The ultrastructure of mouse lung; general architecture of capillary and alveolar walls

The general architecture of capillary and alveolar walls of the mouse lung was studied by means of the electron microscope. In order to minimize tissue damage and to improve the cutting properties of embeddings, several modifications in the tissue processing methods were adopted. These modifications were: fixation by infusion, a prolonged time of dehydration, of impregnation, and of polymerization, the use of acetone for dehydration, ammonium sulfide treatment of the fixed and washed tissue, and an elevated (80 degrees C.) polymerization temperature combined with the use of prepolymerized methacrylate. The generally favorable effects of these modified methods upon preservation and cutting properties of embedded tissue are discussed. Both capillary endothelium and alveolar epithelium were found continuous and without pores. The endothelium was seen to be thinnest in those portions that were adjacent to alveolar air spaces. Two morphological "types" of alveolar epithelial cells were found. One protruded into the alveolar lumen with its thick portion containing the nucleus. The other was often located in a niche of the alveolar wall, and contained peculiar dark inclusions amidst numerous mitochondria. Both were attenuated at their periphery to form the thin epithelial layer. The layer between endothelium and epithelium was designated as basement membrane. It was seen to be generally thin and structureless, but was found thickened in some areas where it also contained collagen fibrils.     

The alveolar macrophage

The alveolar macrophage is one of the few tissue macrophage populations readily accessible to study both in the human and in animals. Since harvesting of these cells by bronchoalveolar lavage was first described in 1961, alveolar macrophages have been extensively investigated. This population is the predominant cell type within the alveolus, and undoubtedly serves as the first line of host defense against inhaled organisms and soluble and particulate molecules. Early studies focussed on this endocytic role and delineated the cells' phagocytic and microbicidal capacities. More recent investigations demonstrated an extensive synthetic and secretory repertoire including lysozyme, neutral proteases, acid hydrolases and O2 metabolites. In addition, the complex immunoregulatory role of the macrophage has also been appreciated. These cells have been shown to produce a wide variety of pro- and anti-inflammatory agents including arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathways, cytokines which modulate lymphocyte function and factors which promote fibroblast migration and replication.     

The alveolar macrophage: the Trojan horse of Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, has a particular strategy for invading the host and crossing the alveolar barrier. B. anthracis survives within alveolar macrophages, after germination within the phagolysosome, then enters the external medium where it proliferates. Recent data have shown that edema toxin and lethal toxin are the major genetic determinants mediating the survival of germinated spores within macrophages. Here, recent advances in the analysis of B. anthracis pathogenesis are summarized and future challenges discussed.     

Modeling the cellular impact of nanoshell-based biosensors using mouse alveolar macrophage cultures

In this study, the relative toxicity of native gold-silica nanoshells (NS) has been compared to nanoshells modified with poly(ethylene glycol)-thiol (PEG-SH) and a Raman-active PEG, p-mercaptoaniline-poly(ethylene glycol) (pMA-PEG), in mouse alveolar macrophage cell cultures (RAW 264.7). The results from toxicity profiling using an MTT assay demonstrate that cell viability post-particle exposure is a function of three factors: nanoshell concentration, surface functionalization, and incubation time. By minimizing particle concentrations and incubation times, cell cultures are able to recover within 24 h of nanoshell removal, indicative of nanoshells having more of a cytostatic versus cytotoxic effect on macrophage cells. The mechanism of the cytostatic effect has been investigated by imaging the presence of reactive oxygen species (ROS) using a fluorescence assay kit (Image-iT? LIVE) after the introduction of NS to the cell cultures. Elevated ROS signals are seen in the cells containing higher concentration of NS, and indicate that the major reason of toxicity may due to the oxidative stress caused by excess NS particles. Raman imaging experiments with pMA-PEG coated nanoshells showed that cells exposed for even short exposure times (～2 h) retained those particles up to 24 h after exposure, while migration experiments suggest that surviving cells retain their nanoshells and may reallocate them to progeny cells upon cell division.     

The isolation of a rat alveolar macrophage lectin

A lectin in rat alveolar macrophage membranes with a high affinity for binding ligands containing L-fucose and N-acetyl-D-glucosamine has been isolated by affinity chromatography on Fuc-BSA-Sepharose (where Fuc is fucosyl and BSA is bovine serum albumin). The lectin was extracted from rat lung homogenates with Triton X-100, absorbed from the extract onto Fuc-BSA-Sepharose in the presence of Ca2+ and eluted by removal of Ca2+. After a second adsorption to and elution from Fuc-BSA-Sepharose, three protein species were detected electrophoretically in fractions that bind Fuc-BSA. One, which was the mannose/N-acetylglucosamine lectin (Mr = 32,000) found earlier in hepatocytes, was removed by adsorption on anti-lectin IgG-Sepharose. Another (Mr = 46,000) was removed by adsorption to Fuc-BSA-Sepharose and elution with galactose. The remaining lectin (Mr = 180,000) bound fucose and N-acetylglucosamine but not galactose. Binding was maximal between pH 6.5 and 9.0 and dependent on Ca2+. Immunocytological analysis with rabbit anti-lectin IgG and fluorescein-labeled goat anti-rabbit IgG revealed the lectin to be in rat alveolar macrophages and nonparenchymal cells of liver. Thus, the lectin appears to be present in macrophages and is likely involved in receptor-mediated endocytosis. It is distinctly different structurally from the hepatocyte lectin with a similar ligand-binding specificity.     

Doxycycline treatment prevents alveolar destruction in VEGF-deficient mouse lung

In vivo lung-targeted VEGF gene inactivation results in pulmonary cell apoptosis, airspace enlargement, and increased lung compliance consistent with an emphysema-like phenotype. The predominant hypothesis for the cause of lung destruction in emphysema is an imbalance between active lung protease and anti-protease molecules. Therefore, we investigated the role of protease (e.g., matrix metalloproteinases--MMPs) and anti-protease (e.g., tissue inhibitors of metalloproteinases--TIMPs) expression in contributing to the lung structural remodeling observed in pulmonary-VEGF-deficient mice. VEGFLoxP mice instilled through the trachea with an adeno-associated virus expressing Cre recombinase (AAV/Cre) manifest airspace enlargement and a greater (P < 0.05) mean linear intercept (MLI: 44.2 +/- 4.2 microm) compared to mice instilled with a control virus expressing LacZ (31.3 +/- 2.5 microm). Airspace enlargement was prevented by the continuous administration of the general MMP inhibitor, doxycycline (Dox) (Cre + Dox: 32.6 +/- 2.5 microm), and MLI values were not different from either control (LacZ + Dox: 30.5 +/- 1.2 microm). In situ magnetic resonance imaging of VEGF gene inactivated mouse lungs revealed uneven inflation, residual trapped gas volumes upon oxygen absorption deflation/re-inflation, and loss of parenchymal structure; effects that were largely prevented by Dox. Five weeks after AAV/Cre infection Western blot revealed a 9.9-fold increase in pulmonary MMP-3, and 2-fold increases in MMP-9 and TIMP-2. However, the increase in MMP-3 was prevented by Dox administration and was associated with a 2-fold increase in serpin b5 (Maspin) expression. These results suggest that doxycycline treatment largely prevents the aberrant lung remodeling response observed in VEGF-deficient mouse lungs and is associated with changes in protease and anti-protease expression.     

M2 macrophage polarisation is associated with alveolar formation during postnatal lung development

Background

Macrophages are traditionally associated with inflammation and host defence, however a greater understanding of macrophage heterogeneity is revealing their essential roles in non-immune functions such as development, homeostasis and regeneration. In organs including the brain, kidney, mammary gland and pancreas, macrophages reside in large numbers and provide essential regulatory functions that shape organ development and maturation. However, the role of macrophages in lung development and the potential implications of macrophage modulation in the promotion of lung maturation have not yet been ascertained.

Methods

Embryonic day (E)12.5 mouse lungs were cultured as explants and macrophages associated with branching morphogenesis were visualised by wholemount immunofluorescence microscopy. Postnatal lung development and the correlation with macrophage number and phenotype were examined using Colony-stimulating factor-1 receptor-enhanced green fluorescent protein (Csf1r-EGFP) reporter mice. Structural histological examination was complemented with whole-body plethysmography assessment of postnatal lung functional maturation over time.Flow cytometry, real-time (q)PCR and immunofluorescence microscopy were performed to characterise macrophage number, phenotype and localisation in the lung during postnatal development. To assess the impact of developmental macrophage modulation, CSF-1 was administered to neonatal mice at postnatal day (P)1, 2 and 3, and lung macrophage number and phenotype were assessed at P5. EGFP transgene expression and in situ hybridisation was performed to assess CSF-1R location in the developing lung.

Results

Macrophages in embryonic lungs were abundant and densely located within branch points during branching morphogenesis. During postnatal development, structural and functional maturation of the lung was associated with an increase in lung macrophage number. In particular, the period of alveolarisation from P14-21 was associated with increased number of Csf1r-EGFP+ macrophages and upregulated expression of Arginase 1 (Arg1), Mannose receptor 1 (Mrc1) and Chemokine C-C motif ligand 17 (Ccl17), indicative of an M2 or tissue remodelling macrophage phenotype. Administration of CSF-1 to neonatal mice increased trophic macrophages during development and was associated with increased expression of the M2-associated gene Found in inflammatory zone (Fizz)1 and the growth regulator Insulin-like growth factor (Igf)1. The effects of CSF-1 were identified as macrophage-mediated, as the CSF-1R was found to be exclusively expressed on interstitial myeloid cells.

Conclusions

This study identifies the presence of CSF-1R+ M2-polarised macrophages localising to sites of branching morphogenesis and increasing in number during the alveolarisation stage of normal lung development. Improved understanding of the role of macrophages in lung developmental regulation has clinical relevance for addressing neonatal inflammatory perturbation of development and highlights macrophage modulation as a potential intervention to promote lung development.     

Purification of the human alveolar macrophage mannose receptor

We report here the first isolation of a mannose receptor from human lung, and identify the alveolar macrophage as the cell of origin. The receptor was purified from detergent-solubilized lung tissue by absorption to mannose- and fucose-Sepharose, and elution with EDTA. The eluted protein had a molecular weight of 175 kD. Maximum binding of 125I-mannan-2 to the isolated receptor occurred at pH 7.5. Binding was inhibited by 40 micrograms/ml mannan (75%); 200 mM mannose (89%); and 200 mM fucose (93%). Galactose (200 mM) had no effect. Polyclonal antibodies raised against the purified receptor reacted with the purified 175 kD protein and a 175 kD protein from detergent extracts of human alveolar macrophages by immunoblot analysis. The antibody immunoprecipitated a 175 kD protein from solubilized 125I-labeled human alveolar macrophage membranes. These studies indicate that the 175 kD protein purified from human lung is the cell surface alveolar macrophage mannose receptor.     

A continuous alveolar macrophage cell line: Comparisons with freshly derived alveolar macrophages

Summary Responses of a recently developed rat alveolar macrophage cell (NR8383.1) line were compared to those of freshly derived alveolar macrophages in vitro. Marked inter- and intraspecies heterogeneity in levels of phagocytosis of unopsonizedPseudomonas aeruginosa or zymosan was noted among freshly derived alveolar macrophages from rats, rabbits, and baboons. In contrast, phagocytic responses of alveolar macrophage cell line were predictable and highly reproducible. Similar results were obtained in measuring oxidative burst, as indicated by the production of H₂O₂ and luminol-enhanced chemiluminescence. Responses were again highly variable in freshly derived alveolar macrophages stimulated with zymosan or phorbol myristic acetate; moreover, freshly derived alveolar macrophages exhibited a wide range of chemiluminescence activity in unstimulated cultures. Results strongly suggest that data derived from the continuous alveolar macrophage culture NR8383.1 can be extrapolated to freshly derived alveolar macrophages of various species, and in many experiments will be useful in avoiding the significant animal-to-animal variance observed among freshly derived cell preparations. This work was supported in part by grant A119811 and SCOR HL23578, from the National Institutes of Health, Bethesda, MD. Portions of these studies appeared as a poster presentation at the American Society for Microbiology, Atlanta, GA, 1987.     

Umbilical cord MSC-derived exosomes improve alveolar macrophage function and reduce LPS-induced acute lung injury

Acute lung injury (ALI) is a severe condition that can progress to acute respiratory distress syndrome (ARDS), with a high mortality rate. Currently, no specific and compelling drug treatment plan exists. Mesenchymal stem cells (MSCs) have shown promising results in preclinical and clinical studies as a potential treatment for ALI and other lung-related conditions due to their immunomodulatory properties and ability to regenerate various cell types. The present study focuses on analyzing the role of umbilical cord MSC (UC-MSC))-derived exosomes in reducing lipopolysaccharide-induced ALI and investigating the mechanism involved. The study demonstrates that UC-MSC-derived exosomes effectively improved the metabolic function of alveolar macrophages and promoted their shift to an anti-inflammatory phenotype, leading to a reduction in ALI. The findings also suggest that creating three-dimensional microspheres from the MSCs first can enhance the effectiveness of the exosomes. Further research is needed to fully understand the mechanism of action and optimize the therapeutic potential of MSCs and their secretome in ALI and other lung-related conditions.     

Complement C5a exacerbates acute lung injury induced through autophagy-mediated alveolar macrophage apoptosis

Intestinal ischemia has a high mortality and often causes acute lung injury (ALI), which is a serious complication, and is accompanied by high mortality up to 40%. An intense local and systemic inflammation occurs during intestinal ischemia/reperfusion (IR)-induced lung injury resulting from activation of immune responses. It has been reported that one component of complement, C5a, is indispensable for the full development of IR-induced lung injury, whereas the detailed molecular mechanism remains to be elucidated. In this study, we found that intestinal IR induced ALI-like symptoms, and C5a receptor (C5aR) expression was upregulated in alveolar macrophages, which are resident macrophages in lung tissue and are important in pulmonary homeostasis. C5a produced during lung injury binds to C5aR in alveolar macrophages, initiates downstream signaling that promotes autophagy, leading to apoptosis of alveolar macrophages. Using Mφ-ATG5^−/− mice, in which the atg5 is deficient specifically in macrophages and autophagy is inhibited, we confirmed that in vivo C5a interacting with C5aR induced autophagy in alveolar macrophages, which promoted alveolar macrophage apoptosis. Further study indicated that autophagy was induced through C5aR-mediated degradation of bcl-2. Taken together, our results demonstrated that C5aR-mediated autophagy induced apoptosis in alveolar macrophages, disrupting pulmonary homeostasis and contributing to the development of ALI. This novel mechanism suggests new therapeutic potential of autophagy regulation in ALI.During diverse clinical procedures, transient ischemia and reperfusion, known as ischemia/reperfusion (IR) clinically, are found in organs or tissues, and cause intense inflammation, both locally and systemically,^{1, 2} which in turn leads to various types of injury, even multiple organ failure, contributing to high mortality. Acute lung injury (ALI) is a common outcome of IR, and usually occurs in patients with intestinal ischemia, leading to high mortality of 60–80%.³ In addition, ALI is a life-threatening complication associated with sepsis, pneumonia, trauma, and many other clinical conditions. Despite improvements in the management of critically ill patients, ALI mortality is approximately 40%, and survivors often do not return to a normal life.⁴ During the IR process, ischemia initiates a local inflammatory response, by releasing pro-inflammatory factors and activating/attracting inflammatory cells, such as neutrophils, macrophages, and lymphocytes.⁵ Oxidative stress resulting from ischemia also contributes to IR injury. Owing to the unique anatomic and physiological features, the lung is susceptible to IR injury through pro-inflammatory cytokines storm.⁶ Only a few pharmacologic treatment options are available for IR-induced ALI, which work by inhibiting inflammation or anti-oxidative effects.⁷ Obviously, more effort is needed to clarify the underlying pathophysiological mechanisms of ALI and find more efficient therapeutic methods.Macrophages are believed to derive from hematopoietic stem cells and are distributed all over the body. Macrophages are of vital importance in immune homeostasis, tissue remodeling, and biological events. Alveolar macrophages are resident lung macrophages, and present the first line of encountering inhaled substances.⁸ Alveolar macrophages have essential roles in maintaining pulmonary homeostasis, without pro-inflammatory effects.⁹ More importantly, alveolar macrophages suppress excessive inflammation, putatively through the strong inhibition of local immune cells, such as T lymphocytes and DCs. For example, rodent alveolar macrophages render inhibition on T-cell activation in the presence of DCs in vitro, through multiple mechanisms, such as releasing the suppressive cytokines, transforming growth factor-β and interleukin-10 (IL-10).^{8, 9, 10, 11, 12} If alveolar macrophages are depleted, the animals display stronger inflammatory responses to otherwise innocuous inhaled antigens.¹³ During ALI, cytokines and chemokines produced by tissue macrophages recruit neutrophils to the injury sites,¹⁴ but the neutrophil recruitment also affects alveolar macrophage activity.^15,16 IL-10 production is induced by macrophages after phagocytosis of apoptotic neutrophils, which in turn suppresses additional cytokine production and inflammation, affecting both pro-inflammatory and anti-inflammatory cellular components of ALI.¹² For these reasons, alveolar macrophages have attracted interest in studies on the mechanisms of ALI.^{8, 9, 10, 11}Complements are key mediators of the first line in protecting hosts from pathogen invasions and have been shown to be involved in IR-induced inflammation. During the ignition and amplification stages, complement activation contributes to inflammation-mediated tissue injury,^{1, 2, 17} which would be significantly diminished if complement factors were depleted.^{18, 19} The complement activation product, C5a, is essential for the full development of injury. C5a has the ability of chemotaxis²⁰ and it can also directly activate neutrophils and macrophages for chemokine production.²¹ C5a receptor (C5aR) signaling is required for C5a to render its effects on the process, as blockade of C5aR signaling will have similar effects to depletion of C5a in the survival of animals with cecal ligation and puncture,²² suggesting that intercepting C5a or C5aR signaling may provide a potential target for therapeutic treatment in inflammatory diseases.²³Although significant effort has been aimed at determining the mechanism of macrophages in ALI, the activity of C5aR on macrophages is unclear. This study aimed to clarify the role of C5aR in macrophage biology during ALI development, and found that elevated C5a induced C5aR signaling in alveolar macrophages, and contributed to autophagy-mediated apoptosis, thus exacerbating the ALI symptoms. This novel mechanism provides a potential role for autophagy regulation in ALI therapeutic applications.