Deletion of the transmembrane transporter ABCG1 results in progressive pulmonary lipidosis

We show that mice lacking the ATP-binding cassette transmembrane transporter ABCG1 show progressive and age-dependent severe pulmonary lipidosis that recapitulates the phenotypes of different respiratory syndromes in both humans and mice. The lungs of chow-fed Abcg1(-/-) mice, >6-months old, exhibit extensive subpleural cellular accumulation, macrophage, and pneumocyte type 2 hypertrophy, massive lipid deposition in both macrophages and pneumocytes and increased levels of surfactant. No such abnormalities are observed at 3 months of age. However, gene expression profiling reveals significant changes in the levels of mRNAs encoding key genes involved in lipid metabolism in both 3- and 8-month-old Abcg1(-/-) mice. These data suggest that the lungs of young Abcg1(-/-) mice maintain normal lipid levels by repressing lipid biosynthetic pathways and that such compensation is inadequate as the mice mature. Studies with A-549 cells, a model for pneumocytes type 2, demonstrate that overexpression of ABCG1 specifically stimulates the efflux of cellular cholesterol by a process that is dependent upon phospholipid secretion. In addition, we demonstrate that Abcg1(-/-), but not wild-type macrophages, accumulate cholesterol ester droplets when incubated with surfactant. Together, these data provide a mechanism to explain the lipid accumulation in the lungs of Abcg1(-/-)mice. In summary, our results demonstrate that ABCG1 plays essential roles in pulmonary lipid homeostasis.     

ABCG1 regulates pulmonary surfactant metabolism in mice and men

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of surfactant. Surfactant synthesis and secretion are restricted to epithelial type 2 (T2) pneumocytes (also called T2 cells). Clearance of surfactant is dependent upon T2 cells and macrophages. ABCG1 is highly expressed in both T2 cells and macrophages. ABCG1-deficient mice accumulate surfactant, lamellar body-loaded T2 cells, lipid-loaded macrophages, B-1 lymphocytes, and immunoglobulins, clearly demonstrating that ABCG1 has a critical role in pulmonary homeostasis. We identify a variant in the ABCG1 promoter in patients with PAP that results in impaired activation of ABCG1 by the liver X receptor α, suggesting that ABCG1 basal expression and/or induction in response to sterol/lipid loading is essential for normal lung function. We generated mice lacking ABCG1 specifically in either T2 cells or macrophages to determine the relative contribution of these cell types on surfactant lipid homeostasis. These results establish a critical role for T2 cell ABCG1 in controlling surfactant and overall lipid homeostasis in the lung and in the pathogenesis of human lung disease.     

A critical role for ABCG1 in macrophage inflammation and lung homeostasis

ATP-binding cassette transporter G1 (ABCG1) effluxes cholesterol from macrophages and plays an important role in pulmonary lipid homeostasis. We hypothesize that macrophages from Abcg1(-/-) mice have increased inflammatory activity, thereby promoting acceleration of pulmonary disease. We herein demonstrate increased numbers of inflammatory cytokines and infiltrating neutrophils, eosinophils, dendritic cells, T cells, and B cells into lungs of Abcg1(-/-) mice before the onset of severe lipidosis. We further investigated the role of macrophages in causing pulmonary disease by performing bone marrow transplantations using B6 and Abcg1(-/-) bone marrow. We found that it was the macrophage, and not pneumocyte type II cells or other nonhematopoietic cells in the lung, that appeared to be the primary cell type involved in the onset of both pulmonary lipidosis and inflammation in the Abcg1(-/-) mice. Additionally, our results demonstrate that Abcg1(-/-) macrophages had elevated proinflammatory cytokine production, increased apoptotic cell clearance, and were themselves more prone to apoptosis and necrosis. However, they were quickly repopulated by monocytes that were recruited to Abcg1(-/-) lungs. In conclusion, we have shown that ABCG1 deletion in macrophages causes a striking inflammatory phenotype and initiates onset of pulmonary lipidosis in mice. Thus, our studies reveal a critical role for macrophage ABCG1 in lung inflammation and homeostasis.     

Systemic inflammation and mortality in chronic obstructive pulmonary disease

Cardiovascular diseases and cancer (especially lung cancer) are leading causes of morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD). Some have implicated systemic inflammation, which is commonly observed in COPD, as the potential mechanistic bridge between COPD and these disorders. This concept has been supported by animal studies especially in rabbits, which have clearly demonstrated the effect of local lung inflammation on systemic inflammation and on the progression of atherosclerosis and by cross-sectional population-based studies, which have shown a significant relationship between systemic inflammation, as measured by circulating C-reactive protein (CRP) and the risk of cardiovascular diseases in COPD patients. These data have been further extended by a recent study that has elucidated the temporal nature of the relationship between systemic inflammation and the risk of cardiovascular events and cancer in COPD patients. This study showed that baseline CRP levels predicted the incidence of cardiovascular events and cancer-specific mortality over 7 to 8 years of follow-up. CRP levels also predicted all-cause mortality. Collectively, these data indicate that systemic inflammation may play an important role in mediating the extra-pulmonary complications of COPD. Systemic inflammation may contribute substantially to the overall morbidity and mortality of COPD patients.     

Systemic inflammation in chronic obstructive pulmonary disease: a population-based study

Background

Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue.

Methods

Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay.

Results

IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6.

Conclusion

AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.     

Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress

Yeast mutants lacking vacuolar proton-translocating ATPase (V-ATPase) subunits (vma mutants) were sensitive to several different oxidants in a recent genomic screen (Thorpe, G. W., Fong, C. S., Alic, N., Higgins, V. J., and Dawes, I. W. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6564-6569). We confirmed that mutants lacking a V(1) subunit (vma2Delta), V(o) subunit, or either of the two V(o) a subunit isoforms are acutely sensitive to H(2)O(2) and more sensitive to menadione and diamide than wild-type cells. The vma2Delta mutant contains elevated levels of reactive oxygen species and high levels of oxidative protein damage even in the absence of an applied oxidant, suggesting an endogenous source of oxidative stress. vma2Delta mutants lacking mitochondrial DNA showed neither improved growth nor decreased sensitivity to peroxide, excluding respiration as the major source of the endogenous reactive oxygen species in the mutant. Double mutants lacking both VMA2 and components of the major cytosolic defense systems exhibited synthetic sensitivity to H(2)O(2). Microarray analysis comparing wild-type and vma2Delta mutant cells grown at pH 5, permissive conditions for the vma2Delta mutant, indicated high level up-regulation of several iron uptake and metabolism genes that are part of the Aft1/Aft2 regulon. TSA2, which encodes an isoform of the cytosolic thioredoxin peroxidase, was strongly induced, but other oxidative stress defense systems were not induced. The results indicate that V-ATPase activity helps to protect cells from endogenous oxidative stress.     

Local and systemic cellular inflammation and cytokine release in chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease caused by repeated exposure to noxious gases or particles. It is now recognized that the disease also features systemic inflammation. The purpose of our study was to compare airway and systemic inflammation in COPD to that seen in healthy subjects and to relate the inflammation with the disease severity.

Methods

Ninety-five COPD patients, encompassing the whole severity spectrum of the disease, were recruited from our outpatient clinic and rehabilitation center and compared to 33 healthy subjects. Induced sputum and blood samples were obtained for measurement of inflammatory cell count. Interleukin (IL)-4, IL-6, IL-10, TNF-α and IFN-γ produced by 24 h sputum and blood cell cultures were measured.

Results

Compared to healthy subjects, COPD exhibited a prominent airway neutrophilic inflammation associated with a marked IL-10, IL-6 and TNF-α release deficiency that contrasted with a raised IFN-γ production. Neutrophilic inflammation was also prominent at blood level together with raised production of IFN-γ, IL-10 and TNF-α. Furthermore, sputum neutrophilia correlated with disease severity assessed by GOLD stages. Likewise the extent of TNF-α release from blood cells also positively correlated with the disease severity but negatively with that of sputum cell culture. Blood release of TNF-α and IL-6 negatively correlated with body mass index. Altogether, our results showed a significant relationship between cellular marker in blood and sputum but poor relationship between local and systemic release of cytokines.

Conclusions

COPD is characterized by prominent neutrophilic inflammation and raised IFN-γ production at both bronchial and systemic level. Overproduction of TNF-α at systemic level correlates with disease severity and inversely with body mass index.     

A model of chronic inflammation and pulmonary emphysema after multiple ozone exposures in mice

Oxidative stress plays a role in the pathophysiology of emphysema through the activation of tissue proteases and apoptosis. We examined the effects of ozone exposure by exposing BALB/c mice to either a single 3-h exposure or multiple exposures over 3 or 6 wk, with two 3-h exposures per week. Compared with air-exposed mice, the increase in neutrophils in bronchoalveolar lavage fluid and lung inflammation index was greatest in mice exposed for 3 and 6 wk. Lung volumes were increased in 3- and 6-wk-exposed mice but not in single-exposed. Alveolar space and mean linear intercept were increased in 6- but not 3-wk-exposed mice. Caspase-3 and apoptosis protease activating factor-1 immunoreactivity was increased in the airway and alveolar epithelium and macrophages of 3- and 6-wk-exposed mice. Interleukin-13, keratinocyte chemoattractant, caspase-3, and IFN-γ mRNA were increased in the 6-wk-exposed group, but heme oxygenase-1 (HO-1) mRNA decreased. matrix metalloproteinase-12 (MMP-12) and caspase-3 protein expression increased in lungs of 6-wk-exposed mice. Collagen area increased and epithelial area decreased in airway wall at 3- and 6-wk exposure. Exposure of mice to ozone for 6 wk induced a chronic inflammatory process, with alveolar enlargement and damage linked to epithelial apoptosis and increased protease expression.     

ABCG1 is deficient in alveolar macrophages of GM-CSF knockout mice and patients with pulmonary alveolar proteinosis

Patients with pulmonary alveolar proteinosis (PAP) display impaired surfactant clearance, foamy, lipid-filled alveolar macrophages, and increased cholesterol metabolites within the lung. Neutralizing autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) are also present, resulting in virtual GM-CSF deficiency. We investigated ABCG1 and ABCA1 expression in alveolar macrophages of PAP patients and GM-CSF knockout (KO) mice, which exhibit PAP-like pulmonary pathology and increased pulmonary cholesterol. Alveolar macrophages from both sources displayed a striking similarity in transporter gene dysregulation, consisting of deficient ABCG1 accompanied by highly increased ABCA1. Peroxisome proliferator-activated receptor gamma (PPARgamma), a known regulator of both transporters, was deficient, as reported previously. In contrast, the liver X receptor alpha, which also upregulates both transporters, was highly increased. GM-CSF treatment increased ABCG1 expression in macrophages in vitro and in PAP patients in vivo. Overexpression of PPARgamma by lentivirus-PPARgamma transduction of primary alveolar macrophages, or activation by rosiglitazone, also increased ABCG1 expression. These results suggest that ABCG1 deficiency in PAP and GM-CSF KO alveolar macrophages is attributable to the absence of a GM-CSF-mediated PPARgamma pathway. These findings document the existence of ABCG1 deficiency in human lung disease and highlight a critical role for ABCG1 in surfactant homeostasis.     

Endotoxin down-regulates ABCG5 and ABCG8 in mouse liver and ABCA1 and ABCG1 in J774 murine macrophages: differential role of LXR

Several of the ATP binding cassette (ABC) transporters have recently been shown to play important roles in reverse cholesterol transport (RCT) and prevention of atherosclerosis. In the liver, ABCG5 and ABCG8 have been proposed to efflux sterols into the bile for excretion. ABCG5 and ABCG8 also limit absorption of dietary cholesterol and plant sterols in the intestine. In macrophages, ABCA1 and ABCG1 mediate cholesterol removal from these cells to HDL. Many of these ABC transporters are regulated by the liver X receptor (LXR). We have previously shown that endotoxin (lipopolysaccharide) down-regulates LXR in rodent liver. In the present study, we examined the in vivo and in vitro regulation of these ABC transporters by endotoxin. We found that endotoxin significantly decreased mRNA levels of ABCG5 and ABCG8 in the liver, but not in the small intestine. When endotoxin or cytokines (tumor necrosis factor and interleukin-1) were incubated with J774 murine macrophages, the mRNA levels of ABCA1 were decreased. This effect was rapid and sustained, and was associated with a reduction in ABCA1 protein levels. Endotoxin and cytokines also decreased ABCG1 mRNA levels in J774 cells. Although LXR is a positive regulator of ABCA1 and ABCG1, we did not observe a reduction in protein levels of LXR or in binding of nuclear proteins to an LXR response element in J774 cells. The decrease in ABCG5 and ABCG8 levels in the liver as well as a reduction in ABCA1 and ABCG1 in macrophages during the host response to infection and inflammation coupled with other previously described changes in the RCT pathway may aggravate atherosclerosis.     

BCG-induced chronic pulmonary inflammation and splenomegaly in mice: suppression of PHA-induced proliferation, delayed hypersensitivity to sheep erythrocytes, and chronic pulmonary inflammation by soluble factors from adherent spleen cells

C57BL/6 (B6), but not CBA, mice develop intense chronic granulomatous inflammation (CGI) in the lungs and spleen in response to an iv injection with killed BCG in an oil-in-saline emulsion (BCG-E). Concomitant with the development of CGI, these mice show diminished responsiveness to PHA and LPS, as well as suppression of antibody synthesis and production of delayed hypersensitivity (DH) to sheep erythrocytes (SRBC). Suppression results from the development of adherent, Thy-1^?, Ig^? spleen cells. The present study shows that cells from inflamed spleens of BCG-E-treated B6 mice elaborate factors in vitro which (a) inhibit PHA-induced proliferation of both normal syngeneic and allogencic cells, (b) suppress DH to SRBC in B6 mice, and (c) diminish the intensity of BCG-E-induced CGI in the lungs and spleens of B6 mice. These factors are produced by adherent Thy-1^? cells in BCG-injected mice but not in similarly treated CBA mice. These factors may be important in understanding the control of immunologically mediated chronic inflammation.     

Genetic inactivation of NPC1L1 protects against sitosterolemia in mice lacking ABCG5/ABCG8

Mice lacking Niemann-Pick C1-Like 1 (NPC1L1) (NPC1L1(-/-)mice) exhibit a defect in intestinal absorption of cholesterol and phytosterols. However, wild-type (WT) mice do not efficiently absorb and accumulate phytosterols either. Cell-based studies show that NPC1L1 is a much weaker transporter for phytosterols than cholesterol. In this study, we examined the role of NPC1L1 in phytosterol and cholesterol trafficking in mice lacking ATP-binding cassette (ABC) transporters G5 and G8 (G5/G8(-/-) mice). G5/G8(-/-) mice develop sitosterolemia, a genetic disorder characterized by the accumulation of phytosterols in blood and tissues. We found that mice lacking ABCG5/G8 and NPC1L1 [triple knockout (TKO) mice] did not accumulate phytosterols in plasma and the liver. TKO mice, like G5/G8(-/-) mice, still had a defect in hepatobiliary cholesterol secretion, which was consistent with TKO versus NPC1L1(-/-) mice exhibiting a 52% reduction in fecal cholesterol excretion. Because fractional cholesterol absorption was reduced similarly in NPC1L1(-/-) and TKO mice, by subtracting fecal cholesterol excretion in TKO mice from NPC1L1(-/-) mice, we estimated that a 25g NPC1L1(-/-) mouse may secrete about 4 mumol of cholesterol daily via the G5/G8 pathway. In conclusion, NPC1L1 is essential for phytosterols to enter the body in mice.     

Effects of acute and chronic nitric oxide inhibition in an experimental model of chronic pulmonary allergic inflammation in guinea pigs

Endogenously produced nitric oxide is a recognized regulator of physiological lung events, such as a neurotransmitter and a proinflammatory mediator. We tested the differences between chronic and acute nitric oxide inhibition by N(omega)-nitro-L-arginine methyl ester (L-NAME) treatment in lung mechanics, inflammation, and airway remodeling in an experimental asthma model in guinea pigs. Both acute and chronic L-NAME treatment reduced exhaled nitric oxide in sensitized animals (P < 0.001). Chronic L-NAME treatment increased baseline and maximal responses after antigen challenge of respiratory system resistance and reduced peribronchial edema and mononuclear cells airway infiltration (P < 0.05). Acute administration of L-NAME increased maximal values of respiratory system elastance and reduced mononuclear cells and eosinophils in airway wall (P < 0.05). Chronic ovalbumin exposure resulted in airway wall thickening due to an increase in collagen content (P < 0.005). Chronic nitric oxide inhibition increased collagen deposition in airway wall in sensitized animals (P < 0.05). These data support the hypothesis that in this model nitric oxide acts as a bronchodilator, mainly in proximal airways. Furthermore, chronic nitric oxide inhibition was effective in reducing edema and mononuclear cells in airway wall. However, airway eosinophilic inflammation was unaltered by chronic L-NAME treatment. In addition, nitric oxide inhibition upregulates collagen deposition in airway walls.     

Association of current smoking with airway inflammation in chronic obstructive pulmonary disease and asymptomatic smokers

Background

Inflammation in the airways and lung parenchyma underlies fixed airway obstruction in chronic obstructive pulmonary disease. The exact role of smoking as promoting factor of inflammation in chronic obstructive pulmonary disease is not clear, partly because studies often do not distinguish between current and ex-smokers.

Methods

We investigated airway inflammation in sputum and bronchial biopsies of 34 smokers with chronic obstructive pulmonary disease (9 Global initiative for Chronic Obstructive Lung Disease stage 0, 9 stage I, 10 stage II and 6 stage III) and 26 asymptomatic smokers, and its relationship with past and present smoking habits and airway obstruction.

Results

Neutrophil percentage, interleukin-8 and eosinophilic-cationic-protein levels in sputum were higher in chronic obstructive pulmonary disease (stage I-III) than asymptomatic smokers. Inflammatory cell numbers in bronchial biopsies were similar in both groups. Current smoking correlated positively with macrophages: in bronchial biopsies in both groups, and in sputum in chronic obstructive pulmonary disease. Pack-years smoking correlated positively with biopsy macrophages only in chronic obstructive pulmonary disease.

Conclusion

Inflammatory effects of current smoking may mask the underlying ongoing inflammatory process pertinent to chronic obstructive pulmonary disease. This may have implications for future studies, which should avoid including mixed populations of smokers and ex-smokers.     

Fen1 mutations result in autoimmunity, chronic inflammation and cancers

Functional deficiency of the FEN1 gene has been suggested to cause genomic instability and cancer predisposition. We have identified a group of FEN1 mutations in human cancer specimens. Most of these mutations abrogated two of three nuclease activities of flap endonuclease 1 (FEN1). To demonstrate the etiological significance of these somatic mutations, we inbred a mouse line harboring the E160D mutation representing mutations identified in human cancers. Selective elimination of nuclease activities led to frequent spontaneous mutations and accumulation of incompletely digested DNA fragments in apoptotic cells. The mutant mice were predisposed to autoimmunity, chronic inflammation and cancers. The mutator phenotype results in the initiation of cancer, whereas chronic inflammation promotes the cancer progression. The current work exemplifies the approach of studying the mechanisms of individual polymorphisms and somatic mutations in cancer development, and may serve as a reference in developing new therapeutic regimens through the suppression of inflammatory responses.     

Differential expression and function of ABCG1 and ABCG4 during development and aging

ABCG1 and ABCG4 are highly homologous members of the ATP binding cassette (ABC) transporter family that regulate cellular cholesterol homeostasis. In adult mice, ABCG1 is known to be expressed in numerous cell types and tissues, whereas ABCG4 expression is limited to the central nervous system (CNS). Here, we show significant differences in expression of these two transporters during development. Examination of β-galactosidase-stained tissue sections from Abcg1^−/−LacZ and Abcg4^−/−LacZ knockin mice shows that ABCG4 is highly but transiently expressed both in hematopoietic cells and in enterocytes during development. In contrast, ABCG1 is expressed in macrophages and in endothelial cells of both embryonic and adult liver. We also show that ABCG1 and ABCG4 are both expressed as early as E12.5 in the embryonic eye and developing CNS. Loss of both ABCG1 and ABCG4 results in accumulation in the retina and/or brain of oxysterols, in altered expression of liver X receptor and sterol-regulatory element binding protein-2 target genes, and in a stress response gene. Finally, behavioral tests show that Abcg4^−/− mice have a general deficit in associative fear memory. Together, these data indicate that loss of ABCG1 and/or ABCG4 from the CNS results in changes in metabolic pathways and in behavior.