fax: (919) 684-4183
Liver Biology and Pathobiology---Pathobiology
Norepinephrine regulates hepatic innate immune system in leptin-deficient mice with nonalcoholic steatohepatitis
Article first published online: 13 AUG 2004
Copyright © 2004 American Association for the Study of Liver Diseases
Volume 40, Issue 2, pages 434–441, August 2004
How to Cite
Li, Z., Oben, J. A., Yang, S., Lin, H., Stafford, E. A., Soloski, M. J., Thomas, S. A. and Diehl, A. M. (2004), Norepinephrine regulates hepatic innate immune system in leptin-deficient mice with nonalcoholic steatohepatitis. Hepatology, 40: 434–441. doi: 10.1002/hep.20320
- Issue published online: 13 AUG 2004
- Article first published online: 13 AUG 2004
- Manuscript Accepted: 28 APR 2004
- Manuscript Received: 2 FEB 2004
- National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Grant Number: RO1 DK 53792
- National Institutes of Health. Grant Number: T32DK07632
- American Gastroenterological Association (AGA) Fellowship/Faculty Transition Award
It is not known why natural killer T (NKT) cells, which modulate liver injury by regulating local cytokine production, are reduced in leptin-deficient ob/ob mice. NKT cells express adrenoceptors. Thus, we hypothesize that the low norepinephrine (NE) activity of ob/ob mice promotes depletion of liver NKT cells, thereby sensitizing ob/ob livers to lipopolysaccharide (LPS) toxicity. To evaluate this hypothesis, hepatic NKT cells were quantified in wild-type mice before and after treatment with NE inhibitors, and in dopamine β-hydroxylase knockout mice (which cannot synthesize NE) and ob/ob mice before and after 4 weeks of NE supplementation. Decreasing NE activity consistently reduces liver NKT cells, while increasing NE has the opposite effect. Analysis of hepatic and thymic NKT cells in mice of different ages demonstrate an age-related accumulation of hepatic NKT cells in normal mice, while liver NKT cells become depleted after birth in ob/ob mice, which have increased apoptosis of hepatic NKT cells. NE treatment inhibits apoptosis and restores hepatic NKT cells. In ob/ob mice with reduced hepatic NKT cells, hepatic T and NKT cells produce excessive T helper (Th)-1 proinflammatory cytokines and the liver is sensitized to LPS toxicity. NE treatment decreases Th-1 cytokines, increases production of Th-2 cytokines, and reduces hepatotoxicity. Studies of CD1d-deficient mice, which lack the receptor required for NKT cell development, demonstrate that they are also unusually sensitive to LPS hepatotoxicity. In conclusion, low NE activity increases hepatic NKT cell apoptosis and depletes liver NKT cells, promoting proinflammatory polarization of hepatic cytokine production that sensitizes the liver to LPS toxicity. (HEPATOLOGY 2004;40:434–441.)
Obesity is strongly associated with nonalcoholic fatty liver disease (NAFLD). Fatty livers are unusually susceptible to injury induced by a secondary inflammatory stress, including that evoked by exposure to endogenous, intestine-derived lipopolysaccarhide (LPS).1 More serious liver injury (steatohepatitis) results, and this eventually leads to cirrhosis in some individuals. Because the transition from steatosis to steatohepatitis dramatically increases the risk of developing liver-related morbidity and mortality, it is important to understand why fatty livers are so vulnerable to inflammatory stress.
To address this issue, our laboratory has been studying genetically obese, leptin-deficient ob/ob mice.2 Similar to obese humans who are usually hyperleptinemic, ob/ob mice are insulin-resistant, have fatty livers, and are exquisitely sensitive to LPS-induced liver injury.3 Previously, we noted that certain populations of liver lymphocytes—specifically natural killer T (NKT) cells—are selectively reduced in the livers of ob/ob mice and suggested that hepatic NKT cell depletion may underlie fatty liver vulnerability to LPS in these animals.4 NKT cells are components of the innate immune system. These cells originate in the thymus but predominately accumulate in the liver, where they regulate local proinflammatory (T helper [Th]-1) and anti-inflammatory (Th-2) cytokine production by other mononuclear cells.5 Certain types of liver injury may result from selective reductions in NKT cell populations. For example, infection with Propionibacterium acnes is thought to sensitize normal livers to subsequent LPS-induced injury by reducing hepatic NKT cells.6
Leptin is now known to have potent immunomodulatory actions.7 Interactions between leptin and its receptors on immune cells regulate immune cell functions,8, 9 including phagocytosis10 and cytokine production.11 Leptin deficiency also sensitizes T cells to corticosteroid-induced apoptosis, which promotes thymic atrophy in ob/ob mice.12 In addition, leptin deficiency profoundly alters the hypothalamic–pituitary–adrenal axis, increasing certain stress-related factors (e.g., corticosteroids)13 while decreasing others (e.g., norepinephrine [NE]).14 It is becoming increasingly apparent that changes in such leptin-regulated, neurohumoral factors directly mediate many features of leptin deficiency. For example, reduced NE causes the hyperostosis and inhibited adipocyte lipolysis that occur in leptin-deficient mice.15 Moreover, targeted disruption of leptin receptor genes in neurons reproduces many aspects of the ob/ob phenotype, strongly suggesting that neuronal factors are necessary to relay leptin-initiated signals to many other cells.16
Previous studies have found that the sympathetic nervous system regulates NKT cells.17 We hypothesize that reduced NE inhibits the hepatic accumulation of NKT cells in leptin-deficient mice. If NE is indeed a major proximal regulator of hepatic NKT cell populations, then changes in NE activity may alter hepatic NKT cell numbers and influence hepatic cytokine production independently of leptin. This has important implications, because most obese humans with NAFLD are not leptin-deficient; consequently, it has been unclear if mechanisms that mediate NAFLD pathogenesis in ob/ob mice have more general relevance. In the present study, we address the importance of NE in hepatic NKT cell regulation and attempt to determine if hepatic NKT cell depletion causes Th-1 polarization of hepatic cytokine-producing cells and enhances sensitivity to liver injury.
Materials and Methods
Adult (8- to 10-week-old) and young (3- and 6-week-old) male C57BL6 ob/ob mice, their lean litter mates, and wild-type C57BL6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). CD1d−/− C57BL6 mice were a gift from Dr. Albert Bendelac (University of Chicago, Chicago, IL). Dopamine β-hydroxylase–deficient C57BL6 mice (DBH−/−) and their heterozygous litter mates (DBH+/−) were from the colony that has been generated by Steven Thomas' laboratory.18 All mice were maintained in a temperature- and light-controlled facility and were permitted ad libitum consumption of water and pellet chow.
Young (3- and 6-week-old) ob/ob mice and their lean litter mates were used to evaluate developmental changes in thymic and hepatic mononuclear cell (HMNC) populations. Older (8- to 10-week-old) ob/ob mice and their lean litter mates were treated with vehicle (pyrogen-free saline), NE (Sigma, St. Louis, MO, 2.5 mg/kg/d via Alzet minipumps [Durect, Cupertino, CA] for 3 weeks), or interleukin (IL) 15 (a gift from Immunex, Seattle, WA, 10 μg/d intraperitoneally for 1 week). In parallel studies, some DBH−/− mice were also treated with vehicle and NE. Wild-type C57BL6 mice (10 to 12 weeks old) were treated with prazosin (Sigma, 0.05 mg/mL in drinking water) or 6-hydroxydopamine (6-OHDA) (Sigma, 100 mg/kg) for 4 weeks. 6-OHDA was injected intraperitoneally daily for 5 consecutive days to induce chemical sympathectomy. Thereafter, mice received 6-OHDA injections three times per week for the remainder of the 4-week treatment period to ensure continued sympathectomy.19 Control mice received either vehicle in drinking water (for the prazosin studies) or via intraperitoneal injection (for the 6-OHDA studies). CD1d−/− C57BL6 mice and wild-type C57BL6 mice were injected intraperitoneally with a single dose of Eschericia coli LPS (Sigma, 50 μg/mouse) and then killed after 0, 1.5, or 6 hours to obtain serum and liver tissue.
All animal experiments fulfilled National Institutes of Health and Johns Hopkins University criteria for the humane treatment of laboratory animals.
Liver Mononuclear Cell Isolation and Cell Surface Labeling.
HMNCs were isolated and labeled using a minor modification of the method we described previously.4 Anti-mouse NK1.1-PE, CD3-FITC, CD4-APC, CD8-PerCP, Annexin-V-PE, and 7-AAD were obtained from Pharmingen (San Diego, CA). After surface labeling, HMNCs were evaluated using flow cytometry (Becton Dickenson, Palo Alto, CA). Data were analyzed using Cell Quest software (Becton Dickenson).
Liver Mononuclear Cell Intracellular Cytokines Labeling.
After isolation, HMNCs were incubated with phorbol 1,2-myristate 1,3-acetate (Sigma, 50 ng/mL), ionomycin (Sigma, 500 ng/mL) and GolgiPlug (Pharmingen, 1 μL/mL). Cells were labeled with surface antibody as decribed above and then permeablized with Cytoperm/Cytofix (Pharmingen) according to the manufacturer's instructions. After permeablization, cells were further labeled for intracellular cytokines such as anti–mouse tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) (Pharmingen). After incubation cells were evaluated using flow cytometry. Data were analyzed as described above.
Liver Mononuclear Cell IL-4 Assay.
After isolation, HMNCs (2 × 105/well) were incubated with anti–CD3 mAb (Pharmingen, 10 μg/mL) overnight at 37°C. IL-4 concentration in the medium was determined via enzyme-linked immunosorbent assay using mouse recombinant IL-4 as standard according the manufacturer's instructions (Biosource, Camarillo, CA).
Serum Alanine Aminotransferase Levels.
Alanine aminotransferase (ALT) levels were measured with a multichannel autoanalyzer in the Clinical Chemistry Laboratory of the Johns Hopkins University Department of Comparative Medicine.
All values are expressed as the mean ± SD. The group means were compared via ANOVA using Microsoft Excel (Microsoft, Redmond, WA).
Selective Depletion of Hepatic, But Not Thymic, NKT Cells During Leptin Deficiency.
Because NKT cells originate in the thymus and migrate to the liver during development,5 we compared thymic and hepatic NKT cell populations in 3- and 6-week-old ob/ob mice and their lean littermates to understand when NKT cells are depleted in congenital leptin deficiency. As reported by others,12 we noted that ob/ob mice develop premature thymic atrophy, which is reflected by dramatic decreases in total thymic mononuclear cell numbers (Fig. 1A). Surprisingly, the NKT cells are relatively spared during thymic involution in ob/ob mice such that the remaining thymic mononuclear cells in these mice are relatively enriched with NKT cells (Fig. 1B). In contrast, there is a selective depletion of NKT cells in the livers of ob/ob mice during the same period. Compared with lean control mice, total hepatic mononuclear cell numbers are not decreased in ob/ob mice at either 3 or 6 weeks after birth (Fig. 1C). However, in the ob/ob group, hepatic NKT cell populations progressively decline during this period (Fig. 1D). These findings suggested to us that extrathymic factors are predominately responsible for the lower levels of hepatic NKT cells in leptin-deficient mice.
Sympathetic Neurotransmitters Regulate Hepatic NKT Cell Populations.
As discussed earlier, NE activity is reduced during leptin deficiency.14 NKT cells express adrenoceptors.20 Furthermore, work by Minagawa and colleagues suggests that sympathetic neurotransmitters regulate the accumulation of NKT cells in the liver after partial hepatectomy.17 However, it is not known how important sympathetic neurotransmitters are for the general maintainence of hepatic NKT cell populations. To address this question, we treated wild-type (leptin-sufficient) mice with prazosin, an α-adrenoceptor blocker, or 6-OHDA to induce chemical sympathectomy. Both agents significantly decreased hepatic NKT cell populations in normal mice (Fig. 2A).
To determine whether or not the effects of α-adrenoceptor blockade and chemical sympathectomy are due to specific inhibition of NE activity, we studied DBH−/− mice, which cannot produce NE.18 Hepatic NKT cells are also significantly decreased in DBH−/− mice (Fig. 2B) compared with their heterozygous littermates. Hepatic NKT cells also increase after supplementing NE in DBH−/− mice (see Fig. 2B). Therefore, either acquired or congenital deficiency of NE causes depletion of liver NKT cells. These findings demonstrate that NE is critically important for normal maintenance of hepatic NKT cell populations.
NE Treatment Increases Hepatic NKT Cell Population in Leptin-Deficient ob/ob Mice.
Leptin deficiency is known to induce multiple hormonal, metabolic, and immunological abnormalities. Therefore, although NE is a major regulator of hepatic NKT cells in leptin-sufficient mice, other factors may be more important in maintaining these cells in the livers of leptin-deficient mice. To address this issue, we treated ob/ob mice with NE or saline vehicle. NE significantly increased hepatic NKT cells in leptin-deficient mice (Fig. 3), demonstrating that this sympathetic neurotransmitter regulates hepatic NKT cell populations independently of leptin. Further analysis of different NK cell–specific surface antigens is planned to define the heterogeneity of the NKT cell population in ob/ob livers before and after NE treatment. Such information will be helpful in determining if various NKT cell subsets differ in their requirements for NE.
NE Treatment Reduces Hepatic NKT Cell Apoptosis in Leptin-Deficient ob/ob Mice.
To gain insight into the mechanisms through which NE increases hepatic NKT cells, we assessed the effects of NE on NKT cell apoptosis. Hepatic expression of TNF-α, a factor that induces NKT cell apoptosis,21 is known to be increased in ob/ob mice.22 Therefore, we suspected that NKT cell apoptosis might be increased in ob/ob livers. To assess this possibility, we evaluated hepatic NKT cell apoptosis using Annexin V. We found that, as predicted, hepatic NKT cell apoptosis is increased significantly in ob/ob mice. Moreover, 3 weeks of NE treatment decreased hepatic NKT cell apoptotic activity to normal levels (Fig. 4). Compared with NE, IL-15, another factor that increases NKT cells,11 has much less of an inhibitory effect on hepatic NKT cell apoptosis (see Fig. 4).
NE Treatment Reverses Hepatic Proinflammatory Cytokine Production During Leptin Deficiency.
The livers of leptin-deficient mice are unusually sensitive to LPS-induced injury, a process that is mediated by proinflammatory cytokines such as TNF-α and IFN-γ. IFN-γ is known to sensitize hepatocytes to TNF-α killing.23 Studies with TNF-α–neutralizing antibodies demonstrate that TNF-α is required for LPS liver injury.24 However, IFN-γ sensitization to TNF-α is also critically important, because mice that are genetically deficient in IFN-γ are completely protected from LPS hepatotoxicity despite persistent TNF-α expression.25 NKT cells produce both IFN-γ and IL-4.5 While the former exacerbates TNF-α toxicity, the latter is a key inducer of anti-inflammatory (Th-2) cytokines, which generally attenuate the toxic effects of TNF-α.26 Therefore, it is difficult to predict the ultimate effects of hepatic NKT cell depletion on hepatic cytokine production and LPS sensitivity.
To address the first issue, we treated ob/ob mice with NE or vehicle, isolated hepatic mononuclear cells, and measured intracellular cytokines. Results from both ob/ob groups were also compared with those of lean control mice. The production of IFN-γ and TNF-α, increased significantly in total liver mononuclear cells from ob/ob mice compared with lean controls. These differences reflect increases in Th-1 cytokine production by several different cell populations, as demonstrated by increased IFN-γ and/or TNF-α expression in hepatic T cells and NK cells (Fig. 5A). Treatment with doses of NE that restores hepatic NKT cell numbers also reduces proinflammatory cytokine production by all of the hepatic mononuclear cell populations that we evaluated (Fig. 5B). On the other hand, the production of IL-4, an anti-inflammatory Th-2 cytokine, is decreased in total liver mononuclear cells from ob/ob mice (Fig. 5C). Treatment with NE significantly increases IL-4 production (see Fig. 5C).
CD1d−/− Mice Are More Susceptible to LPS-Induced Injury.
The previous studies demonstrate that in ob/ob mice, decreases in hepatic NKT cell populations are accompanied by Th-1 polarization of other cytokine-producing cells in the liver. Because proinflammatory cytokines mediate LPS liver injury, the latter suggests a mechanism through which hepatic NKT cell depletion may promote vulnerability to LPS hepatotoxicity in leptin-deficient mice. However, as discussed earlier, leptin-deficient ob/ob mice have multiple immunological abnormalities.7 Therefore, to clarify the significance of NKT cell depletion as a vulnerability factor for LPS hepatotoxicity, we evaluated otherwise normal mice that were genetically deficient in CD1d, a Class I–like molecule that is required for the positive selection of certain NKT cell populations during development.27 Because both ob/ob mice and CD1d−/− mice are relatively depleted of hepatic NKT cells,4, 28 we predicted that CD1d−/− mice would behave like ob/ob mice and exhibit more sensitivity to LPS toxicity than control mice that have normal levels of hepatic NKT cells. To test this hypothesis, we treated CD1d−/− mice with low doses of LPS that are generally well-tolerated by normal mice. After receiving LPS, CD1d−/− mice exhibited significantly more liver injury than control mice, as reflected by two- to threefold higher serum levels of ALT (Fig. 6A). However, CD1d−/− mice appear to be less sensitive to LPS toxicity than ob/ob mice, which have 10-fold greater ALT levels than LPS-treated controls at the same time point (Fig. 6B). This suggests that ob/ob mice have additional factors that enhance their vulnerability to LPS hepatoxicity. Our preliminary comparison of other liver mononuclear cell populations in ob/ob and control mice failed to demonstrate quantitative differences in resident CD4+ or CD8+ T cells or NK cell populations, but we cannot exclude the possibility that strain-related differences in other types of immune cells (e.g., gamma-delta T cells) or hepatocytes themselves might contribute.
NE Treatment Reduces Hepatic Inflammation in ob/ob Mice.
With age, obese ob/ob mice develop intestinal bacterial overgrowth,29 which increases portal endotoxemia and hepatic exposure to endogenous LPS.30 This, in turn, promotes inflammatory cytokine production and steatohepatitis, because treating ob/ob mice with either probiotic (to modify their exposure to endogenous intestinal bacterial products) or anti–TNF-α antibodies (to inhibit TNF-α activity) significantly improves their steatohepatitis.24 Evidence that CD1d−/− mice, which have reduced hepatic NKT cells, exhibit increased sensitivity to LPS-induced hepatotoxicity, suggested to us that reduced hepatic NKT cells might be the underlying cause of LPS sensitivity in ob/ob livers. If so, then NE treatment (which increases hepatic NKT cells and decreases proinflammatory cytokine production in ob/ob mice) is predicted to enhance resistance to LPS hepatoxicity. To evaluate this possibility, we measured serum levels of ALT in NE-treated ob/ob mice, as well as vehicle-treated ob/ob control mice and lean mice. As expected, baseline serum ALT levels were greater in control ob/ob mice, which have spontaneous steatohepatitis, than in lean mice, which have normal livers. Simply treating ob/ob mice with NE returns serum ALT levels to near normal values (see Fig. 6B). Thus, in adult leptin-deficient mice, replenishing NE restores hepatic NKT cell populations, reverses Th-1 polarization of hepatic cytokine-producing cells, and reduces hepatic sensitivity to endogenous, intestinally derived LPS, despite persistent obesity and leptin deficiency.
Chronic inflammation is now recognized as a key mediator of obesity-related diseases, including type 2 diabetes and NAFLD.31 In ob/ob mice, an animal model for obesity-associated diseases, expression of the proinflammatory cytokine TNF-α is increased in white adipose tissue and liver, and both insulin resistance and fatty liver disease are improved significantly by inhibiting TNF-α activity.24 However, the mechanisms that induce and maintain TNF-α activity during obesity are poorly understood. In leptin-deficent ob/ob mice, leptin replacement reverses obesity and obesity-related inflammatory diseases; thus there is no doubt that leptin deficiency promotes chronic inflammation. Paradoxically, however, TNF-α expression and activity are also increased in other animal models of obesity and in obese humans with hyperleptinemia, prompting speculation that excessive (rather than deficient) leptin drives obesity-related inflammation.32 However, the latter concept has been difficult to validate, because some degree of leptin resistance typically develops during chronic hyperleptinemia.33
The poor correlation between serum leptin levels and chronic inflammatory activity suggests that factors other than leptin may be predominately responsible for controlling obesity-related inflammation. This possibility is certainly plausible, because leptin is merely one of the many factors that regulate the hypothalamus and the pituitary and adrenal glands. These tissues produce multiple immunomodulatory hormones and neurotransmitters,34 and recent evidence suggests that such neurohumoral factors directly mediate many aspects of the ob/ob (leptin-deficient) phenotype. For example, leptin deficiency decreases NE,14 and reduced NE is predominately responsible for the hypotension, hyperostosis, and peripheral adiposity that develops in ob/ob mice.35 The present study provides additional support for the concept that reduced NE mediates key features of leptin deficiency by demonstrating that supplemental NE reverses both the depletion of hepatic NKT cell populations and the proinflammatory polarization of hepatic cytokine production that develop during leptin deficiency.
Circulating levels of NE are variable in human obesity. NE levels are increased in some obese populations, particularly those who have overt hypertension.36 On the other hand, other studies show that although many obese individuals have normal “resting” levels of NE, they exhibit significant differences in NE induction following stress.37 In addition, obesity has been associated with cellular resistance to NE and other adrenergic agonists.38 In some tissues, this is due to decreased adrenoceptor expression.39 Indeed, relative resistance to NE may contribute to the pathogenesis of obesity, because sibutramine—a pharmacological agent that prolongs adrenergic activity by inhibiting catecholamine reuptake—is a reasonably effective treatment for obesity.40
When NE activity is decreased, hepatic NKT cell apoptosis is increased and restoring NE reduces apoptosis, even when leptin is absent. Thus, although leptin clearly has immunomodulatory actions,7 NE is a more important viability factor for hepatic NKT cells. Parallel studies in mice with intact genes for leptin and its receptors demonstrate that NE is generally important for regulating hepatic NKT cell populations by showing that complementary experimental approaches that inhibit NE activity also reduce NKT cell accumulation in the livers of leptin-sufficient mice. Evidence that NE regulates hepatic NKT cells independently of leptin suggests that like leptin-deficient ob/ob mice, obese hyperleptinemic mice and humans may also develop hepatic NKT cell depletion. Therefore, hepatic depletion of NKT cells may be involved in a common mechanism that promotes obesity-related liver damage in both hyper- and hypoleptinemic individuals.
The possibility that tissue-specific reductions in NKT cell populations promote organ damage has already been suggested by other groups.41 Indeed, given the strong association between obesity, diabetes, and NAFLD, it is particularly intriguing that NKT cell depletion has been implicated in the pathogenesis of both diabetes and LPS liver injury in certain nonobese animal models for these conditions.6, 41 In those models, organ damage is attributed to relative excesses of proinflammatory cytokines that develop when tissue NKT cell populations are reduced. For example, overabundance of proinflammatory cytokines such as IFN-γ sensitizes hepatocytes to liver injury upon subsequent exposure to doses of LPS that are well-tolerated when IFN-γ is not excessive.25 NKT cell populations normally control proinflammatory Th-1 cytokine activities by promoting the production of anti-inflammatory Th-2 cytokines.42 Thus, when proinflammatory cytokine activity is not tempered by anti-inflammatory cytokines, sustained Th-1 polarization, chronic inflammation, and progressive tissue injury ensue in response to stimuli that typically signal a self-limited inflammatory response. Treatment with NE in ob/ob mice restores hepatic NKT cell population, reverses proinflammatory polarization, and significantly reduces hepatic inflammation.
Thymic selection of NKT cells is blocked during development in CD1d−/− mice. Consequently, in adult CD1d−/− mice, NKT cell populations are reduced in many peripheral tissues, including the liver. As discussed earlier, studies in normal mice infected with P. acnes, as well as in leptin-deficient ob/ob mice, have correlated decreased hepatic NKT cell populations with sensitization to hepatotoxicity from LPS, a potent inducer of proinflammatory cytokines. However, because P. acnes infection and leptin deficiency influence multiple components of the immune system, a cause–effect relationship between hepatic NKT cell depletion and sensitization to LPS liver injury remains speculative. Studies in genetically altered mice with selective depletions of certain NKT cell populations have yielded discrepant results—one group reported that NKT cells protect against LPS toxicity,43 but another group reported the opposite result.44 Present evidence that otherwise healthy, CD1d−/− mice develop worse liver injury after LPS exposure than their littermate controls provides compelling additional support for the importance of reduced hepatic NKT cells in enhancing sensitivity to toxicity from LPS. Our results complement and extend those reported by Emoto et al.,43 who showed that mice that are deficient in Vα14+ NKT cells due to targeted disruption of β2 microglobulin, a factor that is necessary for CD1d activity, are profoundly sensitive to LPS toxicity. Together with our other findings, this result suggests that the decreases in NE that occur in leptin deficiency may mediate progression of obesity-related liver disease, because decreased NE promotes apoptosis of hepatic NKT cells. This in turn reduces NKT cell populations in the liver. Dysregulation of other hepatic cytokine-producing cells ensues, leading to the accumulation of Th-1 polarized lymphocytes. When confronted by a stimulus for proinflammatory cytokine production (e.g., LPS), production of proinflammatory cytokines is relatively unconstrained in the liver, enhancing acute LPS hepatotoxicity while also promoting a sustained (i.e., chronic) inflammatory response.