Obesity is associated with See the related Commentary beginning on page 1785. macrophage accumulation in adipose tissue Stuart P. Weisberg,1 Daniel McCann,1 Manisha Desai,2 Michael Rosenbaum,1 Rudolph L. Leibel,1,3,4 and Anthony W. Ferrante, Jr.3,4 1Division of Molecular Genetics, Department of Pediatrics, 2Department of Biostatistics, 3Deparment of Medicine, and 4Naomi Berrie Diabetes Center, Columbia University, New York, New York, USA Obesity alters adipose tissue metabolic and endocrine function and leads to an increased release of fatty acids, hormones, and proinflammatory molecules that contribute to obesity associated compli- cations. To further characterize the changes that occur in adipose tissue with increasing adiposity, we profiled transcript expression in perigonadal adipose tissue from groups of mice in which adiposity varied due to sex, diet, and the obesity-related mutations agouti (Ay) and obese (Lepob). We found that the expression of 1,304 transcripts correlated significantly with body mass. Of the 100 most significantly correlated genes, 30% encoded proteins that are characteristic of macrophages and are positively cor- related with body mass. Immunohistochemical analysis of perigonadal, perirenal, mesenteric, and sub- cutaneous adipose tissue revealed that the percentage of cells expressing the macrophage marker F4/80 (F4/80+) was significantly and positively correlated with both adipocyte size and body mass. Similar relationships were found in human subcutaneous adipose tissue stained for the macrophage antigen CD68. Bone marrow transplant studies and quantitation of macrophage number in adipose tissue from macrophage-deficient (Csf1op/op) mice suggest that these F4/80+ cells are CSF-1 dependent, bone marrow–derived adipose tissue macrophages. Expression analysis of macrophage and nonmacrophage cell populations isolated from adipose tissue demonstrates that adipose tissue macrophages are responsible for almost all adipose tissue TNF-α expression and significant amounts of iNOS and IL-6 expression. Adipose tissue macrophage numbers increase in obesity and participate in inflam- matory pathways that are activated in adipose tissues of obese individuals. J. Clin. Invest. 112:1796–1808 (2003). doi:10.1172/JCI200319246. Introduction risk factor for myocardial infarction, stroke, type 2 Adiposity, the fraction of total body mass comprised diabetes mellitus, and certain cancers (7–9). of neutral lipid stored in adipose tissue, is closely cor- Changes in adipose tissue mass are associated with related with important physiological parameters such changes in the endocrine and metabolic functions of as blood pressure, systemic insulin sensitivity, and adipose tissue that link increased adiposity to alter- serum triglyceride and leptin concentrations (1–3). ations in systemic physiology. Increased adipocyte vol- Strong positive correlations exist between degree of ume and number are positively correlated with leptin adiposity and several obesity-associated disorders production, and leptin is an important regulator of such as hypertension, dyslipidemia, and glucose intol- energy intake and storage, insulin sensitivity, and meta- erance (2, 4). Visceral fat mass is more closely corre- bolic rate (10–13). Leptin signaling has also been impli- lated with obesity-associated pathology than overall cated in the pathogenesis of arterial thrombosis (14). adiposity (5, 6). Obesity in humans is an independent Adiposity is negatively correlated with production of adiponectin (also known as ACRP30), a hormone that Received for publication June 19, 2003, and accepted in revised form decreases hepatic gluconeogenesis and increases lipid October 13, 2003. oxidation in muscle (15–17). Address correspondence to: Anthony W. Ferrante, Jr., The altered production of proinflammatory mole- Naomi Berrie Diabetes Center, Columbia University, cules (so-called “adipokines”) by adipose tissue has 1150 St. Nicholas Avenue, New York, New York 10032, USA. been implicated in the metabolic complications of Phone: (212) 851-5322; Fax: (212) 851-5331; E-mail: [email protected]. obesity. Compared with adipose tissue of lean indi- Conflict of interest: The authors have declared that no conflict of viduals, adipose tissue of the obese expresses interest exists. increased amounts of proinflammatory proteins such Nonstandard abbreviations used: monocyte chemotactic as TNF-α, IL-6, iNOS (also known as NOS2), TGF-β1, protein-1 (MCP-1); plasminogen activator inhibitor type 1 C-reactive protein, soluble ICAM, and monocyte (PAI-1); stromal vascular cell (SVC); diet-induced obese (DIO); fatty acid–poor BSA (FAP-BSA); colony-stimulating factor 1 receptor chemotactic protein-1 (MCP-1) (18–25), and proco- (Csf1r); thiazolidinedione (TZD). agulant proteins such as plasminogen activator 1796 The Journal of Clinical Investigation | December 2003 | Volume 112 | Number 12 inhibitor type-1 (PAI-1), tissue factor, and factor VII Mice were sacrificed by CO2 asphyxiation at 20–21 (26–28). Obese mice deficient in TNF-α and iNOS are weeks of age during the second and third hour of the more sensitive to insulin than are obese wild-type light cycle. Animals were weighed and adipose tissues mice (21, 29). Proinflammatory molecules have direct (epididymal or parametrial, perirenal, mesenteric, effects on cellular metabolism. For example, TNF-α and inguinal subcutaneous depots), liver, and exten- directly decreases insulin sensitivity and increases sor digitalis longus muscle were removed. Tissues to lipolysis in adipocytes (30, 31). IL-6 leads to hyper- be analyzed by FACS were processed immediately; triglyceridemia in vivo by stimulating lipolysis and other samples were frozen in liquid nitrogen and hepatic triglyceride secretion (32). stored at –75°C prior to RNA extraction and im- Despite the increased production of proinflamma- munohistochemical analysis. All procedures were tory molecules, infiltration of adipose tissue by approved by Columbia University’s Institutional Ani- inflammatory cells has not been described as a com- mal Care and Use Committee. mon feature of obesity. That adipocytes express recep- A total of 24 mice were included in the microarray tors for several proinflammatory molecules (e.g., expression study, four from each of the following TNF-α, IL-6) supports models in which adipocytes groups: C57BL/6J males, C57BL/6J females, high were both the source and target of proinflammatory fat–fed C57BL/6J males, B6.Cg Ay/+ females, B6.V signals. However, recent data suggest that in adipose Lepob/ob males, and B6.V Lepob/ob females. In B6.Cg Ay/+ tissue, proinflammatory molecules, including IL-1β, mice, ectopic overexpression of the agouti transcript PG-E2, TNF-α, and IL-6, are produced by stromal vas- leads to a moderate increase in the mass of adipose tis- cular cells (SVCs) (33, 34). sue and a hyperleptinemic form of obesity (35). Feed- To identify genes whose expression correlated with ing male C57BL/6J mice a high-fat diet also leads to adiposity, we profiled gene expression in perigonadal hyperleptinemic moderate obesity. B6.V Lepob/ob mice adipose tissue from 24 mice in which adiposity varied are leptin-deficient and severely obese (36, 37). A pair due to sex, diet, and the obesity-related mutations of 2-month-old macrophage-deficient (FVB/NJ agouti (Ay) and obese (Lepob).We used an analytical Csf1op/op) and control (FVB/NJ Csf1+/+) female mice were approach that calculated Kendall’s τ, a nonparametric, a gift of E. Richard Stanley (Albert Einstein College of robust correlation statistic for each gene in our data Medicine, New York, New York, USA). Macrophage- set. This analysis identified 1,304 transcripts that were deficient mice are osteopetrotic and toothless; these significantly correlated with body mass, an easily meas- mice were maintained on a powdered chow diet (∼5% ured indirect indicator of adiposity. A large proportion fat content). For transplant experiments, CD45.2+ of these transcripts encoded proteins characteristic of (C57BL/6J) recipient and CD45.1+ (B6.SJL Ptprca macrophages. These results suggested — and histolog- Pep3b/BoyJ) donor mice were purchased from The ical data from both mice and humans confirmed — Jackson Laboratory. These mice are homozygous for that the macrophage content of adipose tissue corre- antigenically distinct forms of the CD45 protein, a lated positively with two indices of adiposity: BMI and protein expressed on all leukocytes. adipocyte size. Expression analysis of macrophage and Human subjects and materials. Healthy lean, overweight, nonmacrophage cell populations isolated from adipose and obese subjects were admitted to the Clinical tissue showed that adipose tissue macrophages are the Research Center at Columbia Presbyterian Medical primary sources of TNF-α and other proinflammatory Center (New York, New York, USA) as part of a longi- molecules in adipose tissue. tudinal study of the metabolic effects of weight per- turbation. The details of this study have been described Methods previously (38–40). The study protocol was approved Animals and animal care. Unless otherwise noted, all by the Institutional Review Board of Columbia Uni- mice were obtained from The Jackson Laboratory (Bar versity, and written informed consent was obtained Harbor, Maine, USA) at 6–8 weeks of age and housed in from each subject. Subjects were fed a liquid formula ventilated Plexiglas cages (one to three animals per diet of 40% fat (corn oil), 45% carbohydrate (glucose cage) within a pathogen-free barrier facility that main- polymer),