Alveolar Macrophage Subpopulations Research Articles

Identification of Siglec-1-negative alveolar macrophages with proinflammatory phenotypes in chronic obstructive pulmonary disease.

Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. However, subpopulations of AMs participating in chronic inflammation have been poorly characterized. We previously reported that Siglec-1 expression on AMs, which is important for bacteria engulfment, was decreased in COPD. Here, we show that Siglec-1-negative AMs isolated from COPD lung tissues exhibit a proinflammatory phenotype and are associated with poor clinical outcomes in patients with COPD. Using flow cytometry, we segregated three subsets of AMs based on the expression of Siglec-1 and their side scattergram (SSC) and forward scattergram (FSC) properties: Siglec-1+SSChiFSChi, Siglec-1-SSChiFSChi, and Siglec-1-SSCloFSClo subsets. The Siglec-1-SSCloFSClo subset number was increased in COPD. RNA sequencing revealed upregulation of multiple proinflammatory signaling pathways and emphysema-associated matrix metalloproteases in the Siglec-1-SSCloFSClo subset. Gene set enrichment analysis indicated that the Siglec-1-SSCloFSClo subset adopted intermediate phenotypes between monocytes and mature alveolar macrophages. Functionally, these cells produced TNF-α, IL-6, and IL-8 at baseline, and these cytokines were significantly increased in response to viral RNA. The increase in Siglec-1-negative AMs in induced sputum is associated with future exacerbation risk and lung function decline in patients with COPD. Collectively, the novel Siglec-1-SSCloFSClo subset of AMs displays proinflammatory properties, and their emergence in COPD airways may be associated with poor clinical outcomes.NEW & NOTEWORTHY Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. We find that Siglec-1-negative alveolar macrophages have a wide range of proinflammatory landscapes and a protease-expressing phenotype. Moreover, this subset is associated with the pathogenesis of COPD and responds to viral stimuli.

Single-cell analysis reveals alterations in cellular composition and cell-cell communication associated with airway inflammation and remodeling in asthma

BackgroundAsthma is a heterogeneous disease characterized by airway inflammation and remodeling, whose pathogenetic complexity was associated with abnormal responses of various cell types in the lung. The specific interactions between immune and stromal cells, crucial for asthma pathogenesis, remain unclear. This study aims to determine the key cell types and their pathological mechanisms in asthma through single-cell RNA sequencing (scRNA-seq).MethodsA 16-week mouse model of house dust mite (HDM) induced asthma (n = 3) and controls (n = 3) were profiled with scRNA-seq. The cellular composition and gene expression profiles were assessed by bioinformatic analyses, including cell enrichment analysis, trajectory analysis, and Gene Set Enrichment Analysis. Cell-cell communication analysis was employed to investigate the ligand-receptor interactions.ResultsThe asthma model results in airway inflammation coupled with airway remodeling and hyperresponsiveness. Single-cell analysis revealed notable changes in cell compositions and heterogeneities associated with airway inflammation and remodeling. GdT17 cells were identified to be a primary cellular source of IL-17, related to inflammatory exacerbation, while a subpopulation of alveolar macrophages exhibited numerous significantly up-regulated genes involved in multiple pathways related to neutrophil activities in asthma. A distinct fibroblast subpopulation, marked by elevated expression levels of numerous contractile genes and their regulators, was observed in increased airway smooth muscle layer by immunofluorescence analysis. Asthmatic stromal-immune cell communication significantly strengthened, particularly involving GdT17 cells, and macrophages interacting with fibroblasts. CXCL12/CXCR4 signaling was remarkedly up-regulated in asthma, predominantly bridging the interaction between fibroblasts and immune cell populations. Fibroblasts and macrophages could jointly interact with various immune cell subpopulations via the CCL8/CCR2 signaling. In particular, fibroblast-macrophage cell circuits played a crucial role in the development of airway inflammation and remodeling through IL1B paracrine signaling.ConclusionsOur study established a mouse model of asthma that recapitulated key pathological features of asthma. ScRNA-seq analysis revealed the cellular landscape, highlighting key pathological cell populations associated with asthma pathogenesis. Cell-cell communication analysis identified the crucial ligand-receptor interactions contributing to airway inflammation and remodeling. Our findings emphasized the significance of cell-cell communication in bridging the possible causality between airway inflammation and remodeling, providing valuable hints for therapeutic strategies for asthma.

Functional characterization of small and large alveolar macrophages in sarcoidosis and idiopathic pulmonary fibrosis compared with non-fibrosis interstitial lung diseases.

Sarcoidosis is a granulomatous disease that mostly affects the lungs. Advanced tissue injury caused by this disease can progress to pulmonary fibrosis with similar characteristics shared with idiopathic pulmonary fibrosis (IPF). The initial presentations of both sarcoidosis and IPF may be shared with other interstitial lung diseases (ILDs). Two populations of macrophages have been described in the alveolar space: small alveolar macrophages (AMs) and large alveolar macrophages. Despite their protective function, these cells may also play a role in the initiation and maintenance of inflammation leading to fibrosis. The aim of this study was the functional characterization of small and large AM subpopulations in sarcoidosis and IPF as a pathology with respectively mild and advanced tissue injury causing fibrosis, in comparison with non-fibrosis ILDs. Activation and adhesion surface markers as well as functions of small and large AMs isolated from bronchoalveolar lavage (BAL) were assessed by Flow Cytometry within patients with confirmed sarcoidosis (n= 14), IPF (n= 6), and non-fibrosis ILDs (n= 9). Our results showed that small AMs are immunologically more active, which may be important for airway inflammation. They are also proportionally more abundant in IPF, and therefore they may be more involved in a fibrosis process associated with the down-regulation of HLA-DR, LeuCAM, and CD62L expression. In Sarcoidosis, the inflammatory process appears to be associated with up-regulation of CD38 expression and oxidative burst activity. A relevant potential of the activation and adhesion markers as well as oxidative burst activity expressed on small and large AMs, in the perspective of differential diagnosis of sarcoidosis and IPF.

Epithelium- and endothelium-derived exosomes regulate the alveolar macrophages by targeting RGS1 mediated calcium signaling-dependent immune response.

Alveolar macrophages (AM) maintain airway immune balance; however, the regulation of heterogeneity of AMs is incompletely understood. We demonstrate that RGS1 coregulates the immunophenotype of AM subpopulations, including pro- and anti-inflammatory, injury- and repair-associated, and pro- and antifibrotic phenotypes, through the PLC-IP3R signal-dependent intracellular Ca2+ response. Flt3+ AMs and Tie2+ AMs had different immune properties, and RGS1 expression in the cells was targeted by exosomes (EXOs) containing miR-223 and miR-27b-3p that were derived from vascular endothelial cells (EnCs) and type II alveolar epithelial cells (EpCs-II), respectively. Imbalance of AMs was correlated with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and pulmonary fibrosis (PF) caused a lack of secretion of CD31+ and CD74+ EXOs derived from EnCs and EpCs-II. Timely treatment with EXOs significantly improved endotoxin-induced ALI/ARDS and bleomycin-induced PF in mice. Thus, EnC- and EpC-II-derived EXOs regulate the immune balance of AMs and can be used as potential therapeutic drugs.

Functional heterogeneity of alveolar macrophage population based on expression of CXCL2.

Alveolar macrophages (AMs) are the major lung-resident macrophages and have contradictory functions. AMs maintain tolerance and tissue homeostasis, but they also initiate strong inflammatory responses. However, such opposing roles within the AM population were not known to be simultaneously generated and coexist. Here, we uncovered heterogeneous AM subpopulations generated in response to two distinct pulmonary fungal infections, Cryptococcus neoformans and Aspergillus fumigatus Some AMs are bona fide sentinel cells that produce chemoattractant CXCL2, which also serves as a marker for AM heterogeneity, in the context of pulmonary fungal infections. However, other AMs do not produce CXCL2 and other pro-inflammatory molecules. Instead, they highly produce anti-inflammatory molecules, including interleukin-10 (IL-10) and complement component 1q (C1q). These two AM subpopulations have distinct metabolic profiles and phagocytic capacities. We report that polarization of pro-inflammatory and anti-inflammatory AM subpopulations is regulated at both epigenetic and transcriptional levels and that these AM subpopulations are generally highly plastic. Our studies have uncovered the role of C1q expression in programming and sustaining anti-inflammatory AMs. Our finding of the AM heterogeneity upon fungal infections suggests a possible pharmacological intervention target to treat fungal infections by tipping the balance of AM subpopulations.

Appearance of Alveolar Macrophage Subpopulations in Correlation With Histopathological Effects in Short-Term Inhalation Studies With Biopersistent (Nano)Materials.

Following inhalation and deposition in the alveolar region at sufficient dose, biopersistent (nano)materials generally provoke pulmonary inflammation. Alveolar macrophages (AMs) are mediators of pulmonary immune responses and were broadly categorized in pro-inflammatory M1 and anti-inflammatory M2 macrophages. This study aimed at identifying AM phenotype as M1 or M2 upon short-term inhalation exposure to different (nano)materials followed by a postexposure period. Phenotyping of AM was retrospectively performed using immunohistochemistry. M1 (CD68+iNOS+) and M2 (CD68+CD206+ and CD68+ArgI+) AMs were characterized in formalin-fixed, paraffin-embedded lung tissue of rats exposed for 6 hours/day for 5 days to air, 100 mg/m3 nano-TiO2, 25 mg/m3 nano-CeO2, 32 mg/m3 multiwalled carbon nanotubes, or 100 mg/m3 micron-sized quartz. During acute inflammation, relative numbers of M1 AMs were markedly increased, whereas relative numbers of M2 were generally decreased compared to control. Following an exposure-free period, changes in iNOS or CD206 expression correlated with persistence, regression, or progression of inflammation, suggesting a role of M1/M2 AMs in the pathogenesis of pulmonary inflammation. However, no clear correlation of AM subpopulations with qualitatively distinct histopathological findings caused by different (nano)materials was found. A more detailed understanding of the processes underlaying these morphological changes is needed to identify biomarkers for different histopathological outcomes.

Identification of an Increased Alveolar Macrophage Subpopulation in Old Mice That Displays Unique Inflammatory Characteristics and Is Permissive to Mycobacterium tuberculosis Infection.

The elderly population is more susceptible to pulmonary infections, including tuberculosis. In this article, we characterize the impact of aging on the phenotype of mouse alveolar macrophages (AMs) and their response to Mycobacterium tuberculosis. Uninfected AMs were isolated from bronchoalveolar lavage of young (3 mo) and old (18 mo) C57BL/6 mice. AMs from old mice expressed higher mRNA levels of CCL2, IFN-β, IL-10, IL-12p40, TNF-α, and MIF than young mice, and old mice contained higher levels of CCL2, IL-1β, IFN-β, and MIF in their alveolar lining fluid. We identified two distinct AM subpopulations, a major CD11c+ CD11b- population and a minor CD11c+ CD11b+ population; the latter was significantly increased in old mice (4-fold). Expression of CD206, TLR2, CD16/CD32, MHC class II, and CD86 was higher in CD11c+ CD11b+ AMs, and these cells expressed monocytic markers Ly6C, CX3CR1, and CD115, suggesting monocytic origin. Sorted CD11c+ CD11b+ AMs from old mice expressed higher mRNA levels of CCL2, IL-1β, and IL-6, whereas CD11c+ CD11b- AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-β and IL-10. CD11c+ CD11b+ AMs phagocytosed significantly more M. tuberculosis, which expressed higher RNA levels of genes required for M. tuberculosis survival. Our studies identify two distinct AM populations in old mice: a resident population and an increased CD11c+ CD11b+ AM subpopulation expressing monocytic markers, a unique inflammatory signature, and enhanced M. tuberculosis phagocytosis and survival when compared with resident CD11c+ CD11b- AMs, which are more immune regulatory in nature.

Type 2 diabetes mellitus induces TNFR1 mediated necroptotic cell death of mice alveolar macrophages infected with Mycobacterium tuberculosis

Abstract Previously, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and found that T2DM mice are susceptible to Mycobacterium tuberculosis (Mtb) infection. We also found that alveolar macrophages from T2DM mice were more permissive to Mtb growth ex vivo compared to non-diabetic controls. In the current study, we determined the defective mechanisms that make T2DM mice alveolar macrophages more susceptible to Mtb infection. Mtb infected alveolar macrophages from T2DM mice produced more TNF-α (973.8 ± 13.3 pg/ml vs. 614.6 ± 27.2 pg/ml, p<0.003) and less apoptotic (4.7 ± 1.8% vs. 28.6 ± 2.3%, p<0.001) compared to Mtb infected non-diabetic control mice. Mtb infected alveolar macrophages from T2DM mice expressed higher levels of TNFR1 (12.8 ± 0.5 vs. 2.09 ± 0.01, p<0.001) and markers of necroptosis RIPK1 (11.5 ± 0.57 vs. 2.94 ± 0.20, p<0.002), RIPK3 (16.1 ± 0.58 vs. 2.89 ± 0.2, p<0.001) and MLKL (9.2 ± 0.56 vs. 1.58 ± 0.72, p<0.02) compared to Mtb infected alveolar macrophages from non-diabetic control mice as determined by real-time PCR. This finding was again proved by Western blot and confocal microscopy. Anti-TNFR1 antibody treatment of alveolar macrophages from T2DM mice before or after Mtb infection reduced RIPK1, RIPK3 and MLKL expression as determined by RT-PCR. Our findings demonstrate that T2DM induces necroptosis of alveolar macrophages upon Mtb infection. Enhanced TNFR1 signaling in T2DM mice alveolar macrophages is responsible for enhanced necroptosis. We are also determining the subpopulations of T2DM mice alveolar macrophages that express higher levels of TNFR1 upon Mtb infection. Studies are underway to determine the in vivo relevance of our current findings to Mtb growth in T2DM mice.

Whole-Lung Irradiation Results in Pulmonary Macrophage Alterations that are Subpopulation and Strain Specific.

Exposure of the lung to radiation produces injury and inflammatory responses that result in microenvironmental alterations, which can promote the development of pneumonitis and/or pulmonary fibrosis. It has been shown that after other toxic insults, macrophages become phenotypically polarized in response to microenvironmental signals, orchestrating the downstream inflammatory responses. However, their contribution to the development of the late consequences of pulmonary radiation exposure remains unclear. To address this issue, fibrosis-prone C57BL/6J mice or pneumonitis-prone C3H/HeJ mice were whole-lung irradiated with 0 or 12.5 Gy and lung digests were collected between 3 and 26 weeks after radiation exposure. CD45(+) leukocytes were isolated and characterized by flow cytometry, and alveolar, interstitial and infiltrating macrophages were also detected. Ly6C, expressed by pro-inflammatory monocytes and macrophages, and mannose receptor (CD206), a marker of alternative activation, were assessed in each subpopulation. While the total number of pulmonary macrophages was depleted at 3 weeks after lung irradiation relative to age-matched controls in both C57 and C3H mice, identification of discrete subpopulations showed that this loss in cell number occurred in the alveolar, but not the interstitial or infiltrating, subsets. In the alveolar macrophages of both C57 and C3H mice, this correlated with a loss in the proportion of cells that expressed CD206 and F4/80. In contrast, in interstitial and infiltrating macrophages, the proportion of cells expressing these markers was increased at several time points after irradiation, with this response generally more pronounced in C3H mice. Radiation exposure was also associated with elevations in the proportion of alveolar and interstitial macrophage subpopulations expressing Ly6C and F4/80, with this response occurring at earlier time points in C57 mice. Although the radiation dose used in this study was not isoeffective for the inflammatory response in the two strains, the differences observed in the responses of these discrete macrophage populations between the fibrosis-prone versus pneumonitis-prone mice nonetheless suggest a possible role for these cells in the development of long-term consequences of pulmonary radiation exposure.

MR imaging and targeting of a specific alveolar macrophage subpopulation in LPS-induced COPD animal model using antibody-conjugated magnetic nanoparticles

PurposeTargeting and noninvasive imaging of a specific alveolar macrophage subpopulation in the lung has revealed the importance for early and better diagnosis and therapy of chronic obstructive pulmonary disease (COPD). In this study, the in vivo effect of pulmonary administration of iron oxide nanoparticles on the polarization profile of macrophages was assessed, and a noninvasive free-breathing magnetic resonance imaging (MRI) protocol coupled with the use of biocompatible antibody-conjugated superparamagnetic iron oxide (SPIO) nanoparticles was developed to enable specific targeting and imaging of a particular macrophage subpopulation in lipopolysaccharide-induced COPD mice model.Materials and methodsEnzyme-linked immunosorbent assay, Real-time polymerase chain reaction, and flow cytometry analysis were performed to assess the biocompatibility of PEGylated dextran-coated SPIO nanoparticles. Specific biomarkers for M1 and M2 macrophages subsets were selected for conjugation with magnetic nanoparticles. MRI protocol using ultra-short echo time sequence was optimized to enable simultaneous detection of inflammation progress in the lung and detection of macrophages subsets. Flow cytometry and immunohistochemistry analysis were finally performed to confirm MRI readouts and to characterize the polarization profile of targeted macrophages.ResultsThe tested SPIO nanoparticles, under the current experimental conditions, were found to be biocompatible for lung administration in preclinical settings. Cluster of differentiation (CD)86- and CD206-conjugated magnetic nanoparticles enabled successful noninvasive detection of M1 and M2 macrophage subpopulations, respectively, and were found to co-localize with inflammatory regions induced by lipopolysaccharide challenge. No variation in the polarization profile of targeted macrophages was observed, even though a continuum switch in their polarization might occur. However, further confirmatory studies are required to conclusively establish this observation.ConclusionCoupling of magnetic iron oxide nanoparticles with a specific antibody targeted to a particular macrophage subpopulation could offer a promising strategy for an early and better diagnosis of pulmonary inflammatory diseases using noninvasive MRI.

SELECTIVE EFFECTS OF PULMONARY SURFACTANT ON VARIOUS SUBPOPULATIONS OF ALVEOLAR MACROPHAGES IN THE MODEL OF EXPERIMENTAL TUBERCULOSIS

Pulmonary surfactant is necessary component for maintenance of high level of phagocytic activity of alveolar macrophages. Tuberculosis inflammation reduces the production of surfactant by type II cells and phagocytic activity of alveolar macrophages. The effects of exogenous pulmonary surfactant on the ultrastructural changes of various subpopulations of alveolar macrophages were studied by TEM-method. For investigations the bronchial alveolar lavage fluid from guinea pigs infected of M. tuberculosis and treated by isoniatid or isoniazid + exogenous pulmonary surfactant were used. It was shown that isoniazid + exogenous pulmonary surfactant normalizes the heterogeneous population of alveolar macrophages providing stimulating effects on their maturation and phagocytic activity more effectively than isoniazid therapy.

Pulmonary Macrophage Subpopulations in the Induction and Resolution of Acute Lung Injury

Macrophages are key orchestrators of the inflammatory and repair responses in the lung, and the diversity of their function is indicated by their polarized states and distinct subpopulations and localization in the lung. Here, we characterized the pulmonary macrophage populations in the interstitial and alveolar compartments during the induction and resolution of acute lung injury induced by Pseudomonas aeruginosa infection. We identified macrophage subpopulations and polarity according to FACS analysis of cell surface protein markers, combined with cell sorting for gene expression using real-time PCR. With these techniques, we validated a novel, alternatively activated (M2) marker (transferrin receptor), and we described three interstitial and alveolar macrophage subpopulations in the lung whose distribution and functional state evolved from the induction to resolution phases of lung injury. Together, these findings indicate the presence and evolution of distinct macrophage subsets in the lung that serve specific niches in regulating the inflammatory response and its resolution. Alterations in the balance and function of these subpopulations could lead to nonresolving acute lung injury.

Alveolar Macrophage Subpopulations in Bronchoalveolar Lavage and Induced Sputum of Asthmatic and Control Subjects

Background. Alveolar macrophages (AM) are the most numerous immune cells in the airways and are involved in the immunological homeostasis of the lung. Intriguingly, their role in asthma remains unclear probably, in part, because of their heterogeneity. Objective. To characterize AM population from bronchoalveolar lavage (BAL) and induced sputum (IS) of asthmatic and normal subjects using specific biomarkers. Methods. Non-asthmatic non-allergic and allergic mild asthmatic subjects were recruited for this study. AM were obtained from BAL and IS and cytospins were prepared. Immunocytochemistry was performed for nine cellular markers (CD68, RFD7, CD14, CD11b, CD83, CD64, CD80, CD86, and FIZZ1). Results. Asthmatic subjects had more AM RFD7+ in BAL compared with IS, whereas control subjects had more AM RFD7+ in IS than in BAL. Consequently, there was an increased number of AM RFD7+ in BAL of asthmatic subjects compared with BAL of control subjects. AM CD11b+ was higher in BAL than in IS in both groups. The expression of FIZZ1, marker of macrophage alternative activation, was similar in asthmatic and normal subjects. Conclusion. The expression of cellular markers on AM differs according to their localization in the lung. Subpopulations of AM may contribute to the inflammatory profile observed in asthmatic subjects.

Delineation of pulmonary alveolar macrophage subpopulations by flow cytometry in normal subjects and in patients with lung cancer.

We have previously described alterations in pulmonary alveolar macrophage (PAM) function in patients with lung cancer when compared with control subjects. This study examined PAM from five patients with lung cancer, seven normal volunteers and nine control patients, to assess whether any differences in surface phenotypic markers were present in lung cancer versus non-cancer subjects, and what changes might be induced with interferon-gamma (IFN-gamma) stimulation. After 3 days' culture with or without IFN-gamma no differences were seen in the percentages of cells staining positively in each group for HLA class I, class II and ICAM-1 (CD54) antigens. However, in 13 out of 14 control subjects, and only one out of the five cancer subjects, dual PAM populations were identified. The second PAM population (PAM-2) was larger and demonstrated a higher expression of class I and ICAM-1 antigens. Unlike the unfractionated PAM population, PAM-2 consistently responded to IFN-gamma stimulation with an increase in both class I (90 +/- 25%) and ICAM-1 (45 +/- 10%) antigens, while there was no change in class II antigen expression. In three subjects PAM-2 was found to induce a significantly greater mitogen response than the rest of the PAM population. If confirmed in a larger group of patients, the absence of PAM-2 in the majority of patients with lung cancer may underlie the functional PAM defects observed in these patients.

Phenotypical and functional characterization of alveolar macrophage subpopulations in the lungs of NO2-exposed rats

BackgroundAlveolar macrophages (AM) are known to play an important role in the regulation of inflammatory reactions in the lung, e.g. during the development of chronic lung diseases. Exposure of rats to NO2 has recently been shown to induce a shift in the activation type of AM that is characterized by reduced TNF-α and increased IL-10 production. So far it is unclear, whether a functional shift in the already present AM population or the occurrence of a new, phenotypically different AM population is responsible for these observations.MethodsAM from rat and mice were analyzed by flow cytometry for surface marker expression and in vivo staining with PKH26 was applied to characterize newly recruited macrophages. Following magnetic bead separation, AM subpopulations were further analyzed for cytokine, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP) mRNA expression using quantitative RT-PCR. Following in vitro stimulation, cytokines were quantitated in the culture supernatants by ELISA.ResultsIn untreated rats the majority of AM showed a low expression of the surface antigen ED7 (CD11b) and a high ED9 (CD172) expression (ED7-/ED9high). In contrast, NO2 exposure induced the occurrence of a subpopulation characterized by the marker combination ED7+/ED9low. Comparable changes were observed in mice and by in vivo labeling of resident AM using the dye PKH26 we could demonstrate that CD11b positive cells mainly comprise newly recruited AM. Subsequent functional analyses of separated AM subpopulations of the rat revealed that ED7+ cells showed an increased expression and production of the antiinflammatory cytokine IL-10 whereas TNF-α production was lower compared to ED7- AM. However, iNOS and IL-12 expression were also increased in the ED7+ subpopulation. In addition, these cells showed a significantly higher mRNA expression for the matrix metalloproteinases MMP-7, -8, -9, and -12.ConclusionNO2 exposure induces the infiltration of an AM subpopulation that, on the one hand may exert antiinflammatory functions by the production of high amounts of IL-10 but on the other hand may contribute to the pathology of NO2-induced lung damage by selective expression of certain matrix metalloproteinases.

Cockade-Like Structures in Alveolar Macrophages in Extrinsic Allergic Alveolitis

Background and Objectives: In immunocytochemical preparations of bronchoalveolar lavage (BAL) cells from patients with extrinsic allergic alveolitis (EAA), we observed the presence of alveolar macrophages with cockade-like structures in their cytoplasm (cockade+ alveolar macrophages). These cockade+ alveolar macrophages may reflect a subpopulation of alveolar macrophages which may show a different predominance in various interstitial lung diseases. In this study we aimed to compare the frequency of cockade+ alveolar macrophages in patients with EAA (n = 14) with the results obtained in patients with sarcoidosis (n = 11), idiopathic interstitial pneumonia (IIP; n = 10) and control subjects (n = 8). We also investigated the expression of the transferrin receptor CD71 on cockade+ alveolar macrophages. Methods: In BAL fluid, the total number of cells and differential counts were determined, and immunocytologic examinations of macrophages and lymphocytes were done using monoclonal antibodies. The percentage of cockade+ alveolar macrophages was determined by counting 300 macrophages in the CD20 field of an immunocytochemical slide. Results: The percentage of cockade+ alveolar macrophages was significantly higher in the EAA group (36 ± 9%) compared to patients with sarcoidosis (12 ± 5%) or IIP (11 ± 10%) and control subjects (3 ± 1%; p < 0.001). The proportion of CD71+ alveolar macrophages was significantly lower in EAA than in the other groups (p < 0.01), and the CD71 antigen was expressed on a significantly lower proportion of cockade+ alveolar macrophages compared to cockade– alveolar macrophages in EAA (p < 0.001). Conclusion: We conclude that cockade+ alveolar macrophages could play a role in the pathogenesis and differential diagnosis EAA.

Phagocytosis of alveolar macrophages after conagenin injection to rats.

Phagocytic functions of rat alveolar macrophages (AM) following intraperitoneal injection of conagenin (CNG) and of AM sub-populations fractionated by Percoll discontinuous gradient centrifugation were investigated. Phagocytosis of opsonized-sheep red blood cells (SRBC) following in vitro incubation with CNG showed a significant increase in a higher density of AM (fraction IV). In addition, phagocytosis was also increased in lower density ones (fractions I and II) by macrophage-activating factor (MAF) co-cultivation. CNG-injected rats for 5 consecutive days showed a dose-dependent increase in phagocytosis of AM compared to the control rats. Although the distribution of AM sub-population in rats injected CNG was not significantly different compared to the control rats, phagocytosis was significantly increased in AM of a lower density fraction (fraction II). These results suggest that CNG directly increases phagocytosis of AM in a higher density fraction, and indirectly enhances phagocytosis in AM of a lower density fraction via increasing MAF-like material production.

Ultrastructural Heterogeneity of the Alveolar Macrophages from Tobacco Smokers with Chronic Bronchitis

Alveolar macrophages recovered by bronchoalveolar lavage from 14 heavy smokers with chronic bronchitis were assessed. Ultrastructural examination revealed marked cellular heterogeneity. Three subpopulations of alveolar macrophages were readily identifiable. These have been termed "young," "mature," and "degrading," reflecting their ultrastructural features. In addition, a majority of the cells were found to be positive by TUNEL staining, indicating DNA damage, but a very small percentage tested positive for Caspase-3, suggesting that apoptosis might not account for the DNA damage in at least some of these cells. A small percentage of proliferating cells were noted.

Changes in Distribution and Morphology of Rat Alveolar Macrophage Subpopulations in Acute Hyperoxic Lung Injury Model

Background : In acute lung injury, alveolar macrophages play a pivotal role in the inflammatory process during the initiation phase and in the reconstruction and fibrosis process during the later phase. Recently, it has been proven that alveolar macrophages are constituted by morphologically, biochemically and immunologically heterogenous cell subpopulations. The possibility of alterations to these characteristics of the alveolar macrophage population during lung disease has been raised. To investigate such a possibility a hyperoxic rat lung model was made to check the distributional and morphological changes of rat alveolar macrophage subpopulation in acute hyperoxic lung injury. Method : Alveolar macrophage were lavaged from normal and hyperoxic lung injury rats and separated by discontinuous gradients of percoll. After cell counts of each density fraction were accessed, the morphomeric analysis of alveolar macrophages was performed on cytocentrifuged preparations by transmission electron micrograph. Result : 1. The total alveolar macrophage cell count significantly increased up to 24 hours after hyperoxic challenge (normal control group , 12 hour group , 24 hour group , p) did not show a significant difference. 2. Alveolar septal thickness significantly increased up to 24 hours after hyperoxic challenge(normal control group , 12 hour group , 24 hour group , p). Number of interstitial macrophage markedly increased at 24 hour group. 3. Hypodense fraction(fraction 1 and fraction 2) of alveolar macrophage showed a significant increase following hyperoxic challenge (.. p. p

Modulation of Adhesion Molecule Profiles on Alveolar Macrophages and Blood Leukocytes

Background: Cell adhesion molecules are believed to be essential for blood cell recruitment to the lung and for the movement of alveolar macrophages (AM) within the lung. Objective: To investigate the expression pattern of L-selectin and β₂ integrins on blood leukocytes and AM, including AM of various maturity. Methods: Flow cytometry was used to study the expression of L-selectin (CD62L) and of the β₂ integrins CD11a, CD11b, and CD11c on AM (including density-defined subpopulations), monocytes (Mo), polymorphonuclear neutrophils (PMN) and lymphocytes (Ly) sampled from healthy individuals, during incubation with and without lipopolysaccharide (LPS). Results: A significantly different modulation pattern of β₂ integrins and L-selectin was demonstrated on Mo and AM, cells of the same differentiation lineage. In contrast to AM, Mo had a marked ability to respond to LPS stimulation by increased expression of CD11a, CD11b and CD11c and decreased expression of L- selectin. These molecules were expressed to a similar degree on AM, whereas the basal levels of CD11b and L-selectin were considerably higher on Mo than on AM. A significantly different expression of CD11a as well as differences in the regulation of L-selectin during incubation were also demonstrated between density-defined subpopulations of AM. CD11a could not be upregulated on PMN, otherwise the modulation patterns of CD11b, CD11c and L-selectin were similar to that on Mo. The expression of CD11a on Ly was 3- to 6-fold higher than on Mo, PMN and AM. The level of CD11b decreased significantly upon incubation (uninfluenced by LPS stimulation), and CD11c was hardly expressed on Ly. The level of L-selectin on Ly was higher than on Mo, AM and PMN and was not decreased during incubation. Conclusion: Developmental origin, degree of cell differentiation (maturity) as well as different environmental conditions all heavily influence the expression and modulation pattern of β₂ integrins and L-selectin on leukocytes and Mo-derived AM.