Bronchoalveolar Lavage Alveolar Macrophages Research Articles

Similar programmed death ligand 1 (PD-L1) expression profile in patients with mild COPD and lung cancer

Programmed Death Ligand 1 (PD-L1) is crucial in regulating the immunological tolerance in non-small cell lung cancer (NSCLC). Alveolar macrophage (AM)-derived PD-L1 binds to its receptor, PD-1, on surveilling lymphocytes, leading to lymphocyte exhaustion. Increased PD-L1 expression is associated with cigarette smoke (CS)-exposure. However, the PD-L1 role in CS-associated lung diseases associated with NSCLC, such as chronic obstructive pulmonary disease (COPD), is still unclear. In two different cohorts of ever smokers with COPD or NSCLC, and ever and never smoker controls, we evaluated PD-L1 expression: (1) via cutting-edge digital spatial proteomic and transcriptomic profiling (Geomx) of formalin-fixed paraffin-embedded (FFPE) lung tissue sections (n = 19); and (2) via triple immunofluorescence staining of bronchoalveolar lavage (BAL) AMs (n = 83). PD-L1 mRNA expression was also quantified in BAL AMs exposed to CS extract. PD-L1 expression was increased in the bronchiolar wall, parenchyma, and vascular wall from mild-moderate (GOLD 1–2) COPD patients compared to severe-very severe (GOLD 3–4) COPD patients and controls. Within all the COPD patients, PD-L1 protein expression was associated with upregulation of genes involved in tumor progression and downregulation of oncosuppressive genes, and strongly directly correlated with the FEV1% predicted, indicating higher PD-L1 expression in the milder vs. more severe COPD stages. In bronchioles, PD-L1 levels were strongly directly correlated with the number of functionally active AMs. In BAL, we confirmed that AMs from patients with both GOLD 1–2 COPD and NSCLC had the highest and similar, PD-L1 expression levels versus all the other groups, independently from active cigarette smoking. Intriguingly, AMs from patients with more severe COPD had reduced AM PD-L1 expression compared to patients with mild COPD. Acute CS extract stimulation increased PD-L1 mRNA expression only in never-and not in ever-smoker AMs. Lungs from patients with mild COPD and NSCLC are characterized by a similar strong PD-L1 expression signature in bronchioles and functionally active AMs compared to patients with severe COPD and controls. Active smoking does not affect PD-L1 levels. These observations represent a new resource in understanding the innate immune mechanisms underlying the link between COPD and lung cancer onset and progression and pave the way to future studies focused on the mechanisms by which CS promotes tumorigenesis and COPD.

Anakinra reduces lung inflammation in experimental acute lung injury.

IntroductionAcute respiratory distress syndrome (ARDS) is a severe form of acute lung injury (ALI) resulting in life‐threatening hypoxaemia. Although ARDS can be caused by a variety of pathogens or major trauma, it is best known as the major cause of mortality in COVID‐19 patients. Since ARDS is often associated with dysregulated inflammatory immune responses, immunomodulatory approaches represent a possible treatment option. The objective of this study was to evaluate the therapeutic potential of interleukin (IL)‐1 blockade using Anakinra in a mouse model of lipopolysaccharide (LPS)‐induced ALI.MethodsWe evaluated the effects of a daily subcutaneous Anakinra treatment in a mouse model of LPS‐induced ALI. We monitored body weight to assess the general health status of the mice. Two days after ALI induction, we evaluated the inflammatory cytokine MIP‐2 as well as protein levels in bronchoalveolar lavage (BAL) fluids. Two and nine days after ALI induction, we evaluated infiltrating leukocytes in BAL fluid and lung tissue.ResultsAnakinra treatment reduced ALI‐induced weight loss compared to nontreated groups. At Day 2, Anakinra treatment reduced levels of MIP‐2 and protein in BAL fluids and reduced frequencies of NK cells and neutrophils in the lung tissue. Nine days after ALI induction, Anakinra treated mice displayed reduced levels of neutrophils and alveolar macrophages in BAL fluids.ConclusionsIL‐1 blockade using Anakinra reduced classical hallmarks of inflammation in a mouse model of ALI. Our data support ongoing and future research on the evaluation of Anakinra as a potential treatment option in ARDS.

Clinical outcomes and lipid-laden macrophages in Electronic cigarette/Vaping product use-Associated Lung Injury (EVALI) in a South Texas population

Objective: To describe the clinical features, proportion of lipid-laden alveolar macrophages in bronchoalveolar lavage (BAL), and short-term and 6-month to 12-month outcomes of patients with Electronic cigarette/Vaping product use-Associated Lung Injury (EVALI).Methods: Retrospective review of clinical characteristics, radiographs, and BAL samples for all patients with a history of vaping who presented with acute hypoxemic respiratory failure to the University Hospital in San Antonio, Texas from 9/2019 to 6/2020 was performed.Results: We report 16 cases (15 men; median age, 30 years [range 19-75]) of EVALI with a history of vaping Tetrahydrocannabinol (THC), nicotine, or both. The most common presenting symptoms were tachycardia, dyspnea, cough, and fever. All patients required supplemental oxygen, including two who required noninvasive positive pressure ventilation, and five who required mechanical ventilation. All 16 patients had bilateral ground-glass opacities (GGO) with peripheral sparing on chest computerizedtomography (CT). Cultures were negative, except for one patient who tested positive for rhinovirus. COVID-19 PCR was done in one individual which was negative. Cytology demonstrated lipid-laden macrophages on Oil-Red-O stain on fresh (i.e., without fixative) BAL in the majority of patients (N = 12) with a mean lipid-saturation percentage of 78% [range, 44%-100%] and the mean Colombo count of 194 [range, 101-359]. Fifteen patients were treated with systemic corticosteroids. The median length of hospital stay was 10 days. At discharge, three patients required supplemental oxygen. Eight of those who had follow-up imaging showed resolution of GGO. One patient had a relapse of symptoms and was again treated with systemic corticosteroids and mycophenolate, with resolution of symptoms. Fourteen patients who were evaluated after discharge denied vaping post-discharge (two patients were not able to be contacted and did not keep follow-up appointments).Conclusions: Successful diagnosis and management of EVALI requires a high clinical suspicion, thorough evaluation to rule out infectious etiologies, and aggressive treatment with systemic corticosteroids, along with sustained abstinence from vaping.

Pulmonary fibrosis in Fra-2 transgenic mice is associated with decreased numbers of alveolar macrophages and increased susceptibility to pneumococcal pneumonia.

Idiopathic pulmonary fibrosis (IPF) is a deadly condition characterized by progressive respiratory dysfunction. Exacerbations due to airway infections are believed to promote disease progression, and presence of Streptococcus in the lung microbiome has been associated with the progression of IPF and mortality. The aim of this study was to analyze the effect of lung fibrosis on susceptibility to pneumococcal pneumonia and bacteremia. The effects of subclinical (low dose) infection with Streptococcus pneumoniae were studied in a well characterized fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of spontaneous, progressive pulmonary fibrosis. Forty-eight hours after transnasal infection with S. pneumoniae, bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood, and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Lung compliance and arterial blood gases were assessed. In contrast to wildtype mice, low dose lung infection with S. pneumoniae in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load, and bacteremia. BAL alveolar macrophages were reduced in Fra-2 TG mice compared to the corresponding WT mice. Proinflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, and CXCL1) were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Lung compliance was decreased in Fra-2 TG mice following low dose infection with S. pneumoniae. Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.

EVALI-ASSOCIATED LIPID LADEN MACROPHAGES: CASE SERIES IN SOUTH TEXAS

SESSION TITLE: Tobacco Cessation and Prevention Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: To describe the clinical features, proportion of lipid-laden alveolar macrophages in bronchoalveolar lavage (BAL), and outcomes of patients with Electronic cigarette/Vaping-Associated Lung Injury (EVALI). METHODS: Clinical characteristics, radiographs, and BAL samples were reviewed for all patients with a history of vaping who presented with acute hypoxemic respiratory failure to the University Hospital in San Antonio, Texas from 9/2019 to 5/2020. RESULTS: We report 10 cases (9 men; median age, 32 years [range, 22-44]), with a history of vaping Tetrahydrocannabinol (THC). The most common signs/symptoms were tachycardia (N=10), dyspnea (N=9), cough (N=6), productive cough (N=3), and fever (N=6) occurring at least 1-28 days (median, 7 days) prior to presentation. All patients required supplemental oxygen, 5 required high-flow nasal cannula, and 3 required mechanical ventilation. Bilateral ground-glass opacities (GGO) with peripheral sparing on computerized tomography (CT) chest were noted in all patients. Sputum and blood cultures were all negative, except for one who had rhinovirus on the respiratory viral panel. Due to high clinical suspicion of EVALI, bronchoscopy was performed and cytology demonstrated lipid-laden macrophages on the oil-red-O stain on BAL in all patients (N=10) with a mean lipid-saturation percentage of 79% [range, 44-100%] and the mean Colombo count of 194 [range, 101-359]. All but one patient was treated with systemic corticosteroids (5 before bronchoscopy, 4 following bronchoscopy). Medium time to steroid administration was 3 days with an outpatient taper. The median length of stay was 10 days [range, 4-25 days]. No patients required supplemental oxygen at discharge. All 7 of those who had follow-up imaging at 6-12 weeks showed resolution of GGO and all 10 patients reported symptom resolution and no further vaping after discharge. CONCLUSIONS: We present a case series describing the diagnosis and successful management of patients with EVALI based on maintaining a high clinical suspicion, appropriate diagnostic testing including labs, imaging, and bronchoscopy with BAL stains for lipid-laden macrophages, as well as routine studies to rule out infectious etiologies, followed by early and aggressive treatment with systemic corticosteroids. CLINICAL IMPLICATIONS: Currently, no particular test has been identified to confirm EVALI. Diagnosis is based on a high clinical suspicion in patients with vaping exposure, as well as obtaining classic imaging and laboratory tests to rule out other potential etiologies. Bronchoscopy and BAL with lipid stains in the appropriate clinical setting may assist clinicians in early diagnosis and in decisions regarding initiating aggressive corticosteroid therapy in these patients. Counseling about vaping cessation is also critical for these patients. DISCLOSURES: Unrestricted grant for CME research relationship with AstraZeneca Please note: $20001 - $100000 Added 05/31/2020 by Sandra Adams, source=Web Response, value=Grant/Research Support Unrestricted grant for CME research relationship with Boehringer Ingelheim Please note: $20001 - $100000 Added 05/31/2020 by Sandra Adams, source=Web Response, value=Grant/Research Support Unrestricted grant for CME research relationship with GlaxoSmithKline Please note: $20001 - $100000 Added 05/31/2020 by Sandra Adams, source=Web Response, value=Grant/Research Support Unrestricted grant for CME research relationship with Sunovion Please note: $20001 - $100000 Added 05/31/2020 by Sandra Adams, source=Web Response, value=Grant/Research Support Support for continuing medical education relationship with Mylan/Theravance Please note: $1001 - $5000 Added 05/31/2020 by Sandra Adams, source=Web Response, value=nebulizer/meds for teaching No relevant relationships by Moeezullah Beg, source=Web Response No relevant relationships by Mahnoor Mir, source=Web Response No relevant relationships by Jay Peters, source=Web Response

Comparison of Oral, Intranasal and Aerosol Administration of Amiodarone in Rats as a Model of Pulmonary Phospholipidosis.

‘Foamy’ alveolar macrophages (FAM) observed in nonclinical toxicology studies during inhaled drug development may indicate drug-induced phospholipidosis, but can also derive from adaptive non-adverse mechanisms. Orally administered amiodarone is currently used as a model of pulmonary phospholipidosis and it was hypothesized that aerosol administration would produce phospholipidosis-induced FAM that could be characterized and used in comparative inhalation toxicology. Han-Wistar rats were given amiodarone via (1) intranasal administration (6.25 mg/kg) on two days, (2) aerosol administration (3 mg/kg) on two days, (3) aerosol administration (10 mg/kg) followed by three days of 30 mg/kg or (4) oral administration (100 mg/kg) for 7 days. Alveolar macrophages in bronchoalveolar lavage were evaluated by differential cell counting and high content fluorescence imaging. Histopathology and mass-spectrometry imaging (MSI) were performed on lung slices. The higher dose aerosolised amiodarone caused transient pulmonary inflammation (p < 0.05), but only oral amiodarone resulted in FAM (p < 0.001). MSI of the lungs of orally treated rats revealed a homogenous distribution of amiodarone and a putative phospholipidosis marker, di-22:6 bis-monoacylglycerol, throughout lung tissue whereas aerosol administration resulted in localization of both compounds around the airway lumen. Thus, unlike oral administration, aerosolised amiodarone failed to produce the expected FAM responses.

Alveolar Macrophage Dysfunction and Increased PD-1 Expression During Chronic SIV Infection of Rhesus Macaques.

HIV infected individuals have been shown to be pre-disposed to pulmonary infections even while receiving anti-retroviral therapy. Alveolar macrophages (AMs) play a critical role in lung innate immunity, but contradictory results have been reported regarding their functionality following HIV infection. Here, using the SIV rhesus macaque model, we document the effect of SIV infection on the phenotypic and functional properties of AMs. Following infection with SIVmac251, AMs in bronchoalveolar lavage (BAL) sampled over 2- to 20-weeks post-infection (wpi) were compared to those in BAL samples from naïve macaques. AM expression of proinflammatory cytokines TNF-α, IL-6, IL-1β, and chemokine RANTES drastically increased 2-wpi compared to AMs of naïve macaques (p < 0.0001 for all), but dropped significantly with progression to chronic infection. Phagocytic activity of AMs 2-and 4-wpi was elevated compared to AMs of naive animals (p = 0.0005, p = 0.0004, respectively) but significantly decreased by 12-wpi (p = 0.0022, p = 0.0019, respectively). By 20-wpi the ability of AMs from chronically infected animals to perform SIV-specific antibody-dependent phagocytosis (ADP) was also diminished (p = 0.028). Acute SIV infection was associated with increased FcγRIII expression which subsequently declined with disease progression. Frequency of FcγRIII+ AMs showed a strong trend toward correlation with SIV-specific ADP, and at 2-wpi FcγRIII expression negatively correlated with viral load (r = −0.6819; p = 0.0013), suggesting a contribution to viremia control. Importantly, PD-1 was found to be expressed on AMs and showed a strong trend toward correlation with plasma viral load (r = 0.8266; p = 0.058), indicating that similar to over-expression on T-cells, PD-1 expression on AMs may also be associated with disease progression. Further, AMs predominantly expressed PD-L2, which remained consistent over the course of infection. PD-1 blockade enhanced SIV-specific ADP by AMs from chronic infection indicating that the PD-1/PD-L2 pathway may modulate functional activity of AMs at that stage. These findings provide new insight into the dynamics of SIV infection leading to AM dysfunction and alteration of pulmonary innate immunity. Our results suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in HIV-infected individuals.

Deletion of PIKfyve alters alveolar macrophage populations and exacerbates allergic inflammation in mice.

Alveolar macrophages (AMs) are specialized tissue-resident macrophages that orchestrate the immune responses to inhaled pathogens and maintain organ homeostasis of the lung. Dysregulation of AMs is associated with allergic inflammation and asthma. Here, we examined the role of a phosphoinositide kinase PIKfyve in AM development and function. Mice with conditionally deleted PIKfyve in macrophages have altered AM populations. PIKfyve deficiency results in a loss of AKT activation in response to GM-CSF, a cytokine critical for AM development. Upon exposure to house dust mite extract, mutant mice display severe lung inflammation and allergic asthma accompanied by infiltration of eosinophils and lymphoid cells. Moreover, they have defects in production of retinoic acid and fail to support incorporation of Foxp3+ Treg cells in the lung, resulting in exacerbation of lung inflammation. Thus, PIKfyve plays a role in preventing excessive lung inflammation through regulating AM function.

Analysis of predictive parameters for the development of radiation-induced pneumonitis.

INTRODUCTION:Prevention and effective treatment of radiation-induced pneumonitis (RP) could facilitate greater use of radiation therapy (RT) for lung cancer. The purpose of this study was to determine clinical parameters useful for early prediction of RP.METHODS:Blood sampling, pulmonary function testing, chest computed tomography, and bronchoalveolar lavage (BAL) were performed in patients with pathologically confirmed lung cancer who had completed ≥60 Gy of RT, at baseline, shortly after RT, and at 1 month posttreatment.RESULTS:By 3 months post-RT, 11 patients developed RP (RP group) and the remaining 11 patients did not (NRP group). RT significantly increased total cell counts and alveolar macrophages in BAL of the NRP group, whereas lymphocyte count was increased in both groups. Matrix metallopeptidase-9 (MMP-9) increased and vascular endothelial growth factor decreased significantly in the BAL fluid (BALF) of the RP group following RT. Serum surfactant protein D (SP-D) increased significantly in the NRP group. SP-D in BALF from the RP group increased significantly with a subsequent increase in serum SP-D. Pulmonary dilution decreased similarly in both groups of patients.CONCLUSIONS:Increased SP-D in BALF, rather than that in serum, could be useful biomarkers in predicting RP. The MMP-9 in BALF might play a role in the pathogenesis of RP. Pulmonary dilution test may not be predictive of the development of RP.

T-cell activation and HLA-regulated response to smoking in the deep airways of patients with multiple sclerosis

Cigarette smoking is a risk factor for multiple sclerosis (MS), and the risk is further multiplied for HLA-DRB1*15+ smokers. To define the smoke-induced immune responses in the lung we performed bronchoscopy with bronchoalveolar lavage (BAL) on smokers and non-smokers, both MS-patients and healthy volunteers. In the BAL, non-smokers with MS showed an increased preformed CD40L expression in CD4+ T-cells while smokers displayed an increase in proliferating (Ki-67+) T-cells. In addition, our results confirm that smoking induces an increase of alveolar macrophages in BAL, and further defined a significant attenuation of this response in carriers of the HLA-DRB1*15 allele, in both MS patients and healthy controls. This first systematic investigation of the immune response in the lungs of smokers and non-smokers diagnosed with MS, thus suggests an MS-associated lung T-cell phenotype, involvement of a specific T-cell response to smoke, and a genetic regulation of the macrophage response.

Brief Glutamine Pretreatment Increases Alveolar Macrophage CD163/Heme Oxygenase-1/p38-MAPK Dephosphorylation Pathway and Decreases Capillary Damage but Not Neutrophil Recruitment in IL-1/LPS-Insufflated Rats.

BackgroundGlutamine (GLN) attenuates acute lung injury (ALI) but its effect on alveolar macrophages is unknown. We hypothesized that GLN pretreatment would induce the anti-inflammatory CD163/heme oxygenase (HO)-1/p38-MAPK dephosphorylation pathway in alveolar macrophages and reduce ALI in rats insufflated with interleukin-1 (IL-1) and lipopolysaccharide (LPS).MethodsMale Sprague-Dawley rats were randomized to the following groups: GLN-IL-1/LPS-, GLN+IL-1/LPS-, GLN-IL-1/LPS+, and GLN+IL-1/LPS+. GLN pretreatment was given via gavage (1g/kg L-alanyl-L-glutamine) daily for 2 days. ALI was subsequently induced by insufflating 50ng IL-1 followed by 5mg/kg E.coli LPS. After 24h, bronchoalveolar lavage (BAL) protein, lactate dehydrogenase (LDH) and neutrophil concentrations were analyzed. BAL alveolar macrophage CD163+ expression, HO-1 and p38-MAPK concentrations were measured, as well as alveolar macrophage tumor necrosis factor (TNF)-α and interleukin (IL)-10 concentrations. Histology and immunofluorescence studies were also performed.ResultsFollowing IL-1/LPS insufflation, GLN pretreated rats had significantly decreased BAL protein and LDH concentrations, but not BAL neutrophil counts, compared to non-GLN pretreated rats. The number of alveolar macrophages and the number of CD163+ macrophages were significantly increased in GLN pretreated IL-1/LPS-insufflated rats compared to non-GLN pretreated, IL-1/LPS-insufflated rats. GLN pretreatment before IL-1/LPS also significantly increased HO-1 concentrations and dephosphorylated p38-MAPK levels but not cytokine levels in alveolar macrophages. Immunofluorescence localized CD163 and HO-1 in alveolar macrophages.ConclusionShort-term GLN pretreatment activates the anti-inflammatory CD163/HO-1/p38-MAPK dephosphorylation pathway of alveolar macrophages and decreases capillary damage but not neutrophil recruitment in IL-1/LPS-insufflated rats.

Binge ethanol exposure alters alveolar macrophage activation in burn-injured mice (INC5P.329)

Abstract Alcohol intoxication is involved in 50% of all burn injuries, resulting in an increased risk of lung infection and failure, relative to burn alone. Our work has shown that in a mouse model of episodic binge ethanol exposure followed by burn injury there is prolonged pulmonary inflammation, relative to burn alone. The magnitude of pulmonary inflammation is mediated in part by alveolar macrophages (AMs), thus we examined the effect of ethanol on AM activation. Thirty minutes after binge ethanol exposure, mice were anesthetized and given a 15% total body surface area dorsal scald injury. At 24 and 72 hrs post-injury bronchoalveolar lavage AM granularity was assessed by flow cytometry using side scatter (SSC). At 24 hrs, 12% of AMs from burn alone had high SSC relative to 1.5% of AMs from sham groups (p&amp;lt;0.05). Only 6% of AMs from combined injured had high SSC. By 72 hrs, 3% of AMs from both burn groups had high SSC. Anti-inflammatory M2 phenotype of high SSC AMs was assessed using M2 marker CD206. At 24 hrs, there was no change in CD206. At 72 hrs, AMs from burn alone had a 43% increase in CD206 relative to sham groups. Giving ethanol prior to burn further elevated the expression of this marker by 46% over burn alone. These data indicate ethanol exposure prior to burn injury can differentially alter AM phenotype at early and later time points.

Alveolar macrophages of GM-CSF knockout mice exhibit mixed M1 and M2 phenotypes

BackgroundActivin A is a pleiotrophic regulatory cytokine, the ablation of which is neonatal lethal. Healthy human alveolar macrophages (AMs) constitutively express activin A, but AMs of patients with pulmonary alveolar proteinosis (PAP) are deficient in activin A. PAP is an autoimmune lung disease characterized by neutralizing autoantibodies to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). Activin A can be stimulated, however, by GM-CSF treatment of AMs in vitro. To further explore pulmonary activin A regulation, we examined AMs in bronchoalveolar lavage (BAL) from wild-type C57BL/6 compared to GM-CSF knockout mice which exhibit a PAP-like histopathology. Both human PAP and mouse GM-CSF knockout AMs are deficient in the transcription factor, peroxisome proliferator activated receptor gamma (PPARγ).ResultsIn sharp contrast to human PAP, activin A mRNA was elevated in mouse GM-CSF knockout AMs, and activin A protein was increased in BAL fluid. Investigation of potential causative factors for activin A upregulation revealed intrinsic overexpression of IFNγ, a potent inducer of the M1 macrophage phenotype, in GM-CSF knockout BAL cells. IFNγ mRNA was not elevated in PAP BAL cells. In vitro studies confirmed that IFNγ stimulated activin A in wild-type AMs while antibody to IFNγ reduced activin A in GM-CSF knockout AMs. Both IFNγ and Activin A were also reduced in GM-CSF knockout mice in vivo after intratracheal instillation of lentivirus-PPARγ compared to control lentivirus vector. Examination of other M1 markers in GM-CSF knockout mice indicated intrinsic elevation of the IFNγ-regulated gene, inducible Nitrogen Oxide Synthetase (iNOS), CCL5, and interleukin (IL)-6 compared to wild-type. The M2 markers, IL-10 and CCL2 were also intrinsically elevated.ConclusionsData point to IFNγ as the primary upregulator of activin A in GM-CSF knockout mice which in addition, exhibit a unique mix of M1-M2 macrophage phenotypes.

Recruitment of Alveolar Macrophages in Bronchoalveolar Lavage (BAL) From Patients With Interstitial Lung Diseases, Analysis of the Mechanisms Involved

SESSION TYPE: ILD Posters

Oil red O stain of alveolar macrophages is an effective screening test for gastroesophageal reflux disease in lung transplant recipients

Gastroesophageal reflux disease (GORD) and microaspiration may be associated with acute graft dysfunction and development of obliterative bronchiolitis (OB) after lung transplantation. The "gold standard" for diagnosis of GORD is the 24-hour esophageal pH-monitoring study, although no simple, non-invasive screening test is routinely employed. Oil red O staining of alveolar macrophages in bronchoalveolar lavage (BAL) fluid identifies exogenous lipid and may be a surrogate marker for microaspiration. In this study we aimed to assess the utility of the lipid index in identifying patients with significant GORD. Our investigation was a prospective analysis of 34 lung transplant patients who were transplanted between April 1999 and July 2006 at a single institution. All patients with recurrent respiratory infections, recurrent acute rejection, unexplained graft dysfunction or newly diagnosed OB had Oil red O staining of alveolar macrophages on BAL specimens at bronchoscopy and 24-hour esophageal pH monitoring. A quantitative assessment called the lipid index was performed resulting in a score from 0 to 400. Abnormal 24-hour pH studies were defined as acid exposure >3.4% in the distal and/or >1% in the proximal esophageal site. Thirty-four patients with a mean age 36.1 years and mean post-operative day of 571 +/- 648 had lipid indices of 143 +/- 94 (range 3 to 341). Twenty-four-hour pH studies revealed a distal mean of 16.1 +/- 6.2% and proximal mean of 6.4 +/- 3.7%. A lipid index >150 was 82.3% sensitive and 76.4% specific for an abnormal 24-hour pH result. Foreign material present on cytology of bronchial fluid seen in 28% of patients showed poor correlation with Oil red O stains and pH studies. The lipid index is an effective, non-invasive screening test that provides direct evidence of esophageal aspiration. Patients with high positive results should proceed to surgical assessment for Nissen fundoplication.

Pulmonary macrophages in rats after hemithoracic irradiation: analysis of morphology and expression of surface antigen

The pathogenesis of radiation pneumonitis is not completely understood. The long latent period and involvement of unirradiated lung tissue may indicate an immune reaction in the injurious process of irradiated lung. To investigate the role of pulmonary macrophages in radiation pneumonitis, morphology and membrane antigen expression of pulmonary macrophages were studied in irradiated rat lung tissue following 4000 R hemithoracic irradiation. Lungs were explanted at 2, 4, 6, 8, 16, and 28 weeks after irradiation. Cryosections of irradiated lung tissue were immunohistochemically studied, and alveolar macrophages in bronchoalveolar lavage fluid were also analyzed using monoclonal antibodies to rat major histocompatibility complex (MHC) antigens and macrophages with flow cytometry. Macrophage subpopulations were analyzed using a panel of monoclonal antibodies to MHC class I (HAM2), MHC class II (OX6) and macrophage differentiation antigens (ED1, ED2, ED3). Alveolar macrophages in bronchoalveolar lavage were morphologically studied by smear and flow cytometry of forward light scatter and 90 degrees light scatter. At 2 weeks after irradiation, when histological changes did not appear, small lymphocyte-like macrophages and large foamy macrophages were observed in both the smear and histograms by flow cytometry. At 4, 6, and 8 weeks after irradiation, these new populations had markedly increased. However, at 16 and 28 weeks after irradiation, the size and shape of alveolar macrophages had returned to normal. In the expression of macrophage membrane antigens, an increase in the frequency of MHC class II+ cells in lavaged cells appeared at 2 weeks after irradiation, and became significant at 4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

AIRWAY INFLAMMATORY RESPONSES TO OXIDATIVE STRESS INDUCED BY PROLONGED LOW-DOSE DIESEL EXHAUST PARTICLE EXPOSURE FROM BIRTH DIFFER BETWEEN MOUSE BALB/C AND C57BL/6 STRAINS

The authors used BALB/c and C57BL/6 mouse strains to search for genetically based differences in response to prolonged (6 months) low-dose (100 μg/m3) diesel exhaust particle (DEP) exposure from birth in terms of airway inflammatory responses. Histopathological assessment showed that inflammatory cells infiltrated the perivascular areas only in C57BL/6 mice. The count of DEP-laden alveolar macrophages in bronchoalveolar lavage (BAL) fluid was significantly greater in BALB/c mice (P <.05) than in C57BL/6 mice. The lymphocyte and eosinophil count in BAL fluid was significantly greater in C57BL/6 mice (P <.05) than in BALB/c mice. Immunoglobulin (Ig) IgG1 and IgG2 levels in serum, and the monocyte chemoattractant protein (MCP)-1 level in BAL fluid were significantly greater in BALB/c mice than in C57BL/6 mice. The interleukin (IL)-12 level in BAL fluid was significantly greater in C57BL/6 mice than in BALB/c mice, but the IL-13 level in BAL fluid was significantly less in BALB/c mice than in C57BL/6 mice. Glutathione S-transferase (GST) mRNA expression and protein production in lung tissues were significantly lower in C57BL/6 mice than in BALB/c mice, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) level in the lung tissues were significantly greater in C57BL/6 mice than in BALB/c mice. In conclusion, prolonged low-dose DEP exposure induces airway inflammatory responses that differ remarkably among mouse strains; these differences are caused by differences in the host defense response to the oxidative stress induced by DEP exposure and may be useful in the development of biomarkers.

223: Quantitative assessment of lipid laden macrophages with oil Red O stain is an effective screening test for GORD

Purpose: Gastro-oesophageal reflux disease (GORD) may be associated with acute graft dysfunction and development of obliterative bronchiolitis (OB) post lung transplantation. The gold standard for diagnosis of GORD is a 24hr oesophageal pH study, although no simple non-invasive screening test is routinely employed. Oil Red O staining of alveolar macrophages in Bronchoalveolar Lavage (BAL) fluid identifies exogenous lipid. Aims: 1. Propose an effective screening test for GORD by quantitative assessment of lipid in alveolar macrophages on BAL. 2. Correlate quantitative assessment of lipid laden macrophages with 24hr pH studies to identify those patients who may require Nissen Fundoplication. Methods and Materials: Retrospective analysis of 97 lung transplant patients from a single institution transplanted between 4/99 and 7/06. All patients with recurrent respiratory infections or acute rejection, unexplained graft loss or newly diagnosed OB had Oil Red O staining of alveolar macrophages on BAL specimens and 24hr pH monitoring. A quantitative assessment was performed resulting in a score from 0 to 400 called the lipid index. One hundred macrophages were examined by two cytologists with the lipid index calculated by the number of cells staining positive for lipid and the quantity per cell. Abnormal 24hr pH studies were defined as acid exposure 3.4% distal and/or 1% proximal oesophageal site. Results: Thirty-four patients of mean age 36.1 yrs and mean postoperative day of 571 648 had lipid indices of 143 94 (3-341). 24hr pH studies revealed a distal mean of 16.1 6.2% and proximal of 6.4 3.7%. A lipid index greater than 175 was 86 % sensitive and 74% specific for an abnormal 24hr pH result. Foreign material present on cytology of BAL seen in 28% of patients showed poor correlation with Oil Red O stains and pH studies. Conclusions: The lipid index is an effective, non-invasive screening test which provides direct evidence of oesophageal aspiration. Patients with high positive results should proceed to assessment for Nissen fundoplication.

Alveolar macrophages participate in the systemic inflammation of alveolar hypoxia

Alveolar hypoxia (AHx, FIO2 0.10) produces widespread inflammation in the systemic rat microcirculation. Dissociation between tissue PO2 and inflammation, plus the observation that plasma from hypoxic rats elicits inflammation in normoxic tissues suggest that AHx inflammation is triggered by an agent released from a distant site and carried by the circulation. These experiments tested the hypothesis that alveolar macrophages (AM) are the source of the agent that triggers AHx inflammation. AM of male rats were depleted by tracheal instillation of clodronate-containing liposomes. Controls received NaCl-containing liposomes. The cremaster muscle was studied with intravital microscopy 4 days after treatment. Clodronate reduced bronchoalveolar lavage AM count by 90%. AHx in controls significantly increased leukocyte-endothelial adhesive interactions, produced degranulation of perivascular mast cells, and increased vascular permeability. None of these effects were seen in AM-depleted AHx rats. While plasma obtained from control AHx rats (FIO2 0.10 for 5 min) induced inflammation in normoxic cremasters, plasma from AM-depleted AHx rats did not have these inflammatory effects. The data suggest that AM are the source of the agent that initiates AHx-induced systemic inflammation. Supported by HL39443 and HD043044.

Stilbazulenyl nitrone (STAZN), a novel azulenyl nitrone antioxidant prevents lung injury following hemorrhagic shock and alters TLR4 distribution in alveolar macrophages

Introduction: Oxidants (OX) produced during shock/resuscitation (SR) contribute to organ failure by priming alveolar macrophages (AM) for increased responsiveness to subsequent inflammatory stimuli such as LPS. STAZN is a second-generation azulenyl nitrone – a potent antioxidant. Since our initial studies showed that OX/SR induces priming of AM by colocalization of LPS receptor - TLR4 within cell signaling domains – the lipid rafts (LR), we hypothesize that STAZN can protect lungs from injury by preventing macrophage LR recruitment of TLR4. Methods: Rats were bled to MAP of 40mmHg, 1h later resuscitated with blood and Ringer’s Lactate (RL) plus intraperitoneal STAZN (30 mg/kg × 2 doses) or vehicle (dimethyl sulfoxide). 1hr post resuscitation intratracheal LPS (3ug/10gm) or saline (SAL) were administered and 4hrs later bronchoalveolar lavage (BAL) fluid was used for differential cell counts. Alternatively, BAL AM were recovered 1hr after resuscitation, LRs were isolated by sucrose gradient ultracentrifugation and presence of TLR4 or GM-1 (LR marker) were detected by dot blotting. Results: Treatment with STAZN and not vehicle alone significantly attenuated LPS induced lung neutrophilia following RL resuscitation by 67% (n = 3, p<0.05). Shock and RL resuscitation induced TLR4 migration into the AM lipid raft fractions (marked by presence of GM-1), an effect inhibited by STAZN. Conclusions: Therapy with STAZN is effective at attenuating lung injury after shock resuscitation through preventing AM lipid raft recruitment of the LPS receptor. Azulenyl nitrones represent a novel and promising class of potent antioxidants for combating organ failure following trauma.