Alveolar Macrophages In Bronchoalveolar Lavage Fluid Research Articles

Cigarette smoke prevents M1 polarization of alveolar macrophages by suppressing NLRP3

Chronic obstructive pulmonary disease (COPD) is an inflammatory condition mainly caused by cigarette smoke (CS). Alveolar macrophages (AMs) contribute to its development, although the polarization of AMs is controversial. This study explored the polarization of AMs and mechanisms underlying their involvement in COPD. AM gene expression data from non-smokers, smokers, and COPD patients were downloaded from the GSE13896 and GSE130928 datasets. Macrophage polarization was evaluated by CIBERSORT and gene set enrichment analysis (GSEA). Polarization-related differentially expressed genes (DEGs) were identified in GSE46903. KEGG enrichment analysis and single sample GSEA were performed. M1 polarization levels were decreased in smokers and COPD patients, whereas M2 polarization did not change. In the GSE13896 and GSE130928 datasets, 27 and 19 M1-related DEGs, respectively, showed expression changes opposite to those in M1 macrophages in smokers and COPD patients compared with the control group. These M1-related DEGs were enriched in NOD-like receptor signaling pathway. Next, C57BL/6 mice were divided into control, lipopolysaccharide (LPS), CS, and LPS + CS groups, and cytokine levels in bronchoalveolar lavage fluid (BALF) and AM polarization were determined. The expression of macrophage polarization markers and NLRP3 was determined in AMs treated with CS extract (CSE), LPS, and an NLRP3 inhibitor. Cytokines levels and the percentage of M1 AMs in BALF were lower in the LPS + CS group than in the LPS group. Exposure to CSE downregulated the expression of M1 polarization markers and NLRP3 induced by LPS in AMs. The present results indicate that M1 polarization of AMs is repressed in smokers and COPD patients, and CS may inhibit LPS-induced M1 polarization of AMs by suppressing NLRP3.

The role of STAT-6/KLF-4/PPAR-γ activation in alveolar macrophage polarization changes in silica-induced pulmonary fibrosis

Objective: To observe the effect of silicon dioxide (SiO(2)) on the polarization of alveolar macrophages (AMs) , and to explore the expressions and the significance of signal transducer and activator of transcription-6 (STAT-6) /Krüppel-like factor-4 (KLF-4) /peroxisome proliferators-activated receptors-γ (PPAR-γ) signaling molecules in AMs. Methods: In November 2020, C57BL/6 mice were randomly divided into crystalline SiO(2) group and normal saline (NS) group, and 12 mice in each group. Mice were intratracheally instillated with 100 μl crystalline SiO(2) suspension (20 mg/ml) or 100 μl NS, and were sacrificed after 28 days. Masson staining was used to observe the degree of pulmonary fibrosis of mice and hydroxyproline (HYP) level were assessed. The proportions of M1-typed and M2-typed AMs in bronchoalveolar lavage fluid (BLAF) were analyzed by flow cytometry. The mRNA relative expression levels of inducible nitric oxide synthase (iNOS) , arginidase-1 (Arg-1) , interleukin (IL) -1β, tumor necrosis factor-α (TNF-α) , IL-6, IL-10, transforming growth factor-β (TGF-β) , STAT-6, KLF-4 and PPAR-γ were detected by real-time fluorescence quantitative PCR. Activities of iNOS and Arg-1, as well as contents of IL-1β, TNF-α, IL-6, IL-10 and TGF-β were assessed by the enzyme-linked immunosorbent. The protein relative expression levels of phosphorylation-signal transducer and activator of transcription-6 (p-STAT-6) , KLF-4 and PPAR-γ were evaluated by immunofluorescence. Results: After 28 days of treatment, the structure of the lung tissue of the mice was destroyed, and the deposition of collagen was significantly increased in the crystalline SiO(2) group. Compared with NS group, HYP level of lung tissue in crystalline SiO(2) group were increased, the proportion of M2-typed AMs in crystalline SiO(2) group was increased, the proportion of M1-typed AMs in crystalline SiO(2) group was decreased, the mRNA relative expressions and contents of Arg-1, IL-10, TGF-β in crystalline SiO(2) group were significantly increased, the mRNA relative expressions and contents of iNOS, IL-1β, TNF-α, IL-6 in crystalline SiO(2) group were significantly decreased, the mRNA of STAT-6, KLF-4, PPAR-γ and the protein relative expression levels of p-STAT-6, KLF-4, PPAR-γ were significantly increased in crystalline SiO(2) group, and the the differences were statistically significant (P<0.05) . Conclusion: Crystalline SiO(2) may mediate the process of pulmonary fibrosis through promote AMs polarization toward M2-typed by activating the STAT-6/KLF-4/PPAR-γ signaling pathway.

Identification of PD-1 ligands: PD-L1 and PD-L2 on macrophages in lung cancer milieu by flow cytometry.

BackgroundThe efficacy of immune checkpoint inhibitors (ICIs) remains unexpected and in some patients the resistance to anti-programmed death-1 (anti-PD-1) and anti-programmed death ligand 1 (anti-PD-L1) agents is observed. One of possible explanation may be PD-L2 activity. PD-1 ligands: PD-L1 and PD-L2 are present on cancer cells but also, not without significance, on alveolar macrophages (AMs) contributing to immune-suppression in the tumor microenvironment. The aim of this study was to analyse PD-L2, PD-L1 expression on AMs in bronchoalveolar lavage fluid (BALF) in relation to PD-1 positive T lymphocytes.MethodsSeventeen patients with lung cancer were investigated. BALF cells from the lung with cancer (clBALF) and from the opposite “healthy” lung (hlBALF) and peripheral blood (PB) lymphocytes were investigated. Flow cytometry method was used.ResultsWe found that 100% of CD68+ AMs from the clBALF were PD-L1 and PD-L2-positive. Unexpectedly, fluorescence minus one (FMO) PD-L1 and PD-L2 stained controls and isotype controls also showed strong autofluorescence. The hlBALF AMs exhibited a similar PD-L1 and PD-L2 autofluorescence. The median proportion of PD-1+ T lymphocytes was higher in the clBALF than the hlBALF and PB (28.9 vs. 23.4% vs. 15.6%, P=0.0281).ConclusionsWe discussed the opportunities of exploring the PD-1-PD-L1/PD-L2 pathway in the lung cancer environment, which may help to find new potential biomarkers for immunotherapy. We concluded that precise identification by flow cytometry of macrophages in the BALF is possible, but our study showed that the autofluorescence of macrophages did not allow to assess a real expression of PD-L2 as well as PD-L1 on AMs.

Increased matrix metalloproteinase-9 to tissue inhibitor of metalloproteinase-1 ratio in smokers with airway hyperresponsiveness and accelerated lung function decline.

BackgroundAirway hyperresponsiveness (AHR) is associated with airway inflammation and a rapid decline in lung function and is a predictor of future risk of COPD among smokers. Alveolar macrophages (AMs) from patients with COPD release a greater amount of matrix metalloproteinase (MMP)-9. We hypothesized that the imbalance between MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) is related to AHR in smokers.Patients and methodsHealthy smokers with AHR (AHR + S) or smokers without AHR (AHR − S; divided according to a methacholine challenge test) and nonsmokers without AHR (AHR − NS) were enrolled. Spirometry was performed during enrollment and repeated after 5 years. Initially, AMs recovered from bronchoalveolar lavage (BAL) fluid were cultured in the presence of p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), MAPK kinase (MEK) 1/2 (the MEK of extracellular signal-regulated kinase [ERK] inhibitor, PD98059), or medium alone for 24 h. The release of MMP-9 and TIMP-1 in culture supernatants was measured by enzyme-linked immunosorbent assay.ResultsA greater reduction in forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC), FEV1 (as a percentage of the predicted value [%pred]), and maximal mid-expiratory flow (MMEF) was observed among AHR + S in the 5-year period. There was a higher proportion of neutrophils and a lower proportion of AMs in BAL fluid recovered from AHR + S. Compared to AMs from AHR − NS and AHR − S, AMs from nonsmokers with AHR (AHR + NS) released more MMP-9 and less TIMP-1, with an increase in MMP-9/TIMP-1 ratios. The MMP-9/TIMP-1 ratio in smokers was positively correlated with the annual decline in FEV1%pred, FVC%pred, and MMEF%pred. Both SB203580 and PD98059 significantly reduced MMP-9, but not TIMP-1, from AMs of smokers.ConclusionAMs of AHR + NS produce excessive MMP-9 over TIMP-1, which may be a predictor of the development of airway obstruction. Inhibition of p38 MAPK and ERK suppresses the generation of MMP-9 by AMs from smokers.

High-dose dexamethasone induced LPS-stimulated rat alveolar macrophages apoptosis.

Prolonged administration of an excessive dose of corticosteroids proved to be harmful for patients with acute lung injury (ALI). A previous study has found that repeated administration of an excessive dose of methylprednisolone reduced alveolar macrophages (AMs) in bronchoalveolar lavage fluid (BALF) with an unknown mechanism. This study aimed to investigate the effect of excessive use of dexamethasone (Dex) on BALF AMs in vitro. Transmission electron microscopy and DNA fragmentation analysis demonstrated that 10−4 and 10−5 M Dex induced lipopolysaccharide-stimulated rat AMs apoptosis with downregulation of tumor necrosis factor-α, interleukin (IL)-12 and upregulation of IL-10, transforming growth factor-β. These results indicated that apoptosis might be a novel contribution involved in the detrimental effect of excessive dose of Dex clinically used to treat ALI.

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.

Preferential Destruction of Interstitial Macrophages over Alveolar Macrophages as a Cause of Pulmonary Disease in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

To our knowledge, this study demonstrates for the first time that the AIDS virus differentially impacts two distinct subsets of lung macrophages. The predominant macrophages harvested by bronchoalveolar lavage (BAL), alveolar macrophages (AMs), are routinely used in studies on human lung macrophages, are long-lived cells, and exhibit low turnover. Interstitial macrophages (IMs) inhabit the lung tissue, are not recovered with BAL, are shorter-lived, and exhibit higher baseline turnover rates distinct from AMs. We examined the effects of SIV infection on AMs in BAL fluid and IMs in lung tissue of rhesus macaques. SIV infection produced massive cell death of IMs that contributed to lung tissue damage. Conversely, SIV infection induced minimal cell death of AMs, and these cells maintained the lower turnover rate throughout the duration of infection. This indicates that SIV produces lung tissue damage through destruction of IMs, whereas the longer-lived AMs may serve as a virus reservoir to facilitate HIV persistence.

TGF-β-induced IL-6 prevents development of acute lung injury in influenza A virus-infected F508del CFTR-heterozygous mice.

As the eighth leading cause of annual mortality in the USA, influenza A viruses are a major public health concern. In 20% of patients, severe influenza progresses to acute lung injury (ALI). However, pathophysiological mechanisms underlying ALI development are poorly defined. We reported that, unlike wild-type (WT) C57BL/6 controls, influenza A virus-infected mice that are heterozygous for the F508del mutation in the cystic fibrosis transmembrane conductance regulator (HETs) did not develop ALI. This effect was associated with higher IL-6 and alveolar macrophages (AMs) at 6 days postinfection (d.p.i.) in HET bronchoalveolar lavage fluid (BALF). In the present study, we found that HET AMs were an important source of IL-6 at 6 d.p.i. Infection also induced TGF-β production by HET but not WT mice at 2 d.p.i. TGF-β neutralization at 2 d.p.i. (TGF-N) significantly reduced BALF IL-6 in HETs at 6 d.p.i. Neither TGF-N nor IL-6 neutralization at 4 d.p.i. (IL-6-N) altered postinfection weight loss or viral replication in either mouse strain. However, both treatments increased influenza A virus-induced hypoxemia, pulmonary edema, and lung dysfunction in HETs to WT levels at 6 d.p.i. TGF-N and IL-6-N did not affect BALF AM and neutrophil numbers but attenuated the CXCL-1/keratinocyte chemokine response in both strains and reduced IFN-γ production in WT mice. Finally, bone marrow transfer experiments showed that HET stromal and myeloid cells are both required for protection from ALI in HETs. These findings indicate that TGF-β-dependent production of IL-6 by AMs later in infection prevents ALI development in influenza A virus-infected HET mice.

Statin-induced lung injury: diagnostic clue and outcome

BackgroundStatin-induced lung injury (SILI) is an uncommon but serious complication of statins. The clinical features and outcome of patients with SILI vary widely. Clinical data relevant to diagnosis and outcome...

Decreased apoptotic rate of alveolar macrophages of patients with idiopathic pulmonary fibrosis.

Introduction. Increased apoptosis of epithelial cells and decreased apoptosis of myofibroblasts are involved in the pathogenesis of IPF. The apoptotic profile of alveolar macrophages (AMs) in IPF is unclear. Aim. To investigate whether AMs of patients with IPF exhibit a different apoptotic profile compared to normal subjects. Methods. We analyzed, by immunohistochemistry, the expression of the apoptotic markers fas, fas ligand , bcl-2, and bax in AM obtained from bronchoalveolar lavage fluid (BALF) of 20 newly diagnosed, treatment-naive IPF patients and of 16 controls. Apoptosis of AM was evaluated by Apoptag immunohistochemistry. IPF patients received either interferon-g and corticosteroids or azathioprine and corticosteroids for six months. Results. BALF AMs undergoing apoptosis were significantly less in IPF patients. No difference was found in the expression of fas or fas ligand, bcl-2 and bax between IPF and control group. No difference was found between the respiratory function parameters of the two treatment groups after six months. A positive correlation was found between the number of bcl-2 positive stained macrophages and DLCO after treatment. Conclusions. The decreased apoptotic rate of AM of patients with IPF is not associated with decreased expression of apoptosis mediators involved in the external or internal apoptotic pathway.

Direct evidence that GM-CSF inhalation improves lung clearance in pulmonary alveolar proteinosis

Autoimmune pulmonary alveolar proteinosis (aPAP) is caused by granulocyte/macrophage-colony stimulating factor (GM-CSF) autoantibodies in the lung. Previously, we reported that GM-CSF inhalation therapy improved alveolar-arterial oxygen difference and serum biomarkers of disease severity in these patients. It is plausible that inhaled GM-CSF improves the dysfunction of alveolar macrophages and promotes the clearance of the surfactant. However, effect of the therapy on components in bronchoalveolar lavage fluid (BALF) remains unclear. To figure out changes in surfactant clearance during GM-CSF inhalation therapy. We performed retrospective analyses of BALF obtained under a standardized protocol from the same bronchus in each of 19 aPAP patients before and after GM-CSF inhalation therapy (ISRCTN18931678, JMA-IIA00013; total dose 10.5-21 mg, duration 12-24 weeks). For evaluation, the participants were divided into two groups, high responders with improvement in alveolar-arterial oxygen difference ≥13 mmHg (n = 10) and low responders with that < 13 mmHg (n = 9). Counts of both total cells and alveolar macrophages in BALF did not increase during the therapy. However, total protein and surfactant protein-A (SP-A) were significantly decreased in high responders, but not in low responders, suggesting that clearance of surfactant materials is correlated with the efficacy of the therapy. Among 94 biomarkers screened in bronchoalveolar lavage fluid, we found that the concentration of interleukin-17 and cancer antigen-125 were significantly increased after GM-CSF inhalation treatment. GM-CSF inhalation decreased the concentration of total protein and SP-A in BALF, and increase interleukin-17 and cancer antigen-125 in improved lung of autoimmune pulmonary alveolar proteinosis.

Expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases

BackgroundInflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases.MethodssCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. In vitro studies were performed to investigate which factors regulate sCD14 release and mCD14 expression.ResultssCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. In vitro, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition.ConclusionsThis study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.

Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice

To determine if nuclear factor-κB (NF-κB) activation may be a key factor in lung inflammation and respiratory dysfunction, we investigated whether NF-κB can be blocked by intratracheal administration of NF-κB decoy oligodeoxynucleotides (ODNs), and whether decoy ODN-mediated NF-κB inhibition can prevent smoke-induced lung inflammation, respiratory dysfunction, and improve pathological alteration in the small airways and lung parenchyma in the long-term smoke-induced mouse model system. We also detected changes in transcriptional factors. In vivo, the transfection efficiency of NF-κB decoy ODNs to alveolar macrophages in BALF was measured by fluorescein isothiocyanate (FITC)-labeled NF-κB decoy ODNs and flow cytometry post intratracheal ODN administration. Pulmonary function was measured by pressure sensors, and pathological changes were assessed using histology and the pathological Mias software. NF-κB and activator protein 1(AP-1) activity was detected by the electrophoretic motility shift assay (EMSA). Mouse cytokine and chemokine pulmonary expression profiles were investigated by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and lung tissue homogenates, respectively, after repeated exposure to cigarette smoke. After 24 h, the percentage of transfected alveolar macrophages was 30.00 ± 3.30%. Analysis of respiratory function indicated that transfection of NF-κB decoy ODNs significantly impacted peak expiratory flow (PEF), and bronchoalveolar lavage cytology displayed evidence of decreased macrophage infiltration in airways compared to normal saline-treated or scramble NF-κB decoy ODNs smoke exposed mice. NF-κB decoy ODNs inhibited significantly level of macrophage inflammatory protein (MIP) 1α and monocyte chemoattractant protein 1(MCP-1) in lung homogenates compared to normal saline-treated smoke exposed mice. In contrast, these NF-κB decoy ODNs-treated mice showed significant increase in the level of tumor necrosis factor-α(TNF-α) and pro-MMP-9(pro-matrix metalloproteinase-9) in mice BALF. Further measurement revealed administration of NF-κB decoy ODNs did not prevent pathological changes. These findings indicate that NF-κB activation play an important role on the recruitment of macrophages and pulmonary dysfunction in smoke-induced chronic lung inflammation, and with the exception of NF-κB pathway, there might be complex mechanism governing molecular dynamics of pro-inflammatory cytokines expression and structural changes in small airways and pulmonary parenchyma in vivo.

Macrophage turnover kinetics in the lungs of mice infected with Streptococcus pneumoniae

Streptococcus pneumoniae is the most prevalent cause of community-acquired pneumonia and is known to induce apoptosis and necrosis in macrophages in vivo. We analyzed the kinetics of alveolar and lung parenchymal macrophage replacement by newly recruited exudate macrophages in vehicle-treated and S. pneumoniae-challenged bone marrow chimeric CD45.1 mice. After lethal irradiation, CD45.1 alloantigen-expressing recipient mice were transplanted with bone marrow cells from CD45.2 alloantigen-expressing donor mice. After only 24 hours of low-dose S. pneumoniae infection, approximately 60% of CD45.1(pos) recipient-type alveolar macrophages (AM) were replaced by CD45.2(pos) donor-type exudate AM in bronchoalveolar lavage fluid, and this increased to more than 80% on Day 7 of infection. In contrast, lung parenchymal macrophages of S. pneumoniae-infected chimeric CD45.1 mice were replaced by only about 10% by 24 hours, although this increased to over 80% by Days 3 to 7 of infection. This dramatic macrophage turnover was accompanied by early induction of apoptosis/necrosis in donor-type exudate AM peaking at 6 hours after infection, whereas peak apoptosis/necrosis induction in recipient-type AM was delayed until Day 7. Collectively, these data for the first time demonstrate that S. pneumoniae infection of the lung triggers a brisk turnover of both resident and recruited mononuclear phagocyte subsets, and suggest an important role of exudate but not resident macrophages in re-establishing alveolar and lung homeostasis.

Macrophage Turnover Kinetics in the Lungs of Mice Infected with Streptococcus pneumoniae

Streptococcus pneumoniae is the most prevalent cause of community-acquired pneumonia and is known to induce apoptosis and necrosis in macrophages in vivo. We analyzed the kinetics of alveolar and lung parenchymal macrophage replacement by newly recruited exudate macrophages in vehicle-treated and S. pneumoniae-challenged bone marrow chimeric CD45.1 mice. After lethal irradiation, CD45.1 alloantigen-expressing recipient mice were transplanted with bone marrow cells from CD45.2 alloantigen-expressing donor mice. After only 24 hours of low-dose S. pneumoniae infection, approximately 60% of CD45.1(pos) recipient-type alveolar macrophages (AM) were replaced by CD45.2(pos) donor-type exudate AM in bronchoalveolar lavage fluid, and this increased to more than 80% on Day 7 of infection. In contrast, lung parenchymal macrophages of S. pneumoniae-infected chimeric CD45.1 mice were replaced by only about 10% by 24 hours, although this increased to over 80% by Days 3 to 7 of infection. This dramatic macrophage turnover was accompanied by early induction of apoptosis/necrosis in donor-type exudate AM peaking at 6 hours after infection, whereas peak apoptosis/necrosis induction in recipient-type AM was delayed until Day 7. Collectively, these data for the first time demonstrate that S. pneumoniae infection of the lung triggers a brisk turnover of both resident and recruited mononuclear phagocyte subsets, and suggest an important role of exudate but not resident macrophages in re-establishing alveolar and lung homeostasis.

Augmented Proliferation of Human Alveolar Macrophages After Allogeneic Bone Marrow Transplantation

After allogeneic bone marrow transplantation (allo-BMT), recipient alveolar macrophages (AM) are gradually replaced by AM of the donor origin. An influx of mononuclear phagocytes of donor origin to the lung is responsible for the repopulation, but the detailed kinetics remain unclear. We therefore studied 24 BMT recipients who underwent bronchoalveolar lavage (BAL) from 24 to 83 days after BMT. AM cell number, size, morphology, proliferating ability, and genotype of AM were measured. Before day 50, the number and size of AM in BAL fluid were similar to those of normal nonsmokers. However, after day 50, the mean number of AM increased threefold and the mean cell size decreased due to the increase of small AM. These small cells are presumably of donor origin based on DNA fingerprinting analysis and based on fluorescence in situ hybridization for the Y chromosome in a sex-mismatched case. Immunohistochemistry and cell cycle analysis demonstrated that the increase in AM number coincided with a remarkable increase of AM expressing proliferating cell nuclear antigen, suggesting that small AM are proliferating. This is the first report representing that augmented proliferation of donor AM in situ may contribute to the reconstitution of AM population after BMT.

Augmented Proliferation of Human Alveolar Macrophages After Allogeneic Bone Marrow Transplantation

AbstractAfter allogeneic bone marrow transplantation (allo-BMT), recipient alveolar macrophages (AM) are gradually replaced by AM of the donor origin. An influx of mononuclear phagocytes of donor origin to the lung is responsible for the repopulation, but the detailed kinetics remain unclear. We therefore studied 24 BMT recipients who underwent bronchoalveolar lavage (BAL) from 24 to 83 days after BMT. AM cell number, size, morphology, proliferating ability, and genotype of AM were measured. Before day 50, the number and size of AM in BAL fluid were similar to those of normal nonsmokers. However, after day 50, the mean number of AM increased threefold and the mean cell size decreased due to the increase of small AM. These small cells are presumably of donor origin based on DNA fingerprinting analysis and based on fluorescence in situ hybridization for the Y chromosome in a sex-mismatched case. Immunohistochemistry and cell cycle analysis demonstrated that the increase in AM number coincided with a remarkable increase of AM expressing proliferating cell nuclear antigen, suggesting that small AM are proliferating. This is the first report representing that augmented proliferation of donor AM in situ may contribute to the reconstitution of AM population after BMT.

Ethanol Inhibits Inducible Nitric Oxide Synthese Transcription and Post‐Transcriptional Processes in Vivo

The effects of ethanol (ETOH) on post-transcriptional regulation of inducible nitric oxide synthase (iNOS) in vivo has not been demonstrated. We examined the effect of ETOH on iNOS mRNA, protein, and the production of the nitrate and nitrite anion (RNI) in rat lung alveolar macrophages (AM) in vivo when stimulated by lipopolysaccharide (LPS) and dibutyryl cyclic AMP (DB-cAMP). Sprague-Dawley rats (225-250 g) (n = 5-7/gp) were given intratracheal LPS (0.6 mg/kg) or DB-cAMP (0.1 and 1 mg/kg) 30 min after ETOH (4 mg/kg, intraperitoneally (ip)) or phosphate-buffered sterile saline (PBS) (5 ml/kg, ip) or pyrrolidine dithiocarbamate (PDTC 10 mg/kg, intratracheally) or 15 min after diethyl dithiocarbamate (DETC) (5 mg/kg, intratracheally). At selected times after administration of LPS or DB-cAMP, the animals were anesthetized, the lungs with the heart attached were removed and the lungs subjected to bronchoalveolar lavage (BAL). The BAL fluid was assayed for tumor necrosis factor alpha (TNF alpha) and RNI. The BAL fluid AM were isolated and analyzed for iNOS mRNA and protein with competitor-equalized polymerase chain reaction (PCR) and Western blots, respectively. The ex vivo incubates of AM were assayed for RNI and TNF alpha. LPS and DB-cAMP each increased iNOS mRNA and protein in AM and RNI in the BAL fluid and ex vivo incubates of AM. However, the peak of the increase of iNOS mRNA occurred at 2 hr for DB-cAMP and 4 to 6 hr for LPS. Only LPS increased the concentrations of TNF alpha in BAL fluid and ex vivo incubates of AM. ETOH attenuated LPS-mediated up-regulation of iNOS mRNA and TNF alpha and iNOS protein and RNI produced by the AM. In contrast, pretreatment of rats with ETOH did not affect DB-cAMP-mediated increases of iNOS mRNA in AM at any time but suppressed the amount of iNOS protein and RNI produced in DB-cAMP-stimulated AM. DETC, but not PDTC, attenuated LPS-mediated up-regulation of iNOS mRNA without effects on that produced by DB-cAMP. Since ETOH and DETC, but not PDTC, suppressed LPS-mediated but not DB-cAMP-mediated transcription of iNOS, we conclude that two distinct pathways exist for induction of iNOS mRNA by these agonists. ETOH and DETC may inhibit the LPS-mediated activation pathway by acting as antioxidants. Also, since ETOH inhibited DB-cAMP-mediated increases in iNOS protein without affecting iNOS mRNA ETOH also acts at a post-transcriptional or translational site to inhibit iNOS protein in rat AM in vivo.

Exposure to peat dust: acute effects on lung function and content of bronchoalveolar lavage fluid.

Mechanised production of peat for fuel consumption is associated with high concentrations of organic dust, which is inhaled by the peat workers. In the present study 17 workers at two peat bogs in northern Sweden were examined. Personal sampling of total dust and the respirable fraction was performed during several workshifts. Dynamic spirometry was carried out before and at the end of shifts. Bronchoscopy with bronchoalveolar lavage (BAL) was performed in six subjects at the end of the working season and the results were compared with unexposed reference subjects. Peat workers using modern machines with ventilated cabins containing air filters were found to be exposed to low concentrations of peat dust. The recorded dust concentrations were below the threshold limit value for organic dust (5 mg/m3 air) in all but one worker. The respirable fraction of peat dust recorded in the breathing zone of the workers correlated significantly with a decrease in forced expiratory volume in one second (FEV1). The effect on lung function in non-asthmatic peat workers was, however, small. The concentration of lysozyme positive alveolar macrophages in BAL fluid was significantly lower in the peat workers compared with reference subjects. An inverse correlation was found between the mentioned cells and exposure to the respirable fraction of the peat dust. Furthermore, one particularly dust exposed worker had pronounced increases in alveolar macrophages, fibronectin concentration, and mast cells in BAL fluid.