The role of the liver X receptor in chronic obstructive pulmonary disease

Background

There is a need for novel anti-inflammatory therapies to treat COPD. The liver X receptor (LXR) is a nuclear hormone receptor with anti-inflammatory properties.

Methods

We investigated LXR gene and protein expression levels in alveolar macrophages and whole lung tissue from COPD patients and controls, the effect of LXR activation on the suppression of inflammatory mediators from LPS stimulated COPD alveolar macrophages, and the effect of LXR activation on the induction of genes associated with alternative macrophage polarisation.

Results

The levels of LXR mRNA were significantly increased in whole lung tissue extracts in COPD patients and smokers compared to non-smokers. The expression of LXR protein was significantly increased in small airway epithelium and alveolar epithelium in COPD patients compared to controls. No differences in LXR mRNA and protein levels were observed in alveolar macrophages between patient groups. The LXR agonist GW3965 significantly induced the expression of the LXR dependent genes ABCA1 and ABCG1 in alveolar macrophage cultures. In LPS stimulated alveolar macrophages, GW3965 suppressed the production of CXCL10 and CCL5, whilst stimulating IL-10 production.

Conclusions

GW3965 did not significantly suppress the production of TNFα, IL-1β, or CXCL8. Our major finding is that LXR activation has anti-inflammatory effects on CXC10, CCL5 and IL-10 production from alveolar macrophages.     

Autoregulation of the human liver X receptor alpha promoter

Previous work has implicated the nuclear receptors liver X receptor alpha (LXR alpha) and LXR beta in the regulation of macrophage gene expression in response to oxidized lipids. Macrophage lipid loading leads to ligand activation of LXRs and to induction of a pathway for cholesterol efflux involving the LXR target genes ABCA1 and apoE. We demonstrate here that autoregulation of the LXR alpha gene is an important component of this lipid-inducible efflux pathway in human macrophages. Oxidized low-density lipoprotein, oxysterols, and synthetic LXR ligands induce expression of LXR alpha mRNA in human monocyte-derived macrophages and human macrophage cell lines but not in murine peritoneal macrophages or cell lines. This is in contrast to peroxisome proliferator-activated receptor gamma (PPAR gamma)-specific ligands, which stimulate LXR alpha expression in both human and murine macrophages. We further demonstrate that LXR and PPAR gamma ligands cooperate to induce LXR alpha expression in human but not murine macrophages. Analysis of the human LXR alpha promoter led to the identification of multiple LXR response elements. Interestingly, the previously identified PPAR response element (PPRE) in the murine LXR alpha gene is not conserved in humans; however, a different PPRE is present in the human LXR 5'-flanking region. These results have implications for cholesterol metabolism in human macrophages and its potential to be regulated by synthetic LXR and/or PPAR gamma ligands. The ability of LXR alpha to regulate its own promoter is likely to be an integral part of the macrophage physiologic response to lipid loading.     

T-0901317, a synthetic liver X receptor ligand,inhibits development of atherosclerosis in LDL receptor-deficient mice

Liver X receptors (LXR alpha and LXR beta) are nuclear receptors, which are important regulators of cholesterol and lipid metabolism. LXRs control genes involved in cholesterol efflux in macrophages, bile acid synthesis in liver and intestinal cholesterol absorption. LXRs also regulate genes participating in lipogenesis. To determine whether the activation of LXR promotes or inhibits development of atherosclerosis, T-0901317, a synthetic LXR ligand, was administered to low density lipoprotein receptor (LDLR)(-/-) mice. T-0901317 significantly reduced the atherosclerotic lesions in LDLR(-/-) mice without affecting plasma total cholesterol levels. This anti-atherogenic effect correlated with the plasma concentration of T-0901317, but not with high density lipoprotein cholesterol, which was increased by T-0901317. In addition, we observed that T-0901317 increased expression of ATP binding cassette A1 in the lesions in LDLR(-/-) mice as well as in mouse peritoneal macrophages. T-0901317 also significantly induced cholesterol efflux activity in peritoneal macrophages. These results suggest that LXR ligands may be useful therapeutic agents for the treatment of atherosclerosis.     

Rhinovirus regulation of IL-1 receptor antagonist in vivo and in vitro: a potential mechanism of symptom resolution.

Rhinovirus (RV) upper respiratory tract infections are prototypic transient inflammatory responses. To address the mechanism of disease resolution in these infections, we determined if RV stimulated the production of the IL-1 receptor antagonist (IL-1ra) in vivo and in vitro. In contrast to IL-1alpha and IL-1beta, immunoreactive IL-1ra was readily detected in the nasal washings of normal human volunteers. Symptomatic RV infection caused a small increase in IL-1alpha, a modest increase in IL-1beta, and an impressive increase in IL-1ra. Maximal induction of IL-1alpha and IL-1beta was transiently noted 48 h after RV infection. In contrast, maximal induction of IL-1ra was prolonged appearing 48-72 h after RV infection. These time points corresponded to the periods of peak symptomatology and the onset of symptom resolution, respectively. Western analysis of nasal washings demonstrated that RV stimulated the accumulation of intracellular IL-1ra type I in all and secreted IL-1ra in a subset of volunteers. Unstimulated normal respiratory epithelial cells contained intracellular IL-1ra type I mRNA and protein. RV infection increased the intracellular levels and extracellular transport of this IL-1ra moiety without causing significant changes in the levels of IL-1ra mRNA. IL-1ra may play an important role in the resolution of RV respiratory infections. RV stimulates epithelial cell IL-1ra elaboration, at least in part, via a novel translational and/or posttranslational mechanism.     

Regulated expression of the apolipoprotein E/C-I/C-IV/C-II gene cluster in murine and human macrophages. A critical role for nuclear liver X receptors alpha and beta

Lipid-loaded macrophage "foam cells" accumulate in the subendothelial space during the development of fatty streaks and atherosclerotic lesions. To better understand the consequences of such lipid loading, murine peritoneal macrophages were isolated and incubated with ligands for two nuclear receptors, liver X receptor (LXR) and retinoic acid receptor (RXR). Analysis of the expressed mRNAs using microarray technology led to the identification of four highly induced genes that encode apolipoproteins E, C-I, C-IV, and C-II. Northern blot analysis confirmed that the mRNA levels of these four genes were induced 2-14-fold in response to natural or synthetic ligands for LXR and/or RXR. The induction of all four mRNAs was greatly attenuated in peritoneal macrophages derived from LXRalpha/beta null mice. The two LXR response elements located within the multienhancers ME.1 and ME.2 were shown to be essential for the induction of apoC-II promoter-reporter genes by ligands for LXR and/or RXR. Finally, immunohistochemical studies demonstrate that apoC-II protein co-localizes with macrophages within murine arterial lesions. Taken together, these studies demonstrate that activated LXR induces the expression of the apoE/C-I/C-IV/C-II gene cluster in both human and murine macrophages. These results suggest an alternative mechanism by which lipids are removed from macrophage foam cells.     

Decreased nuclear hormone receptor expression in the livers of mice in late pregnancy

During the third trimester of pregnancy, there is an increase in serum triglyceride and cholesterol levels. The mechanisms accounting for these changes in lipid metabolism during pregnancy are unknown. We hypothesized that, during pregnancy, the expression of nuclear hormone receptors involved in regulating lipid metabolism would decrease. In 19-day pregnant mice, serum triglyceride and non-HDL cholesterol levels were significantly increased, whereas total cholesterol was slightly decreased, because of a decrease in the HDL fraction. Peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta, and PPARgamma, liver X receptor (LXR)alpha and LXRbeta, farnesoid X receptor (FXR), and retinoid X receptor (RXR)alpha, RXRbeta, and RXRgamma mRNA levels were significantly decreased in the livers of 19-day pregnant mice. Additionally, the expressions of thyroid receptor (TR)alpha, pregnane X receptor, sterol regulatory element-binding proteins (SREBP)-1a, SREBP-1c, SREBP-2, and liver receptor homolog 1 were also decreased, whereas the expression of TRbeta, constitutive androstane receptor, and hepatic nuclear factor 4 showed no significant change. mRNA levels of the PPAR target genes carnitine-palmitoyl transferase 1alpha and acyl-CoA oxidase, the LXR target genes SREBP1c, ATP-binding cassettes G5 and G8, the FXR target gene SHP, and the TR target genes malic enzyme and Spot14 were all significantly decreased. Finally, the expressions of PPARgamma coactivator (PGC)-1alpha and PGC-1beta, known activators of a number of nuclear hormone receptors, were also significantly decreased. The decreases in expression of RXRs, PPARs, LXRs, FXR, TRs, SREBPs, and PGC-1s could contribute to the alterations in lipid metabolism during late pregnancy.     

Interleukin-1 (IL-1) receptor antagonist binds to the 80-kDa IL-1 receptor but does not initiate IL-1 signal transduction.

The interleukin-1 receptor antagonist (IL-1ra) is a protein capable of inhibiting receptor binding and biological activities of IL-1 without inducing an IL-1-like response. Equilibrium binding and kinetic experiments show that IL-1ra binds to the 80-kDa IL-1 receptor on the murine thymoma cell line EL4 with an affinity (KD = 150 pM) approximately equal to that of IL-1 alpha and IL-1 beta for this receptor. However, IL-1ra is unable to induce two early events associated with IL-1 activity. Surface-bound IL-1ra does not undergo receptor-mediated internalization, and IL-1ra does not activate the protein kinase activity responsible for down-modulation of the EGF receptor on the murine 3T3 fibroblast cell line. The failure to induce general, early responses characteristic of IL-1 indicates that IL-1ra is unlikely to act as an agonist on any cell expressing the 80-kDa receptor.     

Stat3-dependent induction of interleukin-3 receptor expression in leukemia inhibitory factor-stimulated M1 mouse leukemia cells

M1 mouse leukemia cells differentiate to macrophages/monocytes by the stimulation of interleukin-6 (IL-6)/leukemia inhibitory factor (LIF). To identify new LIF-induced genes, we have performed representational difference analysis using M1 cells and cloned mouse interleukin-3 (IL-3) receptor beta subunit gene. The mRNA expression of both IL-3 receptor (IL-3R) alpha and beta subunits is upregulated after 1 h stimulation of LIF and remains to be elevated along the differentiation of M1 cells. This induction is almost completely suppressed in M1 cells expressing a dominant negative form of Stat3. Furthermore, we show that IL-3-induced Stat5 phosphorylation increases in LIF-stimulated M1 cells. These results suggest that Stat3 may play a role in the differentiation of myeloid cells by regulating IL-3R expression.     

Bovine interleukin-1 expression by cultured mammary epithelial cells (MAC-T) and its involvement in the release of MAC-T derived interleukin-8

MAC-T cells, an established bovine mammary epithelial cell line, were utilized to investigate both expression of interleukin-1 (IL-1) mRNA and secretion of IL-1 after Escherichia coli lipopolysaccharide (E. coli LPS) stimulation. In addition, recombinant human IL-1beta, recombinant human IL-1 receptor antagonist (IL-1ra) and a neutralizing goat antibody against type I human IL-1 receptor were used to study the involvement of IL-1 in the release of IL-8. The expression of MAC-T derived IL-1alpha mRNA was correlated to production of IL-1alpha protein as measured by an IL-1alpha sandwich ELISA. Secretion of IL-1alpha was dose- and time-dependent, with a maximal level of 600 pg/ml detectable upon 2-h stimulation with 20 microg of LPS per ml. IL-1ra and the neutralizing antibody significantly blocked the ability of IL-1beta to stimulate secretion of IL-8 by MAC-T cells. During this study, we have demonstrated that MAC-T cells secrete IL-1 in response to LPS stimulation and IL-1 is an important mediator for the release of the bovine IL-8 by MAC-T cells. These results further indicate the potential importance of mammary epithelial cells as a source of immunoregulation in the mammary gland via cytokine elaboration.     

Natural and recombinant interleukin 1 receptor antagonist does not inhibit human T-cell proliferation induced by mitogens, soluble antigens or allogeneic determinants.

Interleukin 1 receptor antagonist (IL-1ra) has been found in glycosylated form in the urine of febrile patients or of children with rheumatoid arthritis, and in the supernatant of monocytes cultured in the presence of immune complexes. It blocks competitively the binding of IL-1 alpha and beta to their receptors. Produced amongst others by mononuclear cell lines and matured monocytes and alveolar macrophages, it prevents prostaglandin E2 and collagenase production by fibroblasts and synovial cells. In mice, IL-1ra improves survival after lethal endotoxemia. In this study, both natural and recombinant human IL-1ra (rhIL-1ra) were tested in an allogeneic T-cell reaction, and in mitogen- or antigen-induced lymphocyte proliferation. Neither the natural nor the rhIL-1ra blocked T-cell proliferation, but rhIL-1ra abolished the effect of exogenous IL-1 beta. This was not due to a loss of bioactivity of IL-1ra in culture, as the IL-1ra of the supernatant still completely inhibited 125I-IL-1 alpha binding to EL 4-6.1 cells and markedly reduced PGE2 production during antigen presentation. We conclude that IL-1ra alone, even at high concentrations, is not sufficient to block human T-cell proliferation in vitro.     

Interleukin-1 gene cluster polymorphisms in cerebral infarction

Interleukin-1 (IL-1) has pleiotropic actions in the central nervous system. During the past decade, a growing corpus of evidence has indicated an important role of this cytokine in the development of brain damage following cerebral ischaemia. The expression of IL-1 in the brain is dramatically increased during the early and chronic stage of infarction. The IL-1 gene cluster on chromosome 2ql4 contains three related genes (IL-1alpha, IL-1beta, and IL-1 receptor antagonist, IL-1ra) located within a 430-kb region. Polymorphisms in the genes encoding IL-1alpha, IL-1beta, and IL-1ra have been associated with several inflammatory diseases. Therefore we hypothesized that these cytokines might be good candidates for cerebral infarction (CI). We ascertained these genotypes in 363 CI patients and 640 controls matched for age and gender. A significant increase was found for the IL-1alpha (-889) allele 2 carriers in CI patients compared with controls (chi2 = 5.633, P = 0.018, odds ratio (OR) = 1.5). Furthermore, the IL-1alpha (-889) allele 2 carriers increased the relative risk for CI in the subjects without the IL-1ra allele 2 (chi2 = 7.989, P = 0.005, OR = 1.7). There was no significant association between IL-1beta (+3,953) polymorphism and CI. These results suggest that IL-1alpha-889 and IL-1ra polymorphisms are effective in the development of CI in Koreans.     

Interleukin 1 receptor antagonist in human epidermis and cultured keratinocytes.

Interleukin 1 (IL-1), present in high amounts in normal human skin without any sign of inflammation, suggests a complex mechanism by which its bioactivity is regulated. The specific receptor antagonist of IL-1 (IL-1ra) was analyzed in human skin, sweat and cultured keratinocytes. Extracts of both skin and cultured keratinocytes blocked the binding of [125I]IL-1 to its receptor whereas sweat did not. The inhibitory activity was cell-associated, was not secreted by cultured keratinocytes, and IL-1ra mRNA was identified in these cells. There was an inverse relationship between the level of IL-1ra and that of IL-1 alpha and beta since extracts of differentiating keratinocytes (DK) and higher IL-1ra levels and expressed more mRNA for IL-1ra than non-differentiated keratinocytes (NDK), whereas NDK contained 4 times more IL-1 alpha and beta proteins than DK. This association of cell differentiation with a shift in agonist/antagonist ratio might be related to important autocrine or paracrine functions of IL-1 in normal and inflamed human skin.