1 Endocrine Unit, Division of Endocrinology, Diabetes and Nutrition, Department of Internal Medicine, University Hospital, Geneva, Switzerland
2 Division of Endocrinology, Diabetology, and Metabolism, Department of Medicine, University Hospital, Lausanne, Switzerland
3 Hans Wilsdorf Laboratory, Division of Immunology and Allergy, Department of Internal Medicine, University Hospital, Geneva, Switzerland
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ABSTRACT |
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The interleukin-1 receptor antagonist (IL-1Ra) is a member of the interleukin (IL)-1 family that binds to IL-1 receptors without inducing a cellular response, thereby antagonizing the effects of IL-1 and -1ß (1). Three forms of IL-1Ra have been described, two of which are expressed as intracellular proteins (icIL-1RaI and icIL-1RaII) and one of which is secreted (sIL-1Ra) (2). An understanding of the function(s) of the intracellular forms of IL-1Ra is still elusive (3), whereas it is clear that sIL-1Ra inhibits IL-1
and -1ß actions on target cells. Because of its beneficial effects in many models of disease, sIL-1Ra has been used as a therapeutic agent in human patients and was recently approved by the U.S. Food and Drug Administration for treating rheumatoid arthritis (4,5). Furthermore, IL-1Ra knockout mice have an enhanced susceptibility to septic shock and demonstrate an increased predisposition to the spontaneous development of inflammatory disorders, an observation that illustrates the functional relevance of this anti-inflammatory factor (68).
The production of sIL-1Ra is induced by many of the stimuli that usually induce IL-1 in the same cell (rev. in 2). However, we and others have recently demonstrated that the anti-inflammatory cytokine interferon-ß (IFN-ß) and leptin can induce sIL-1Ra production without the simultaneous increase in IL-1 in human monocytes and monocytic cell lines (9,10). This finding can be explained by the observation that factors involved in the signaling pathways of these stimuli, such as serine/threonine phosphatases, differentially regulate IL-1ß and IL-1Ra production in monocytes-macrophages (11). The premise that leptin is able to induce sIL-1Ra has been further extended by the finding that serum sIL-1Ra levels are sevenfold higher in subjects with hyperleptinemic obesity, as compared to nonobese subjects (12). Furthermore, sIL-1Ra levels dropped after weight loss was induced by intestinal bypass surgery. Interestingly, in these patients, the serum sIL-1Ra concentrations correlated better with BMI and the degree of insulin resistance than with serum leptin levels, suggesting that metabolic regulators other than leptin may control the production of sIL-1Ra. Based on these observations, the present study was undertaken to investigate 1) whether tissues other than liver and spleen are able to express and produce sIL-1Ra, 2) whether IL-1Ra production is increased in these tissues in obesity and after an inflammatory stimulus, and 3) what are the putative metabolic factors regulating the production of IL-1Ra. Our results demonstrate that white adipose tissue (WAT) is a major source of sIL-1Ra and that its expression in this tissue is markedly increased in obesity and inflammation.
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RESEARCH DESIGN AND METHODS |
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Human adipose tissue.
After authorization by the ethical commission of the Center Hospitalier Universitaire Vaudois and informed consent from the patients were received, subcutaneous and visceral adipose tissue samples were obtained from 28 morbidly obese women with a median BMI of 45 kg/m2 (range 3765 kg/m2) and with no comorbidity who were undergoing gastric banding or gastric bypass surgery. Subcutaneous adipose tissue samples were also obtained from 10 patients (9 women, 1 man) with a BMI <30 kg/m2 undergoing abdominal plastic surgery. Tissues were immediately frozen in liquid nitrogen.
RNA preparation and mRNA quantification.
Total RNA was prepared from 500 mg of human WAT, 250 mg of mouse WAT, or 50 mg of rodent liver, heart, spleen, muscle, brown adipose tissue (BAT), or hypothalamus (HT) by using the TRIzol reagent (Invitrogen, Basel, Switzerland), according to manufacturers instructions. Total RNA (5 µg) was reverse transcribed using 800 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen), in the presence of 0.3 units/µl RNAsin (Promega, Madison, WI), 7.5 µmol/l of random primers (oligo(dN)6), 1.2 mmol/l dNTP, and 12 µmol/l of dithiothreitol.
The expression of RNAs for human sIL-1Ra, icIL-1Ra, and 28S, as well as for mouse sIL-1Ra, IL-1ß, tumor necrosis factor- (TNF-
), and 36B4 RNA, was determined by quantitative real-time PCR using a LightCycler (Roche Diagnostics, Rotkreuz, Switzerland) with the DNA Master SYBR Green I or Fast Start DNA Master SYBR Green I (Roche Molecular Biochemicals, Rotkreuz, Switzerland) kits as appropriate. Each of the transcript levels was quantitated in at least two independent LightCycler runs in each individual sample. The PCR products of sIL-1Ra and icIL-1Ra were verified by automated sequencing (ABI PRISM 3100; Perkin-Elmer/Cetus, Norwalk, CT) and cloned into the T/A cloning vector pCR3.1 according to the manufacturers instructions (Invitrogen, Groningen, the Netherlands).
Preparation of adipose tissue homogenates and cytokine determination.
Human WAT (50 µg) was treated in a glass/glass homogenizer (Dounce) in 500 µl of lysis buffer containing 50 mmol/l Tris base, 150 mmol/l NaCl, 2 mmol/l EDTA, 2 mmol/l EGTA, 40 mmol/l ß-glycerophosphate, 50 mmol/l NaF, 10 mmol/l sodium pyrophosphate, 10% glycerol, 1% Triton X-100 (pH 7.4), and a protease inhibitor cocktail (Complete Mini EDTA-Free; Roche Diagnostics). After being centrifuged at 700g for 30 min, the supernatants were used for the determination of IL-1Ra, IL-6, and leptin. IL-1Ra and IL-6 concentrations in the serum, WAT homogenates, and supernatants of WAT explants were determined with the Quantikine enzyme-linked immunosorbent assay kits (R&D Systems Europe Ltd, Abingdon, UK), measuring the total amounts of these cytokines. IL-1 and IL-1ß were determined with kits from Immunotech (Marseille, France), and TNF-
was determined using antibodies from Biosource International (Lucernachem, Lucerne, Switzerland). Leptin levels were determined using a radioimmunometric assay kit from Linco Research (St. Louis, MO). The performance of these kits in quantifying cytokines in protein extracts from tissues was validated by the addition of known amounts of recombinant cytokines.
The measurement of serum sIL-1Ra levels in rodents is influenced by the interference from serum proteins, and no adequately validated kit is currently available.
Explant preparation and culture.
Human subcutaneous WAT, obtained from patients undergoing abdominoplasty, was prepared as previously described (13). Briefly, tissue was placed in PBS (prewarmed at 37°C and containing penicillin [100 units/ml] and streptomycin [100 mg/ml]), and connective tissue and blood vessels were removed by dissection before mincing the tissue into 5- to 10-mg pieces. Minced tissue fragments were filtered through a nylon mesh (350 µm) and washed with medium (M199, supplemented with 5% fetal bovine serum [FBS] and P+S; Invitrogen). Then 0.51 g of minced tissue was placed into 10 ml M199 (5% FBS, penicillin, and streptomycin) for 18 h before being stimulated for 48 h with the appropriate compounds. At the end of the stimulation period, the supernatants were collected for cytokine determination and the explants were recovered and placed directly into TRIzol reagent (Invitrogen) for immediate RNA preparation.
Statistics.
Results are expressed as means ± SE. The nonparametric Mann-Whitney U test was used for comparison between two groups of mice (n = 45), and the paired Students t test was used for the human visceral and subcutaneous adipose tissues (n = 28). These tests and the linear regression analysis were performed with SYSTAT 10.01 (SPSS, Chicago, IL).
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RESULTS |
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Expression of sIL-1Ra mRNA and protein in human adipose tissue.
To test whether sIL-1Ra was also upregulated in human adipose tissue, samples of subcutaneous WAT were obtained from 10 patients with a BMI <30 kg/m2. In addition, visceral and subcutaneous WAT was obtained from 28 obese patients undergoing gastric banding or bypass surgery. Protein extracts and total RNA were prepared for assessing the presence of IL-1Ra and other cytokines at mRNA and protein level.
Adipose tissue homogenates were assayed for their protein contents in IL-1Ra, Il-1, Il-1ß, TNF-
, IL-6, and leptin using validated immunoassays. IL-1Ra protein was present in all subcutaneous tissue samples, with an average content of 2.8 ± 0.5 pg/mg of tissue in nonobese patients and a significantly increased content (3.1-fold) in the obese group (Fig. 3A). Using quantitative real-time PCR, we also determined the respective amounts of mRNA for sIL-1Ra and icIL-1Ra using a standard curve with known amounts of the cloned cDNA for either form. As was seen in the rodents, sIL-1Ra mRNA was significantly increased in WAT from obese patients (6.6-fold) (Fig. 3B), whereas the intracellular form remained unchanged. Moreover, the expression of sIL-1Ra was 8- and 98-fold higher than that of icIL-1Ra in nonobese and obese subcutaneous WAT, respectively (Fig. 3B). Therefore, the increased concentrations of IL-1Ra protein present in the tissues from obese patients most likely represented the secreted form of the protein. sIL-1Ra mRNA was equally expressed in visceral and subcutaneous adipose tissue, with a significant correlation between the two within patients (r2 = 0.42, P < 0.001; data not shown). There was no significant difference in the protein content for IL-1Ra or TNF-
between subcutaneous and visceral WAT; however, IL-1ß was slightly increased in visceral WAT and IL-1
was undetectable (Fig. 3C, left panel). In contrast, IL-6 protein was nearly fivefold more abundant in visceral than in subcutaneous WAT (Fig. 3C, left panel), whereas the leptin content was slightly lower in visceral fat (Fig. 3C, right panel). The IL-1Ra protein content of subcutaneous WAT correlated with serum IL-1Ra levels (r2 = 0.45; P < 0.001) and, to a lesser extent, with BMI (r2 = 0.2; P < 0.02). However, serum leptin levels did not correlate with its abundance in WAT (data not shown). For comparison, the IL-6 protein content per weight unit of WAT from the obese patients was 5- to 10-fold lower than for IL-1Ra, but was 3- to 4-fold higher for leptin (Fig. 3C). The ratio of the protein content of IL-1Ra to IL-1ß was increased 4.8-fold in WAT from obese patients (Fig. 3D), suggesting that the metabolic effects of locally produced IL-1 might be inhibited by IL-1Ra in adipose tissue.
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Taken together, our results suggest that adipose tissue is a quantitatively relevant source of serum sIL-1Ra.
Human adipose tissue in explants secrete IL-1Ra.
Because WAT expresses and produces IL-1Ra, we wished to test whether explants from human WAT were capable of secreting IL-1Ra. To this end, we incubated human adipose explants from subcutaneous WAT for 48 h with various stimulators known to induce IL-1Ra secretion in monocytes, such as phorbol myristate acetate (PMA), IFN-ß, and the peroxisome proliferator-activated receptor- (PPAR-
) ligand rosiglitazone, which, as we have recently shown, regulates IL-1Ra in human monocytes (15). WAT explants secreted 14 ± 3.6 ng · g explant-1 · 48 h-1 of IL-1Ra in the basal state; this secretion was significantly enhanced by either PMA or IFN-ß (to 39 ± 8.4 or 73 ± 21.2 ng · g explant-1 · 48 h-1, respectively) (Fig. 4A). In contrast to what was seen in monocytes, rosiglitazone treatment resulted in an inhibition of PMA-stimulated secretion. With regard to the steady-state levels of IL-1Ra mRNA, the regulation of expression was similar to that of its secretion, with PMA increasing the basal level 4.6-fold and a strong upregulation being obtained with IFN-ß (29-fold) (Fig. 4B). Taken together, these results show that human WAT is a quantitatively important and regulated source of sIL-1Ra.
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DISCUSSION |
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The expression of IL-1Ra increases nearly 40-fold in WAT of obese rodents; the question then arises as to which factors mediate this marked augmentation. The fact that the increased expression of IL-1Ra is accompanied to a similar extent by the augmentation of TNF- mRNA, but not IL-1ß mRNA, argues against a general inflammatory state associated with obesity. This suggestion is also substantiated by human studies in which IL-6 serum levels were not increased beyond the upper limit of the normal range (12,21). Hence, it appears that metabolic factors selectively enhance the expression of the IL-1Ra gene in adipose tissue. Indeed, we have recently shown that PPAR-
ligands enhance the expression and secretion of IL-1Ra by stimulated human monocytes. However, our experiments with human WAT explants have shown a strong stimulatory effect of PMA and IFN-ß, whereas the PPAR-
ligand inhibited the PMA-induced secretion of IL-1Ra, indicating a tissue-specific regulation. This finding was in keeping with the observation that in morbidly obese humans, the serum sIL-1Ra levels are less well correlated with BMI than with the degree of insulin resistance (12), the latter being mainly attributable to an altered insulin sensitivity of skeletal muscle (17). Furthermore, we found the expression of sIL-1Ra to be markedly increased in skeletal muscle from obese mice, suggesting that this tissue may also contribute to circulating IL-1Ra levels in obesity. Our experiments with human WAT explants show, however, that PPAR-
ligands are unable to induce the secretion of IL-1Ra, whereas PMA and IFN-ß are potent inducers. Because IFN-ß not only is a fibroblast-derived interferon, but also is produced by other cell types such as macrophages, epithelial, endothelial, and lymphoid cells (22), it is tempting to speculate that stromal cells and preadipocytes might be involved in regulating IL-Ra secretion through a paracrine mechanism. Based on our data obtained from rodents, it appears that the regulation of cytokine expression in WAT is multifactorial, although 1) marked site-specific differences in the basal and obesity-related expression of IL-1Ra exist (this is also true for IL-1ß and TNF-
, the latter being markedly upregulated in epididymal fat of ob/ob mice, as previously described [14)], but with an opposite effect in inguinal WAT) and 2) in response to LPS, both the maximal induction of sIL-1Ra in WAT as well as the time course are markedly different in WATe and WATi, with the former showing a markedly prolonged enhancement of sIL-1Ra expression compared to any of the other tissues examined. However, the factors responsible for this difference are unknown.
The functional consequences of the increased production of IL-1Ra in obesity remain speculative. However, beside the anti-inflammatory, and hence potentially antiatherogenic, effects of IL-1Ra (7,23), the question arises whether this cytokine antagonist also has metabolic activities. Although this issue remains largely unexplored, data from Luheshi et al. (24) indicate that IL-1Ra induces an acquired resistance to leptin in rodents; it has been hypothesized that this mechanism is also operative in humans (12). Perhaps even more important, IL-1ß was shown to stimulate lipolysis, glucose transport, and adipocyte maturation in adipose tissue, as well as inhibit lipogenesis (25), an effect that is likely mediated through the IL-1 receptor type 1 and the IL-1 receptor accessory protein (2630), both of which we have shown to be expressed in human WAT (unpublished data). Hence, it can be speculated that the auto- and paracrine effects of the increased IL-1Ra:IL-1ß ratio may inhibit these effects, contributing to an increase in body fat and weight gain. Whether IL-1Ra has any additional metabolic effects and/or an antiatherogenic protective effect in human obesity remains to be demonstrated.
Taken together, these findings imply that adipose tissue is an important source of sIL-1Ra, whose secretion can be induced by inflammatory stimuli as well as obesity-associated factors. This, in turn, implies that adipose tissue might serve as a novel target for developing therapeutic strategies for inflammatory autoimmune diseases. Moreover, because IL-1 appears to mediate, at least in part, the central effects of leptin on appetite regulation and thermogenesis, it can be speculated that the upregulation of IL-1Ra in obesity results in a partial blockade of the hypothalamic actions of leptin, resulting in an acquired resistance to leptin (12,24).
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ACKNOWLEDGMENTS |
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We are grateful to Philippe Cettour-Rose for his expertise in performing the animal experiments, as well as to Dr. Pascale Roux-Lombard and the technician of the Laboratory of Clinical Immunology and Allergology, Immunology Division of our hospital for cytokine determination in human serum. We also gratefully acknowledge Drs. Brigitte Pittet and Navid Alizadeh for providing some of the human samples of adipose tissue and Roswitha Rem for her critical reading and corrections of the manuscript.
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FOOTNOTES |
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Received for publication 6 January 2003 and accepted in revised form 12 February 2003.
C.E.J.-A. and E.S. contributed equally to this work.
BAT, brown adipose tissue; FBS, fetal bovine serum; HT, hypothalamus; icIL-1Ra, intracellular IL-1Ra; IFN-ß, interferon-ß; IL, interleukin; IL-1Ra, interleukin-1 receptor antagonist; LPS, lipopolysaccharide; PMA, phorbol myristate acetate; PPAR-, peroxisome proliferator-activated receptor-
; sIL-1Ra, secreted IL-1Ra; TNF-
, tumor necrosis factor-
; WAT, white adipose tissue; WATe, epididymal WAT; WATi, inguinal WAT
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REFERENCES |
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