Anagha Malur

Deletion of PPARγ in Alveolar Macrophages Is Associated with a Th-1 Pulmonary Inflammatory Response

Peroxisome proliferator-activated receptor γ (PPARγ) is constitutively expressed at high levels in healthy alveolar macrophages, in contrast to other tissue macrophages and blood monocytes. PPARγ ligands have been shown to down-regulate IFN-γ-stimulated inducible NO synthase (iNOS) in macrophages. Because NO is an important inflammatory mediator in the lung, we hypothesized that deletion of alveolar macrophage PPARγ in vivo would result in up-regulation of iNOS and other inflammatory mediators. The loss of PPARγ in macrophages was achieved by crossing floxed (+/+) PPARγ mice and a transgenic mouse containing the CRE recombinase gene under the control of the murine M lysozyme promoter (PPARγKO). Alveolar macrophages were harvested by bronchoalveolar lavage (BAL). Lymphocytes (CD8:CD4 ratio = 2.8) were increased in BAL of PPARγKO vs wild-type C57BL6; p ≤ 0.0001. Both iNOS and IFN-γ expression were significantly elevated (p ≤ 0.05) in BAL cells. Th-1 associated cytokines including IL-12 (p40), MIP-1α (CCL3), and IFN inducible protein-10 (IP-10, CXCL10) were also elevated. IL-4 and IL-17A were not detected. To test whether these alterations were due to the lack of PPARγ, PPARγ KO mice were intratracheally inoculated with a PPARγ lentivirus construct. PPARγ transduction resulted in significantly decreased iNOS and IFN-γ mRNA expression, as well as reduced BAL lymphocytes. These results suggest that lack of PPARγ in alveolar macrophages disrupts lung homeostasis and results in a Th1-like inflammatory response.

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 γ (PPARγ), a known regulator of both transporters, was deficient, as reported previously. In contrast, the liver X receptor α, 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 PPARγ by lentivirus-PPARγ 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 PPARγ pathway. These findings document the existence of ABCG1 deficiency in human lung disease and highlight a critical role for ABCG1 in surfactant homeostasis.

An open-label trial of rituximab therapy in pulmonary alveolar proteinosis

Rituximab, a monoclonal antibody directed against the B-lymphocyte antigen CD20, has shown promise in several autoimmune disorders. Pulmonary alveolar proteinosis (PAP) is an autoimmune disorder characterised by autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF). An open-label, proof-of-concept phase II clinical trial was conducted in 10 PAP patients. The intervention consisted of two intravenous infusions of rituximab (1,000 mg) 15 days apart. Bronchoalveolar lavage (BAL) fluid and peripheral blood samples were collected. The primary outcome was improvement in arterial blood oxygenation. Both arterial oxygen tension and alveolar–arterial oxygen tension difference in room air improved in seven out of the nine patients completing the study. Lung function and high-resolution computed tomography scans, which were secondary outcomes, also improved. Peripheral blood CD19+ B-lymphocytes decreased from mean±sem 15±2% to <0.05% (n=10) 15 days post-therapy. This decrease persisted for 3 months in all patients; at 6 months, CD19+ B-cells were detected in four out of seven patients (5±2%). Total anti-GM-CSF immunoglobulin (Ig)G levels from baseline to 6 months were decreased in BAL fluids (n=8) but unchanged in sera (n=9). In this PAP cohort: 1) rituximab was well-tolerated and effectively ameliorated lung disease; and 2) reduction in anti-GM-CSF IgG levels in the lung correlated with disease changes, suggesting that disease pathogenesis is related to autoantibody levels in the target organ.

Nitric oxide regulation of asthmatic airway inflammation with segmental allergen challenge.

BACKGROUND Despite evidence of increased nitric oxide (NO) in asthmatic compared with healthy individuals, the role of NO in airway inflammation is unclear. OBJECTIVE The purpose of the study was to determine the in vivo effects of localized allergen challenge on airway NO levels and transcription factor activation. METHODS In this study localized allergen challenge was used as a model of asthmatic exacerbation to determine the relationship of NO to airway inflammation. RESULTS With allergen challenge, asthmatic patients had a rise in airway NO levels, whereas NO levels in healthy controls did not change. The increased NO in asthma with allergen challenge compared with healthy control subjects was associated with an increase in inflammatory cytokines (GM-CSF and macrophage inflammatory protein-1) in epithelial lining fluid and eosinophilic infiltrate in bronchoalveolar lavage fluid (BAL) and biopsy specimens. To investigate the mechanisms of cytokine gene expression, activation of the transcription factors activator protein-1 and nuclear factor-kappaB (NF-kappaB) in cells from BAL were evaluated. Activator protein-1 was not activated before or after local allergen challenge. In contrast, NF-kappaB activation was less in BAL cells from asthmatic patients with increased NO in comparison with controls. CONCLUSION Our studies are the first to suggest an inverse correlation between NF-kappaB and airway NO in a localized segmental allergen challenge model in allergic asthmatic patients. The current study demonstrates that activation of the inflammatory response (eg, cytokines, cellular infiltrate) in allergic asthmatic patients is temporally associated with increased airway NO. We propose that NO that is up-regulated by cytokines is part of an autoregulatory feedback loop (ie, allergen challenge stimulates inflammatory cytokine production, which in turn stimulates NO production, and NO down-regulates cytokine production).

The Prostacyclin Analogue Treprostinil Blocks NFκB Nuclear Translocation in Human Alveolar Macrophages

Primary pulmonary hypertension (PPH) is characterized by increased pulmonary arterial pressure and vascular resistance. We and others have observed that inflammatory cytokines and infiltrates are present in the lung tissue, but the significance is uncertain. Treprostinil (TRE), a prostacyclin analogue with extended half-life and chemical stability, has shown promise in the treatment of PPH. We hypothesize that TRE might exert beneficial effects in PPH by antagonizing inflammatory cytokine production in the lung. Here we show that TRE dose-dependently inhibits inflammatory cytokine (tumor necrosis factor-α, interleukin-1β, interleukin-6, and granulocyte macrophage colony-stimulating factor) secretion and gene expression by human alveolar macrophages. TRE blocks NFκB activation, but IκB-α phosphorylation and degradation are unaffected. Moreover, TRE does not affect the formation of the NFκB·DNA complex but blocks nuclear translocation of p65. These results are the first to illustrate the anti-cytokine actions of TRE in down-regulating NFκB, not through its inhibitory component or by direct binding but by blocking nuclear translocation. These data indicate that inflammatory mechanisms may be important in the pathogenesis of PPH and cytokine antagonism by blocking NFκB may contribute to the efficacy of TRE therapy in PPH.

Novel Murine Model of Chronic Granulomatous Lung Inflammation Elicited by Carbon Nanotubes.

Lung granulomas are associated with numerous conditions, including inflammatory disorders, exposure to environmental pollutants, and infection. Osteopontin is a chemotactic cytokine produced by macrophages, and is implicated in extracellular matrix remodeling. Furthermore, osteopontin is up-regulated in granulomatous disease, and osteopontin null mice exhibit reduced granuloma formation. Animal models currently used to investigate chronic lung granulomatous inflammation bear a pathological resemblance, but lack the chronic nature of human granulomatous disease. Carbon nanoparticles are generated as byproducts of combustion. Interestingly, experimental exposures to carbon nanoparticles induce pulmonary granuloma-like lesions. However, the recruited cellular populations and extracellular matrix gene expression profiles within these lesions have not been explored. Because of the rapid resolution of granulomas in current animal models, the mechanisms responsible for persistence have been elusive. To overcome the limitations of previous models, we investigated whether a model using multiwall carbon nanoparticles would resemble chronic human lung granulomatous inflammation. We hypothesized that pulmonary exposure to multiwall carbon nanoparticles would induce granulomas, elicit a macrophage and T-cell response, and mimic other granulomatous disorders with an up-regulation of osteopontin. This model demonstrates: (1) granulomatous inflammation, with macrophage and T-cell infiltration; (2) resemblance to the chronicity of human granulomas, with persistence up to 90 days; and (3) a marked elevation of osteopontin, metalloproteinases, and cell adhesion molecules in granulomatous foci isolated by laser-capture microdissection and in alveolar macrophages from bronchoalveolar lavage. The establishment of such a model provides an important platform for mechanistic studies on the persistence of granuloma.

Targeted PPARγ deficiency in alveolar macrophages disrupts surfactant catabolism

Surfactant accumulates in alveolar macrophages of granulocyte-macrophage colony-stimulating factor (GM-CSF) knockout (KO) mice and pulmonary alveolar proteinosis (PAP) patients with a functional loss of GM-CSF resulting from neutralizing anti–GM-CSF antibody. Alveolar macrophages from PAP patients and GM-CSF KO mice are deficient in peroxisome proliferator-activated receptor-γ (PPARγ) and ATP-binding cassette (ABC) lipid transporter ABCG1. Previous studies have demonstrated that GM-CSF induces PPARγ. We therefore hypothesized that PPARγ promotes surfactant catabolism through regulation of ABCG1. To address this hypothesis, macrophage-specific PPARγ (MacPPARγ) knockout mice were utilized. MacPPARγ KO mice develop foamy, lipid-engorged Oil Red O positive alveolar macrophages. Lipid analyses revealed significant increases in the cholesterol and phospholipid contents of MacPPARγ KO alveolar macrophages and extracellular bronchoalveolar lavage (BAL)–derived fluids. MacPPARγ KO alveolar macrophages showed decreased expression of ABCG1 and a deficiency in ABCG1-mediated cholesterol efflux to HDL. Lipid metabolism may also be regulated by liver X receptor (LXR)–ABCA1 pathways. Interestingly, ABCA1 and LXRβ expression were elevated, indicating that this pathway is not sufficient to prevent surfactant accumulation in alveolar macrophages. These results suggest that PPARγ mediates a critical role in surfactant homeostasis through the regulation of ABCG1.

An inverse relationship between peroxisome proliferator–activated receptor γ and allergic airway inflammation in an allergen challenge model

BACKGROUND Peroxisome proliferator-activated receptor gamma (PPAR-gamma) expression has not been evaluated in bronchoalveolar lavage (BAL) cells from allergic asthmatic patients. OBJECTIVE To determine whether inappropriate down-regulation of PPAR-gamma in alveolar macrophages may contribute to persistent airway inflammation in allergic asthma. METHODS We used segmental allergen challenge as a model of in vivo experimental allergic asthmatic exacerbation and airway inflammation. PPAR-y gene expression was evaluated at baseline and 24 hours later in asthmatic patients and controls using real-time polymerase chain reaction. Immunofluorescence was used to determine cellular location of the PPAR-gamma protein. RESULTS We demonstrate for the first time to our knowledge that PPAR-gamma messenger RNA and protein, which are highly expressed in alveolar macrophages of healthy individuals, are significantly reduced in asthmatic patients after segmental allergen challenge. In allergic asthmatic patients (n=9), PPAR-gamma gene expression decreased significantly from baseline to postchallenge BAL (median decrease, 45%; P = .008). Furthermore, immunofluorescence staining demonstrated that PPAR-gamma protein was associated with alveolar macrophages and not with inflammatory eosinophils and neutrophils. CONCLUSION Results implicate down-regulation of PPAR-gamma in BAL cells as a potential factor in dysregulation of lung homeostasis in asthmatic patients. The present findings suggest that PPAR-gamma agonists could have a future role in asthma therapy and warrant further study.

Restoration of PPARγ reverses lipid accumulation in alveolar macrophages of GM-CSF knockout mice

Pulmonary alveolar proteinosis (PAP) is a lung disease characterized by a deficiency of functional granulocyte macrophage colony-stimulating factor (GM-CSF) resulting in surfactant accumulation and lipid-engorged alveolar macrophages. GM-CSF is a positive regulator of PPARγ that is constitutively expressed in healthy alveolar macrophages. We previously reported decreased PPARγ and ATP-binding cassette transporter G1 (ABCG1) levels in alveolar macrophages from PAP patients and GM-CSF knockout (KO) mice, suggesting PPARγ and ABCG1 involvement in surfactant catabolism. Because ABCG1 represents a PPARγ target, we hypothesized that PPARγ restoration would increase ABCG1 and reduce macrophage lipid accumulation. Upregulation of PPARγ was achieved using a lentivirus expression system in vivo. GM-CSF KO mice received intratracheal instillation of lentivirus (lenti)-PPARγ or control lenti-eGFP. Ten days postinstillation, 79% of harvested alveolar macrophages expressed eGFP, demonstrating transduction. Alveolar macrophages showed increased PPARγ and ABCG1 expression after lenti-PPARγ instillation, whereas PPARγ and ABCG1 levels remained unchanged in lenti-eGFP controls. Alveolar macrophages from lenti-PPARγ-treated mice also exhibited reduced intracellular phospholipids and increased cholesterol efflux to HDL, an ABCG1-mediated pathway. In vivo instillation of lenti-PPARγ results in: 1) upregulating ABCG1 and PPARγ expression of GM-CSF KO alveolar macrophages, 2) reducing intracellular lipid accumulation, and 3) increasing cholesterol efflux activity.

Differential regulation of human blood monocyte and alveolar macrophage inflammatory cytokine production by nitric oxide

BACKGROUND Nitric oxide (NO) has been associated with airway inflammation in asthma. Our previous work suggests that NO functions in an anti-inflammatory capacity through downregulation of stimulated cytokine secretion by normal human alveolar macrophages. Functional differences between alveolar macrophages and blood monocytes are thought to be related to maturation. OBJECTIVE The purpose of this study was to determine the effect of NO on stimulated cytokine production by monocytes from asthmatics and normal healthy controls. METHODS Monocytes and alveolar macrophages were obtained from normal volunteers (n = 13) and asthmatics with atopy (n = 7). Monocyte and alveolar macrophage cultures were stimulated with 0.5 microgram/mL lipopolysaccharide +/- 1.0 mM DETA NONOate (releases NO in culture with t1/2 = 20 hours at 37 degrees C) and incubated for 24 hours. Cell-free supernatants were collected and assayed by ELISA for tumor necrosis factor-alpha (TNF) and granulocyte macrophage colony stimulating factor (GM-CSF). RESULTS Nitric oxide did not inhibit TNF production in monocytes of asthmatics and normals (mean +/- SEM % TNF stimulation = 19.6 +/- 9.7). Similar to previous results, NO did inhibit alveolar macrophages (% TNF suppression = 60.6 +/- 4.4). To determine whether this differential effect of NO on the two cell populations was related to maturation, monocytes were matured by culture for 7 days. The in vitro matured monocytes demonstrated 51.7 +/- 7.9% suppression of TNF. For each cell population, the responses of the asthmatics and healthy controls were not different. The differential effect is not cytokine specific since similar results were obtained with GM-CSF. CONCLUSION These results demonstrate a differential effect of NO on monocyte and alveolar macrophages cytokine regulation and this effect may be related to the state of maturation.