0.379, P=0.006), and negatively correlated with HDL-C levels (R=−0.300, P=0.034) (Figure 5a). Moreover, they were also strongly correlated with insulin resistance-related parameters such as increase in fasting blood glucose (R=0.311, P=0.028), insulin levels (R=0.357, P=0.011), HOMA-IR index (R=0.390, P=0.005) and A1C% (R=0.301, P=0.034) (Figure 5b). These data indicate that adipose tissue s4-1BB is a novel inflammatory biomarker reflecting obesity-related metabolic risk factors in humans.
Obesity-related inflamed adipose tissue is characterized by increased expression of many inflammatory cytokines and inflammatory receptors. An important role of 4-1BB, a member of TNF superfamily and inflammatory receptor, has been reported in various inflammatory and metabolic diseases.9, 20, 23, 24, 25, 42 We have now demonstrated that it is also associated with human obesity. All the changes we observed are consistent with those seen in obese mice.9, 20 Moreover, the upregulation of 4-1BB transcripts in adipose tissue was positively correlated with BMI index, and inflammatory cytokine levels (MCP-1, TNF-α and IL-6) and serum CRP, and negatively correlated with the level of an anti-inflammatory adipokine (adiponectin). This association of 4-1BB with inflammatory markers in human obese adipose tissue is similar to that of other inflammatory receptors such as CD40 and HVEM.8, 13 As cell–cell crosstalk through inflammatory receptor–ligand pairs, including 4-1BB/4-1BBL, initiates adipose inflammation,9 the upregulation of 4-1BB transcripts in human adipose tissue may contribute to initiating and/or promoting human adipose inflammation. It is also tempting to speculate that 4-1BB-mediated signals may combine with other inflammatory receptors to amplify adipose inflammation in human obesity.
Inflammatory receptors and ligands are released in soluble form by cleavage from the cell surface, and this is considered an important mechanism by which cells can either enhance or inhibit the signals delivered by their respective membrane-bound counterparts.43, 44, 45 S4-1BB can be released by activated lymphocytes and generated by proteolytic cleavage,46 and levels of s4-1BB increase in various inflammatory diseases.26, 27, 28, 29, 30 As obesity-induced inflamed adipose tissue contains increased numbers of activated T cells,20, 47 which can release s4-1BB, we hypothesized that s4-1BB protein is increased in human inflamed adipose tissue. Along with the upregulation of 4-1BB transcripts, we found for the first time that s4-1BB levels in the adipose tissue of obese subjects were significantly elevated compared with those from lean subjects. More importantly, we showed that s4-1BB is released by human stromal vascular fraction-derived adipocytes and adipose tissue explants, and by T cells treated with obesity-related factors such as FFA or lipopolysaccharide. This indicates that the adipocytes and T cells in inflamed adipose tissue are sources of s4-1BB, and suggests that the s4-1BB functions as an autocrine/paracrine factor. S4-1BB levels in the adipose tissue were positively correlated not only with BMI, but also with levels of inflammatory markers such as MCP-1/IL-6 in adipose tissue and serum CRP. These findings indicate that adipose s4-1BB directly participates in obesity-induced adipose inflammation in humans and may be used as a biomarker to estimate obesity-induced adipose inflammation.
It has been suggested that the shedding of some inflammatory receptors is a way to modulate inflammatory or costimulatory responses induced by local cell–cell interactions.48 For example, soluble tumor necrosis factor receptors (sTNFRs) function as inhibitors of their ligand by antagonizing the activities of the membrane-bound receptors,49 whereas sIL-6R acts as an agonist to enhance inflammatory responses.50 It has been shown that s4-1BB antagonizes the stimulatory activities of membrane-bound 4-1BB in T cells, reducing cell activation,46, 51 although the potential of s4-1BB to interact with 4-1BBL remains unclear. Interestingly, we found that s4-1BB directly induced the migration of monocytes and T cells, which express 4-1BBL, and that the chemotactic activity was blocked by neutralizing anti-4-1BBL antibody, indicating that the binding of s4-1BB to its ligand is responsible for its chemotactic activity. Similarly, activation of T cells with s4-1BB increased the release of inflammatory cytokines, and this again was blocked by the presence of TKS-1. These findings indicate that s4-1BB is capable of delivering a reverse inflammatory signal to 4-1BBL-expressing cells such as macrophages and activated T cells, thus acting like an endocrine factor to promote obesity-induced adipose inflammation.
Finally, it should be noted that the elevated 4-1BB transcripts and s4-1BB levels are closely associated with several risk factors for the metabolic complications of human obesity such as atherosclerosis and insulin resistance.20, 23, 52 For example, 4-1BB is detected in human atherosclerotic plaques and promotes plaque inflammation.52 In addition, 4-1BB deficiency reduces atherosclerosis in hyperlipidemic mice,23 as well as ameliorating systemic insulin resistance in obese mice;20 thus, metabolic parameters such as levels of fasting plasma glucose, insulin, triglyceride and cholesterol were significantly lower in high-fat diet-fed 4-1BB-deficient mice than those of high-fat diet-fed wild-type mice.20 In this study, despite the unbalanced ratio of males to females in the lean versus obese subjects, we observed that levels of s4-1BB in obese adipose tissue were positively correlated with atherosclerotic parameters such as diastolic BP, systolic BP and triglycerides, and negatively correlated with HDL-cholesterol. In addition, the elevated s4-1BB levels were also positively correlated with serum insulin resistance parameters such as fasting blood glucose, insulin levels and HOMA-IR index. Although some metabolic parameters in obese subjects sustain in the metabolic cutoff point, which defines risk of metabolic abnormalities among Asians,53, 54 the positive correlation between s4-1BB levels in adipose tissue and the metabolic parameters in the circulation suggests that elevation of s4-1BB may reflect not only obesity-induced inflammation but also metabolic dysregulation. More importantly, when we further classified the obese subjects according to the criteria for metabolic syndrome,36, 37 we observed that s4-1BB levels in adipose tissue were significantly higher in the obese subjects with metabolic syndrome than those without it, indicating that s4-1BB could serve as a novel biomarker and/or therapeutic target for both obesity-induced inflammation and metabolic syndrome in humans. In addition, we have also observed some positive data for serum s4-1BB levels: levels of s4-1BB in serum were elevated in the obese subjects (data not shown). As blood s4-1BB level would be more attractive as a biomarker than adipose tissue level, further study is needed to elucidate the relationship between blood s4-1BB levels and inflammatory and/or metabolic parameters.
In conclusion, we for the first time demonstrate that levels of 4-1BB transcripts and s4-1BB are elevated in human obese adipose tissue, and that they are closely correlated with higher adiposity, adipose inflammatory markers and metabolic parameters (Supplementary Figure S1). Moreover, s4-1BB displayed chemotactic activity for human monocytes and T cells, and enhanced inflammatory cytokine release from these cells, indicating that it is able to promote adipose inflammation in human obesity. We conclude that 4-1BB may be of diagnostic value and/or be a therapeutic target for obesity-induced inflammation and metabolic syndrome in human.
Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM . Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance. J Clin Invest 1995; 95: 2409.
Rasouli N, Kern PA . Adipocytokines and the metabolic complications of obesity. J Clin Endocrinol Metab 2008; 93: s64–s73.
Grundy SM . Obesity, metabolic syndrome, and cardiovascular disease. J Clin Endocrinol Metab 2004; 89: 2595–2600.
Wisse BE . The inflammatory syndrome: the role of adipose tissue cytokines in metabolic disorders linked to obesity. J Am Soc Nephrol 2004; 15: 2792–2800.
Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr . Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003; 112: 1796–1808.
Shoelson SE, Lee J, Goldfine AB . Inflammation and insulin resistance. J Clin Invest 2006; 116: 1793–1801.
Suganami T, Nishida J, Ogawa YA . Paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor α. Arterioscler Thromb Vac Biol 2005; 25: 2062–2068.
Poggi M, Jager J, Paulmyer-Lacroix O, Peiretti F, Gremeaux T, Verdier M et al. The inflammatory receptor CD40 is expressed on human adipocytes: contribution to crosstalk between lymphocytes and adipocytes. Diabetologia 2009; 52: 1152–1163.
Tu TH, Kim C-S, Goto T, Kawada T, Kim B-S, Yu R . 4-1BB/4-1BBL interaction promotes obesity-induced adipose inflammation by triggering bidirectional inflammatory signaling in adipocytes/macrophages. Mediators Inflamm 2012; 2012: 972629.
Brake DK, Smith EB, Mersmann H, Smith CW, Robker RL . ICAM-1 expression in adipose tissue: effects of diet-induced obesity in mice. Am J Physiol Cell Physiol 2006; 291: C1232–C1239.
Lumeng CN, Maillard I, Saltiel AR . T-ing up inflammation in fat. Nat Med 2009; 15: 846–847.
Kim HM, Jeong CS, Choi HS, Kawada T, Yu R . LIGHT/TNFSF14 enhances adipose tissue inflammatory responses through its interaction with HVEM. FEBS Lett 2011; 33: 144–152.
Bassols J, Moreno JM, Ortega F, Ricart W, Fernandez-Real JM . Characterization of herpes virus entry mediator as a factor linked to obesity. Obesity 2010; 18: q239–246.
Metkar SS, Naresh K, Manna PP, Srinivas V, Advani S, Nadkarni J . Circulating levels of TNFα and TNF receptor superfamily members in lymphoid neoplasia. Am J Hematol 2000; 65: 105–110.
Seijkens T, Kusters P, Engel D, Lutgens E . CD40–CD40L: linking pancreatic, adipose tissue and vascular inflammation in type 2 diabetes and its complications. Diab Vasc Dis Res 2013; 10: 115–122.
Desideri G, Ferri C . Effects of obesity and weight loss on soluble CD40L levels. JAMA 2003; 289: 1781–1782.
Missiou A, Wolf D, Platzer I, Ernst S, Walter C, Rudolf P et al. CD40L induces inflammation and adipogenesis in adipose cells-a potential link between metabolic and cardiovascular disease. Thromb Haemost 2010; 103: 788.
Vinay DS, Choi BK, Bae JS, Kim WY, Gebhardt BM, Kwon BS . CD137-deficient mice have reduced NK/NKT cell numbers and function, are resistant to lipopolysaccharide-induced shock syndromes, and have lower IL-4 responses. J Immunol 2004; 173: 4218–4229.
Kim D-H, Chang W-S, Lee Y-S, Lee K-A, Kim Y-K, Kwon BS et al. 4-1BB engagement costimulates NKT cell activation and exacerbates NKT cell ligand-induced airway hyperresponsiveness and inflammation. J Immunol 2008; 180: 2062–2068.
Kim C-S, Kim JG, Lee B-J, Choi M-S, Choi H-S, Kawada T et al. Deficiency for costimulatory receptor 4-1BB protects against obesity-induced inflammation and metabolic disorders. Diabetes 2011; 60: 3159–3168.
Shao Z, Schwarz H . CD137 ligand, a member of the tumor necrosis factor family, regulates immune responses via reverse signal transduction. J Leukoc Biol 2011; 89: 21–29.
Lippert U, Zachmann K, Ferrari DM, Schwarz H, Brunner E, Latif A et al. CD137 ligand reverse signaling has multiple functions in human dendritic cells during an adaptive immune response. Eur J Immunol 2008; 38: 1024–1032.
Jeon HJ, Choi J-H, Jung I-H, Park J-G, Lee M-R, Lee M-N et al. CD137 (4–1BB) deficiency reduces atherosclerosis in hyperlipidemic mice. Circulation 2010; 121: 1124–1133.
Seo SK, Park HY, Choi JH, Kim WY, Kim YH, Jung HW et al. Blocking 4-1BB/4-1BB ligand interactions prevents herpetic stromal keratitis. J Immunol 2003; 171: 576–583.
Kwon B . CD137-CD137 ligand interactions in inflammation. Immune Netw 2009; 9: 84–89.
Shao Z, Schäffler A, Hamer O, Dickopf J, Goetz A, Landfried K et al. Admission levels of soluble CD137 are increased in patients with acute pancreatitis and are associated with subsequent complications. Exp Mol Pathol 2012; 92: 1–6.
Dongming L, Zuxun L, Liangjie X, Biao W, Ping Y . Enhanced levels of soluble and membrane-bound CD137 levels in patients with acute coronary syndromes. Clin Chim Acta 2010; 411: 406–410.
Jung HW, Choi SW, Choi JI, Kwon BS . Serum concentrations of soluble 4-1BB and 4-1BB ligand correlated with the disease severity in rheumatoid arthritis. Exp Mol Med 2004; 36: 13–22.
Michel J, Langstein J, Hofstädter F, Schwarz H . A soluble form of CD137 (ILA/4‐1BB), a member of the TNF receptor family, is released by activated lymphocytes and is detectable in sera of patients with rheumatoid arthritis. Eur J Immunol 1998; 28: 290–295.
Sharief M . Heightened intrathecal release of soluble CD137 in patients with multiple sclerosis. Eur J Neurol 2002; 9: 49–54.
Yan J, Gong J, Liu P, Wang C, Chen G . Positive correlation between CD137 expression and complex stenosis morphology in patients with acute coronary syndromes. Clin Chim Acta 2011; 412: 993–998.
Yan J, Wang C, Chen R, Yang H . Clinical implications of elevated serum soluble CD137 levels in patients with acute coronary syndrome. Clinics 2013; 68: 193.
Organization WH. The Asia-Pacific Perspective: Redefining Obesity and its Treatment. World Health Organization: Geneva, Switzerland, 2000.
Friedewald WT, Levy RI, Fredrickson DS . Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499–502.
Matthews D, Hosker J, Rudenski A, Naylor B, Treacher D, Turner R . Homeostasis model assessment: insulin resistance and β-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28: 412–419.
Alberti KGMM Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA et al. Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation 2009; 120: 1640–1645.
Alberti K, Zimmet P, Shaw J . Metabolic syndrome—a new world-wide definition. A Consensus Statement from the International Diabetes Federation. Diabet Med 2006; 23: 469–480.
Shao Z, Schäffler A, Hamer O, Dickopf J, Goetz A, Landfried K et al. Admission levels of soluble CD137 are increased in patients with acute pancreatitis and are associated with subsequent complications. Exp Mol Pathol 2011; 92: 1–6.
Olsson I, Gatanaga T, Gullberg U, Lantz M, Granger G . Tumour necrosis factor (TNF) binding proteins (soluble TNF receptor forms) with possible roles in inflammation and malignancy. Eur Cytokine Netw 1993; 4: 169.
Jones SA, Horiuchi S, Topley N, Yamamoto N, Fuller GM . The soluble interleukin 6 receptor: mechanisms of production and implications in disease. FASEB J 2001; 15: 43–58.
Heaney ML, Golde DW . Soluble receptors in human disease. J Leukoc Biol 1998; 64: 135–146.
Kim W, Kim J, Jung D, Kim H, Choi H-J, Cho HR et al. Induction of lethal graft-versus-host disease by anti-CD137 monoclonal antibody in mice prone to chronic graft-versus-host disease. Biol Blood Marrow Transplant 2009; 15: 306–314.
Cartier A, Côté M, Bergeron J, Alméras N, Tremblay A, Lemieux I et al. Plasma soluble tumour necrosis factor-α receptor 2 is elevated in obesity: specific contribution of visceral adiposity. Clin Endocrinol (Oxf) 2010; 72: 349–357.
Choi JW, Kim SK . Relationships of soluble APO-1 (Fas/CD95) concentrations, obesity, and serum lipid parameters in healthy adults. Ann Clin Lab Sci 2005; 35: 290–296.
Anderson DR, Poterucha JT, Mikuls TR, Duryee MJ, Garvin RP, Klassen LW et al. IL-6 and its receptors in coronary artery disease and acute myocardial infarction. Cytokine 2013; 62: 395–400.
Michel J, Schwarz H . Expression of soluble CD137 correlates with activation-induced cell death of lymphocytes. Cytokine 2000; 12: 742–746.
Nishimura S, Manabe I, Nagasaki M, Eto K, Yamashita H, Ohsugi M et al. CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity. Nat Med 2009; 15: 914–920.
Salih HR, Schmetzer HM, Burke C, Starling GC, Dunn R, Pelka-Fleischer R et al. Soluble CD137 (4-1BB) ligand is released following leukocyte activation and is found in sera of patients with hematological malignancies. J Immunol 2001; 167: 4059–4066.
Van Zee KJ, Kohno T, Fischer E, Rock CS, Moldawer LL, Lowry SF . Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor alpha in vitro and in vivo. Proc Natl Acad Sci USA 1992; 89: 4845–4849.
Hirano T . Interleukin 6 and its receptor: ten years later. Int Rev Immunol 1998; 16: 249–284.
Furtner M, Straub R, Krüger S, Schwarz H . Levels of soluble CD137 are enhanced in sera of leukemia and lymphoma patients and are strongly associated with chronic lymphocytic leukemia. Leukemia 2005; 19: 883–885.
Olofsson PS, Söderström LÅ, Wågsäter D, Sheikine Y, Ocaya P, Lang F et al. CD137 is expressed in human atherosclerosis and promotes development of plaque inflammation in hypercholesterolemic mice. Circulation 2008; 117: 1292–1301.
He M, Li ETS, Harris S, Huff MW, Yau CY, Anderson GH . Canadian global village reality: anthropometric surrogate cutoffs and metabolic abnormalities among Canadians of East Asian, South Asian, and European descent. Can Fam Physician 2010; 56: e174–e182.
Rakugi H, Ogihara T . The metabolic syndrome in the Asian population. Curr Sci Inc 2005; 7: 103–109.
This work has supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No.2012R1A2A4A01002702), and an NRF grant funded by the Korean government (MSIP) (No.2008-0062618).
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on International Journal of Obesity website
About this article
Cite this article
Tu, T., Kim, C., Kang, J. et al. Levels of 4-1BB transcripts and soluble 4-1BB protein are elevated in the adipose tissue of human obese subjects and are associated with inflammatory and metabolic parameters. Int J Obes 38, 1075–1082 (2014) doi:10.1038/ijo.2013.222
- adipose tissue
- metabolic syndrome
Dendritic cell maturation in the corneal epithelium with onset of type 2 diabetes is associated with tumor necrosis factor receptor superfamily member 9
Scientific Reports (2018)
Journal of Diabetes Research (2017)
Enhancing the safety of antibody-based immunomodulatory cancer therapy without compromising therapeutic benefit: Can we have our cake and eat it too?
Expert Opinion on Biological Therapy (2016)
4-1BBL signaling promotes cell proliferation through reprogramming of glucose metabolism in monocytes/macrophages
FEBS Journal (2015)