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Serum samples from 74 obese women were assayed for 25-hydroxyvitamin D [25(OH)D] concentrations using an automated immunoassay [Architect (Abbott)] and ELISA (Alpco Diagnostics), and results were compared with the LC/MS/MS reference method (Quest Diagnostics). The Architect values were significantly lower (mean difference: -13 nmol/L; 95% limits: -54; 28 nmol/L) and the ELISA values were significantly higher (mean difference: 24 nmol/L; 95% limits: -36; 84 nmol/L) than the LC/MSIMS values. The slope of the Passing-Bablok regression line relative to LC/MSIMS was 0.5 [95% confidence interval (CI): 0.41; 0.60] and 1.17 (95% CI: 0.87; 1.56) for Architect and ELISA, respectively, with an intercept of approximately 16 for both assays. Using 50 nmol/L as the cut-point for deficiency, Architect and ELISA misclassified 20 and 27% of the subjects, respectively. In subjects with low circulating 25-hydroxylated ergocalciferol [25(OH)D2] (<10 nmol/L), a Bland-Altman plot and Kappa statistics (Kappa = 0.73; 95% CI: 0.49-0.97) showed good agreement between Architect and LC/MS/MS. However, in subjects with high circulating 25(OH)D2 (>or=10 nmol/L), Architect demonstrated poor agreement (Kappa = 0.40; 95% CI: 0.16-0.65). ELISA demonstrated a higher degree of overestimation in women with minimal 25(OH)D2 than those with high 25(OH)D2, suggesting that ELISA overestimates 25-hydroxylated cholecalciferol [25(OH)D3] but underestimates 25(OH)D2.

Context: Adipose tissue macrophage (ATM) infiltration is a major pathway for obesity-induced insulin resistance but has not been studied as a mechanism for insulin resistance in PCOS., Objective: We tested whether polycystic ovary syndrome (PCOS) is associated with increased ATM infiltration, especially of inflammatory subtype identified by the CD11c marker., Design and Setting: We conducted a case-control study at an academic medical center in the United States., Participants and Interventions: Fourteen PCOS and 14 control women of similar age and body mass index (BMI) underwent a gluteal fat biopsy. Markers of ATM, integrins, TNF-α, and adiponectin, were analyzed by quantitative RT-PCR using a standard curve method. Crown-like structures (CLS) were identified by immunohistochemistry. Abdominal magnetic resonance imaging and frequently sampled i.v. glucose tolerance test were performed to assess abdominal fat and insulin sensitivity (SI)., Main Outcome: Women with PCOS were compared with control women of similar age and BMI for ATM markers, CLS density, adipose tissue expression of inflammatory cytokines and adiponectin, SI, and abdominal fat depots., Results: Women with PCOS had an increase in CD11c expression (P = 0.03), CLS density (P = 0.001), α5 expression (P = 0.009), borderline increase in TNF-α expression (P = 0.08), and a decrease in adiponectin expression (P = 0.02) in gluteal adipose tissue. Visceral (P = 0.009) and sc abdominal fat (P = 0.005) were increased in PCOS. SI was lower in PCOS (P = 0.008)., Conclusions: PCOS is associated with an increase in CD11c expression and CLS density and a decrease in adiponectin expression in sc adipose tissue. Additionally, PCOS is associated with higher central abdominal fat depots independent of BMI. These alterations are present among mostly nonobese women and could represent mechanisms for insulin resistance.

Obesity increases severity of acute pancreatitis and risk of pancreatic cancer. Pancreatitis and obesity are associated with elevated IL-6, a cytokine involved in inflammation and tumorigenesis. We studied the role of IL-6 in the response of lean and obese mice to pancreatitis induced by IL-12 + IL-18. Lean and diet-induced obese (DIO) WT and IL-6 KO mice and ob/ob mice pretreated with anti-IL-6 antibodies were evaluated at Days 1, 7, and 15 after induction of pancreatitis. Prolonged elevation of IL-6 in serum and visceral adipose tissue was observed in DIO versus lean WT mice, whereas circulating sIL-6R declined in DIO but not lean mice with pancreatitis. The severe inflammation and lethality of DIO mice were also observed in IL-6 KO mice. However, the delayed resolution of neutrophil infiltration; sustained production of CXCL1, CXCL2, and CCL2; prolonged activation of STAT-3; and induction of MMP-7 in the pancreas, as well as heightened induction of serum amylase A of DIO mice, were blunted significantly in DIO IL-6 KO mice. In DIO mice, production of OPN and TIMP-1 was increased for a prolonged period, and this was mediated by IL-6 in the liver but not the pancreas. Results obtained in IL-6 KO mice were confirmed in ob/ob mice pretreated with anti-IL-6 antibodies. In conclusion, IL-6 does not contribute to the increased severity of pancreatitis of obese mice but participates in delayed recovery from acute inflammation and may favor development of a protumorigenic environment through prolonged activation of STAT-3, induction of MMP-7, and sustained production of chemokines.

IL-17A is a proinflammatory cytokine critical for host defense and involved in the pathogenesis of autoimmune disorders. Obesity is associated with chronic low-grade inflammation but also with a heightened acute inflammatory response. We investigated the effect of obesity on IL-17A production using the model of ZY-induced peritonitis. Compared with lean controls, administration of ZY induced a significantly exacerbated inflammatory response in obese leptin-deficient ob/ob mice and in mice with diet-induced obesity (DIO). Levels of IL-17A in the peritoneal fluid in response to ZY were elevated significantly in ob/ob and DIO mice compared with lean animals. Reconstitution of ob/ob mice with exogenous leptin did not alter production of IL-17A significantly in response to ZY. Peritoneal cells and adipose tissue obtained from ZY-injected obese mice expressed significantly higher levels of IL-17A mRNA compared with lean mice. Approximately 2% of peritoneal Ly6G(+) neutrophils from ZY-injected obese mice expressed IL-17A protein, compared with 0.2% of cells obtained from lean mice. Neutralization of IL-17 in ob/ob mice inhibited neutrophil recruitment and production of neutrophil-attracting CXC chemokines and IL-6, without affecting macrophage infiltration or levels of IL-10 and the chemokine CCL2. In contrast, neutralization of IL-6 did not affect production of IL-17A or chemokines while reducing production of the acute-phase protein serum amyloid A significantly. These data demonstrate that neutrophil-derived IL-17A is increased in obese mice during acute inflammation and contributes to exacerbation of inflammatory responses.

Leptin-deficient ob/ob mice are resistant to dextran sulfate sodium (DSS)-induced colitis and Concanavalin A (Con A)-induced hepatitis. However, the signal transduction pathways involved have not been identified. The present study investigated the effect of leptin-induced STAT3 signaling in the DSS and Con A models. Mice carrying a leptin receptor (LEPR) gene mutant for Y1138 (s/s mice), with abrogated leptin-induced STAT3 signaling, were compared with wild-type (WT) and LEPR-deficient db/db mice. Administration of DSS to s/s mice resulted in a clinical score and colon shortening of intermediate severity compared with disease induced in WT and db/db mice-the latter group having the lowest disease severity. A comparable degree of inflammatory infiltrate and epithelial damage was observed in the colon of WT and s/s mice, and these parameters were reduced in db/db mice. Levels of IFN-gamma, IL-6, IL-10, and TNF-alpha were comparable in the colon of s/s and db/db mice, and a similar trend was observed for CXCL2. s/s and WT mice developed severe liver disease in response to Con A, whereas db/db mice were protected. However, Con A-induced serum IL-6 and TNF-alpha levels in s/s mice mimicked levels observed in db/db rather than WT mice. In conclusion, lack of leptin-induced STAT3 signaling is associated with reduced cytokine production following DSS and Con A administration, but it appears to sensitize mice to the effects of proinflammatory mediators.

Adipokines, cytokines mainly produced by adipocytes, are active participants in the regulation of inflammation. Administration of zymosan (ZY) was used to investigate the regulation and role of adipokines during peritonitis in mice. Injection of ZY led to a significant increase in leptin levels in both serum and peritoneal lavage fluid, whereas a differential trend in local vs. systemic levels was observed for both resistin and adiponectin. The role of leptin in ZY-induced peritonitis was investigated using leptin-deficient ob/ob mice, with and without reconstitution with exogenous leptin. Leptin deficiency was associated with delayed resolution of peritoneal inflammation induced by ZY, because ob/ob mice had a more pronounced cellular infiltrate in the peritoneum as well as higher and prolonged local and systemic levels of IL-6, TNFalpha, IL-10, and chemokine (C-X-C motif) ligand 2 compared with wild-type mice. Reconstitution with exogenous leptin exacerbated the inflammatory infiltrate and systemic IL-6 levels in ob/ob mice while inhibiting production of TNFalpha, IL-10, and chemokine (C-X-C motif) ligand 2. In contrast with the important role of leptin in regulating each aspect of ZY-induced peritonitis, adiponectin deficiency was associated only with a decreased inflammatory infiltrate, without affecting cytokine levels. These findings point to a complex role for adipokines in ZY-induced peritonitis and further emphasize the interplay between obesity and inflammation.

Adiponectin (APN) is an adipocyte-derived protein that regulates insulin sensitivity and displays antiinflammatory activities in a variety of experimental models. The present study aimed at investigating the effect of APN deficiency on the inflammatory response to endotoxin (lipopolysaccharide, LPS) and Concanavalin A (ConA) in vivo in mice. Administration of a high dose of LPS (100 microg/mouse) induced production of comparable amounts of IL-6, TNFalpha, and interferon-gamma in wild-type (WT) and APN knockout (KO) mice. Furthermore, LPS-induced hypoglycemia, anorexia, and body weight loss did not differ between WT and APN KO mice. Administration of a low dose of LPS (100 or 10 ng/g) in association with d-galactosamine induced equivalent mortality rates, hepatotoxicity, and serum IL-6 in WT and APN KO mice. Finally, ConA-induced cytokine production and hepatotoxicity were not significantly different between WT and APN KO mice. These data indicate that--despite its well-described role as an antiinflammatory molecule--endogenous APN does not play a critical role in modulating the inflammatory responses to LPS and ConA in mice.

The role of leptin in the immune system has been well established. While adipocytes represent the major source, leptin production by lymphocytes, infiltrating at the site of inflammation, was recently demonstrated. However, the significance of this locally released leptin remains unresolved. In the present study, two models in which absence of leptin-signalling is associated with protection were employed: the model of ConA-induced hepatitis and the CD4(+)CD45Rb(high) transfer model of colitis. For the ConA model, scid mice were reconstituted with either WT or leptin-deficient (ob/ob) CD4(+) T cells. Eight weeks post transfer, ConA was injected and serum ALT, TNFalpha, leptin as well as liver mononuclear cell activation and histological signs of inflammation were evaluated. No difference between recipients of WT or ob/ob cells was observed for any of the parameters evaluated. In the second model, either WT or ob/ob CD4(+)CD45Rb(high) cells were transferred into scid mice. No histological differences were detected, although recipients of ob/ob cells showed higher weight loss compared to recipients of WT cells. Spontaneous production of IL-6 from colon cultures obtained from recipients of ob/ob cells was reduced compared to recipients of WT cells, whereas stimulation of lamina propria lymphocytes with leptin resulted in a higher IFNgamma release in recipients of ob/ob cells compared to recipients of WT cells. In conclusion, the present study provides evidence that T cell-derived leptin does not play a major role in the regulation of the inflammatory process, indicating that the adipose tissue is the critical player in the immune-modulating effects of leptin.

A considerable amount of research has focused on elucidating the mechanisms by which cytokines synthesized by cells of the innate immune system participate in the life-threatening multiple-organ failure of endotoxic shock. We show here that alphabeta T cells, which are archetypes of the adaptive cellular immune response, suppress the proinflammatory cascade triggered during the early stages of lipopolysaccharide (LPS)-induced endotoxemia. The absence of alphabeta T cells led to the fulminant death of LPS-challenged mice, coinciding with a massive release of the proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma and a marked reduction in the synthesis of the immunosuppressive cytokine transforming growth factor (TGF)-beta. Cytotoxic T lymphocyte antigen (CTLA)-positive alphabeta T cells emerging shortly after LPS challenge appear to control TGF-beta synthesis. The neutralization of either TGF-beta or CTLA4 resulted in similar increases in IFN-gamma and TNF-alpha serum concentrations in LPS-challenged mice. These observations suggest that suppressor alphabeta T lymphocytes protect against the proinflammatory cascade unleashed during the innate stages of endotoxemia.

Concanavalin A-induced hepatotoxicity was compared in lipodystrophic aP2-nSREBP-1c transgenic mice (LD mice) lacking adipose tissue, obese leptin-deficient ob/ob mice, and lean wild-type (WT) mice. Serum leptin and adiponectin were low in LD mice, whereas ob/ob mice had undetectable leptin, but high adiponectin. Protection from hepatotoxicity was observed in ob/ob, but not in LD mice, despite low cytokine levels and reduced T cell activation and hepatic natural killer T cells in both groups. Administration of adiponectin protected LD mice from hepatotoxicity without altering cytokine levels. In contrast, administration of leptin heightened disease susceptibility by restoring cytokine production. Neutralization of TNF alpha protected LD mice from liver damage. Increased in vivo susceptibility to the hepatotoxic effect of TNF alpha was observed in LD mice. In vitro, adiponectin protected primary hepatocytes from TNF alpha-induced death, whereas leptin had no protective effect. In conclusion, although leptin increases susceptibility to hepatotoxicity by regulating cytokine production and T cell activation, adiponectin protects hepatocytes from TNF alpha-induced death.

Leptin-deficient (ob/ob) mice are resistant in different models of autoimmunity and inflammation, suggesting that leptin regulates immunity and inflammation. To investigate whether leptin deficiency modulates the spontaneous intestinal inflammation observed in interleukin (IL)-10-deficient mice, double IL-10- and leptin-deficient [IL-10 knockout (KO) ob/ob] mice were generated and compared with single IL-10 KO mice for colitis severity. Body weight in IL-10 KO ob/ob mice was significantly reduced compared with that of ob/ob mice. However, when compared with wild-type or IL-10 KO mice, IL-10 KO ob/ob mice were still markedly obese. IL-10 KO and IL-10 KO ob/ob mice developed colitis with a comparable time-course and severity in terms of macroscopic and histologic scores. Likewise, production of interferon-gamma, IL-6, and IL-13 from colon cultures and splenocytes did not differ among these two groups. Conversely, rates of apoptosis were higher in lamina propria lymphocytes obtained from the colon of IL-10 KO ob/ob compared with IL-10 KO mice. In conclusion, although leptin deficiency has been associated with resistance in models of autoimmunity and inflammation induced by exogenous stimuli, leptin appears not to play a significant role in the spontaneous colitis of IL-10 KO mice, although it modulates survival of intestinal lymphocytes.

Interleukin (IL)-18 binding protein (IL-18BP) is a natural inhibitor of the pleiotropic cytokine IL-18. To study the role of IL-18BP in modulating inflammatory responses in vivo, mice transgenic for human IL-18BP isoform a (IL-18BP-Tg) were generated. The transgene was expressed at high levels in each organ examined. High levels of bioactive human IL-18BPa were detectable in the circulation of IL-18BP-Tg mice, which were viable, fertile, and had no tissue or organ abnormality. The high levels of IL-18BP in the transgenic mice were able to completely neutralize the interferon-gamma (IFN-gamma)-inducing activity of exogenously administered IL-18. Following administration of endotoxin, with or without prior sensitization with heat-inactivated Propionibacterium acnes, IL-18BP-Tg mice produced significantly lower serum levels of IFN-gamma and macrophage-inflammatory protein-2 compared with nontransgenic littermates. Significantly reduced production of IFN-gamma in response to endotoxin was also observed in cultures of IL-18BP-Tg splenocytes. Finally, IL-18BP-Tg mice were completely protected in a model of hepatotoxicity induced by administration of concanavalin A. These results indicate that high endogenous levels of IL-18BP in trangenic mice effectively neutralize IL-18 and are protective in response to different inflammatory stimuli.

Interferon (IFN)-gamma-inducing factor was previously termed interleukin (IL)-18. Although IL-12 is also an IFN-gamma-inducing factor, the activity of IL-18 (but not IL-12) in models of sepsis and death is dependent on the intracellular cysteine protease IL-1beta converting enzyme (caspase-1). Caspase-1 is required for cleavage of the inactive precursor form of IL-18 into an active cytokine, and caspase-1-deficient mice are resistant to lethal endotoxemia. The absence of IFN-gamma (but not IL-1beta) in caspase-1-deficient mice is responsible for this resistance. However, the role of IFN-gamma in murine defense against gram-negative infection is inconsistent. Mice deficient in IFN-gamma are not resistant to lethal endotoxemia but are resistant when treated with neutralizing antibodies to IL-18 and challenged with a lethal injection of some endotoxins. Anti-IL-18 treatment also reduces neutrophil accumulation in liver and lungs. Neutralizing IL-18 with the IL-18 binding protein protects mice against endotoxin- and ischemia-induced hepatic damage. Thus, blockade of IL-18 appears to be a viable clinical target to combat the pathologic consequences of sepsis via IFN-gamma mechanisms.

Leptin, the product of the ob gene, is a pleiotropic molecule that regulates food intake as well as metabolic and endocrine functions. Leptin also plays a regulatory role in immunity, inflammation, and hematopoiesis. Alterations in immune and inflammatory responses are present in leptin- or leptin-receptor-deficient animals, as well as during starvation and malnutrition, two conditions characterized by low levels of circulating leptin. Both leptin and its receptor share structural and functional similarities with the interleukin-6 family of cytokines. Leptin exerts proliferative and antiapoptotic activities in a variety of cell types, including T lymphocytes, leukemia cells, and hematopoietic progenitors. Leptin also affects cytokine production, the activation of monocytes/macrophages, wound healing, angiogenesis, and hematopoiesis. Moreover, leptin production is acutely increased during infection and inflammation. This review focuses on the role of leptin in the modulation of the innate immune response, inflammation, and hematopoiesis.

A novel murine model of intrauterine infection/inflammation-induced preterm birth based on direct endoscopic intracervical inoculation is described. Using this model, we investigated infection-induced premature pregnancy loss in normal and interleukin (IL) 1beta-deficient mice. Seventy-four CD-1, HS, C57BL/6J wild type (IL-1beta+/+), and C57BL/6J IL-1beta-deficient (IL-1beta-/-) mice were inoculated intracervically using a micro-endoscope, at a time corresponding to 70% of average gestation. Intracervical injection of lipopolysaccharide (LPS) or Escherichia coli reliably induced premature birth: 100% of mice intracervically injected with LPS and 92% of mice with a positive endometrial E. coli culture delivered prematurely within 36 h after inoculation. No losses were observed in mice inoculated with saline. Pregnancy loss was associated with increased uterine tissue cyclooxygenase-2 gene expression and uterine content of IL-1beta, tumor necrosis factor alpha, macrophage inflammatory protein-1alpha, and IL-6, as well as elevation of nuclear factor-kappaB activity in uterine tissues. Although IL-1beta-/- mice exhibited decreased uterine cytokine production in response to bacteria and LPS, IL-1beta deficiency did not affect the rate of pregnancy loss. This model using direct intracervical bacterial or LPS inoculation is useful for studying preterm pregnancy loss in genetically altered mice in order to develop novel interventions for infection-associated preterm labor.

Interleukin-18 (IL-18) is a newly described cytokine, formerly called interferon-gamma (IFN-gamma)-inducing factor. In a simple 24-h human whole blood culture, IFN-gamma was produced by the combination of lipopolysaccharide (LPS) plus IL-18. To liberate cytokines in the leukocyte and red cell compartments, the detergent Triton X-100 was added to the entire blood culture. The combination of low concentrations of LPS plus IL-18 induced a 3- to 5-fold greater production of IFN-gamma than did either stimulant alone. Tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8 were also produced. The presence of IL-10 completely suppressed the production of IFN-gamma and reduced that of TNF-alpha, IL-6, and IL-8. Thus, IFN-gamma, TNF-alpha, IL-8, and IL-6 are produced in a single whole blood culture, making correlations in the synthesis of a T helper type 1 cytokine and proinflammatory cytokines with disease activity possible in a single culture.

Initially described in 1989 as interferon-gamma (IFN-gamma) inducing factor (IGIF), interleukin-18 (IL-18) is a novel pro-inflammatory cytokine that is clearly more than an inducer of IFN-gamma. The cytokine possesses several biological properties such as activation of nuclear factor-kappaB (NF-kappaB), Fas ligand expression, the induction of both CC and CXC chemokines, and increased production of competent human immunodeficiency virus. Most activities are due to a receptor complex that recruits the IL-1 receptor-activating kinase (IRAK), leading to translocation of NF-kappaB. This property and others support the concept that IL-18 is related to the IL-1 family. Indeed, one of the IL-18 receptor chains is the IL-1 receptor-related protein, a member of the IL-1R family. In addition, IL-18 is structurally similar to IL-1beta and like IL-1beta is first synthesized as a leaderless precursor requiring the IL-1beta converting enzyme for cleavage into an active molecule. The biology of IL-18 is reviewed in the overview and the implication for a role for this cytokine in disease is presented.

Interleukin-1 (IL-1) plays a crucial role in the development of the pathophysiological responses to infection and inflammation. However, the relative contributions of IL-1 alpha and IL-1 beta remain to be clarified. IL-1 beta-deficient mice are a powerful tool to investigate the specific role of IL-1 beta in various experimental conditions. In this report, we summarize the response of IL-1 beta deficient mice to two different inflammatory stimuli, turpentine and endotoxin. Although IL-1 beta-deficient mice respond normally to the systemic administration of lipopolysaccharide (LPS), they do not develop an acute-phase response in the localized tissue damage model of turpentine injection. The results obtained using the IL-1 beta-deficient mice are compared here with those observed in the IL-1 beta-converting enzyme-deficient, IL-6-deficient, tumour necrosis factor-receptor p55-deficient, and interferon-gamma-receptor-deficient mice.

Glucocorticoids (GC) are well known inhibitors of tumour necrosis factor (TNF) production. We investigated the role of endogenous GC in the regulation of TNF production in mice treated with lipopolysaccharide (LPS) using a pretreatment with dexamethasone (DEX) to down-regulate the hypothalamus-pituitary-adrenal axis (HPA). Short-term DEX pretreatment (up to 12 h before LPS) inhibited TNF production, but earlier (24-48 h) pretreatments potentiated it. This up-regulating effect was not observed in adrenalectomized mice or when GC synthesis was inhibited with cyanoketone (CK). This effect could not be explained only by the suppression of LPS-induced corticosterone (CS) levels induced by DEX, since a 48-h pretreatment potentiated TNF production without affecting LPS-induced CS levels. On the other hand, mice chronically pretreated with DEX were still responsive to its inhibitory effect on TNF production, thus ruling out the possibility of a decreased responsiveness to GC.