Particle-laden Alveolar Macrophages Research Articles

Centrality of Myeloid-Lineage Phagocytes in Particle-Triggered Inflammation and Autoimmunity.

Exposure to exogenous particles found as airborne contaminants or endogenous particles that form by crystallization of certain nutrients can activate inflammatory pathways and potentially accelerate autoimmunity onset and progression in genetically predisposed individuals. The first line of innate immunological defense against particles are myeloid-lineage phagocytes, namely macrophages and neutrophils, which recognize/internalize the particles, release inflammatory mediators, undergo programmed/unprogrammed death, and recruit/activate other leukocytes to clear the particles and resolve inflammation. However, immunogenic cell death and release of damage-associated molecules, collectively referred to as “danger signals,” coupled with failure to efficiently clear dead/dying cells, can elicit unresolved inflammation, accumulation of self-antigens, and adaptive leukocyte recruitment/activation. Collectively, these events can promote loss of immunological self-tolerance and onset/progression of autoimmunity. This review discusses critical molecular mechanisms by which exogenous particles (i.e., silica, asbestos, carbon nanotubes, titanium dioxide, aluminum-containing salts) and endogenous particles (i.e., monosodium urate, cholesterol crystals, calcium-containing salts) may promote unresolved inflammation and autoimmunity by inducing toxic responses in myeloid-lineage phagocytes with emphases on inflammasome activation and necrotic and programmed cell death pathways. A prototypical example is occupational exposure to respirable crystalline silica, which is etiologically linked to systemic lupus erythematosus (SLE) and other human autoimmune diseases. Importantly, airway instillation of SLE-prone mice with crystalline silica elicits severe pulmonary pathology involving accumulation of particle-laden alveolar macrophages, dying and dead cells, nuclear and cytoplasmic debris, and neutrophilic inflammation that drive cytokine, chemokine, and interferon-regulated gene expression. Silica-induced immunogenic cell death and danger signal release triggers accumulation of T and B cells, along with IgG-secreting plasma cells, indicative of ectopic lymphoid tissue neogenesis, and broad-spectrum autoantibody production in the lung. These events drive early autoimmunity onset and accelerate end-stage autoimmune glomerulonephritis. Intriguingly, dietary supplementation with ω-3 fatty acids have been demonstrated to be an intervention against silica-triggered murine autoimmunity. Taken together, further insight into how particles drive immunogenic cell death and danger signaling in myeloid-lineage phagocytes and how these responses are influenced by the genome will be essential for identification of novel interventions for preventing and treating inflammatory and autoimmune diseases associated with these agents.

Lung Toxicity Analysis of Nano-Sized Kaolin and Bentonite: Missing Indications for a Common Grouping.

Kaolin and bentonite (nanoclay NM-600) are nanostructured aluminosilicates that share a similar chemical composition, platelet-like morphology, and high binding capacity for biomolecules. To investigate if these material-based criteria allow for a common grouping, we prepared particle suspensions of kaolin and bentonite with a similar hydrodynamic diameter and administered them to NR8383 alveolar macrophages in vitro and also to a rat lung using quartz DQ12 as a reference material. Bentonite was far more bioactive in vitro, indicated by a lower threshold for the release of enzymes, tumor necrosis factor α, and H2O2. In addition, in the lung, the early effects of bentonite exceeded those of kaolin and even those of quartz, due to strongly increased numbers of inflammatory cells, and elevated concentrations of total protein and fibronectin within the bronchoalveolar lavage fluid. The pro-inflammatory effects of bentonite decreased over time, although assemblies of particle-laden alveolar macrophages (CD68 positive), numerous type-2 epithelial cells (immunopositive for pro-surfactant protein C), and hypertrophic lung epithelia persisted until day 21. At this point in time, kaolin-treated lungs were completely recovered, whereas quartz DQ12 had induced a progressive inflammation. We conclude that bentonite is far more bioactive than equally sized kaolin. This argues against a common grouping of aluminosilicates, previously suggested for different kaolin qualities.

Diesel exhaust inhalation induces heat shock protein 70 expression in vivo

Exposure to urban air pollution is an independent risk factor for increased cardiovascular diseases. Heat shock protein 70 (HSP70) has been implicated in the pathogenesis of vascular dysfunction and cardiovascular diseases. This study has been designed to determine whether inhalation of urban air induces HSP70 expression in the lung and blood as well as the association of HSP70 and air pollution-induced vascular dysfunction. Apolipoprotein E (Apo-E) deficient mice were exposed to diesel exhaust (DE) either acutely (3 days, 200 or 400 µg/m3 for 6 h/day) or chronically (7 weeks, 200 or 400 µg/m3 for 6 h/day). HSP70 was measured in the lung using immunohistochemistry, and in the plasma by ELISA. Abdominal aorta rings were used to determine vascular functional responses. Chronic DE-exposure increased the fraction of HSP70 positive alveolar macrophages (AM) that was related to the fraction of particle-laden AM in the lung (r2 = 0.48, p < 0.01). Chronic DE-exposure increased plasma HSP70 levels and reduced blood vessel responses to phenylephrine (PE). The fraction of particle-laden HSP70 positive AM was associated with abnormal vasoconstriction responses to PE induced by DE-exposure (r2 = 0.12, p = 0.02). Our results show that chronic inhalation of DE increases HSP70 expression in the lung and systemic circulation, and we postulate that HSP70 possibly contributes to air pollution induced vascular dysfunction and cardiovascular diseases.

Inhalation toxicity study of disk-shaped potassium octatitanate particles (Terracess TF) in rats following 90 days of aerosol exposure

Since fibrous particles such as asbestos and some man-made fibers (MMF) have been known to produce carcinogenic or fibrogenic effects, disk-shaped potassium octatitanate (POT) particles (trade name: Terracess TF) were manufactured as nonfibrous particles. A 90-day inhalation toxicity study of Terracess TF was performed to evaluate comparative inhalation toxicity of the disk shape with a fibrous shape that was previously evaluated. Four groups of 20 male and 15 female rats each were exposed to Terracess TF aerosols at concentrations of 0, 2, 10, or 50 mg/m3 for 90 days. Ten male and 10 female rats per group were sacrificed at 90 days of exposure. After 90 days of exposure, 5 male rats per group were sacrificed at 3 wk of recovery period and 4–5 male rats per group or 5 female rats per group were sacrificed at 15 wk of recovery for lung clearance and histopathology. The mass median aerodynamic equivalent diameter (MMAED) of the aerosols of test materials ranged from 2.5 to 2.9 μm. There were no test-substance-related adverse effects on clinical observations. At the end of the 90-day exposure, a slight increase in lung-to-body weight ratios was observed at 50 mg/m3 in male but not in female rats. However, lung weights were within normal limits after 3- or 15-wk recovery periods. Microscopically, inhaled Terracess TF particles were mostly phagocytized by free alveolar macrophages (AMs) in the alveolar airspaces and alveolar walls maintained normal structure at 2 and 10 mg/m3. At 50 mg/m3, some alveoli were distended and filled with aggregates of particle-laden AMs. The alveolar walls showed slight type II pneumocyte hyperplasia, but neither proliferative inflammation nor alveolar fibrosis was present at 50 mg/m3. The clearance half-times for Terracess TF were estimated to be in the order of 6 to 9 mo for the 50-mg/m3 group and 2 to 3 mo for the 10- and 2-mg/m3 groups. The lung responses and lung clearance rate were comparable to those of “nuisance” type dusts at these concentrations. Based on interpretation that aggregated particle-laden AMs in alveoli was considered to be an early histopathological sign of lung overloading, an effect level was considered to be 50 mg/m3 and no-observed-adverse-effect level (NOAEL) was 10 mg/m3. This experiment clearly demonstrated that particle morphology was considered to be an important factor to determine inhaled particle toxicity.

Extrapulmonary translocation of intratracheally instilled fine and ultrafine particles via direct and alveolar macrophage-associated routes

Translocation of inhaled ultrafine particles from the lungs into the blood may impair cardiovascular function. We administered ultrafine (20-nm) and fine (200-nm) gold colloid or fluorescein-labeled polystyrene particles to mice intratracheally and examined their localization in the lung and extrapulmonary organs. Fifteen minutes after instillation, dispersed and agglomerated 20-nm gold colloid particles were observed on the surface of endothelial cells, on the alveolar surface, in endocytotic vesicles of alveolar epithelial cells, and in the basement membrane of the lung. A small but noteworthy amount of gold was detected in the liver, kidney, spleen, and heart by inductively coupled plasma-mass spectrometry. After administration of 20- or 200-nm fluorescent particles, free particles were detected infrequently in blood vessels, on the endocardial surface, and in the kidney and liver only in the mice that received 20-nm particles, whereas phagocytes containing 20- or 200-nm particles were found in the extrapulmonary tissues. Fluorescent particle-laden alveolar macrophages administered intratracheally translocated from alveoli to extrapulmonary organs via the blood circulation. Thus, small amounts of ultrafine particles are transported across the alveolar wall into the blood circulation via endocytotic pathways, but particle-laden alveolar macrophages translocate both ultrafine and fine particles from the lungs to the extrapulmonary organs.

Anthracosis in the lungs and associated lymph nodes in sheep and its potential role in the occurrence of pneumonia

The aim of the present study was to examine gross and histopathological findings in the lungs and associated lymph nodes of sheep with anthracosis and to correlate its potential role in the occurrence of pneumonia in the region of Kars, Turkey. In the present study, the lungs, mediastinal and bronchial lymph nodes of 2000 sheep slaughtered in two local abattoirs were examined for anthracosis throughout the year 2000. Anthracosis was found in the lungs and associated lymph nodes of 45 (2.25%) sheep, with no prominent gross pigmentation in the lungs. Twelve (26.6%) out of 45 lungs suspected of anthracosis were found to have dark red consolidation. Black pigmented areas were prominent on the cut surfaces of the lymph nodes. Histopathologically, focal accumulation of cytoplasmic carbon particle-laden alveolar macrophages was observed particularly around the terminal respiratory bronchioles. Sinusoidal macrophages with phagocytosed particles were predominantly located in the medullar region of the lymph nodes. The pigment particles were found to be resistant to all bleaching methods. To prevent anthracosis resulting from exposure to smoke, which may predispose animals to pneumonia as well as tuberculosis, it may be essential to construct barns at a distance from factories emitting smoke and from houses using stoves and clay ovens for heating and cooking, etc., and to ensure good ventilation.

Lung clearance of particles following excessive deposition of fly ash in golden hamsters.

Male golden hamsters were exposed to coal fly ash (FA) at the concentration of 0 (control), 1, 2, or 20 mg/m3 for 3 or 6 months (20 hr/day, 7 days/week). They were sacrificed at 0, 1, 3 and 6 months after exposure to undergo morphological investigation of the lung clearance of particles ingested by alveolar macrophages (AMs) following excessive deposition of FA particles. Particle-laden AMs in a cluster decreased in inverse proportion to the dust burden after the cessation of exposure. AMs with well-spread lamellipodia frequently appeared on the degenerated AMs in a cluster. In addition, neutrophils also agglomerated mainly in the alveoli proximal to the terminal bronchioles and ingested a small amount of particles. These results suggest that AMs and neutrophils might recruit into alveoli enclosing AMs in necrosis and reingest released particles. Macrophages in bronchioles generally ingested fewer particles than those clustered in alveoli. This finding suggests that AMs ingesting a small amount of released particles could move on the mucociliary escalator of the terminal bronchioles. On the other hand, AMs ingesting a large amount of particles would agglomerate in alveoli due to the loss of their migrating ability. The latter agglomeration of particle-laden AMs might be responsible for the retardation of lung clearance in proportion to the lung burden of particles.

石炭灰吸入によるゴールデンハムスター肺胞マクロファージの集積

To investigate the possible mechanisms of the accumulation of particle-laden alveolar macrophages (AMs) in alveoli, male golden hamsters were exposed to coal fly ash (FA) at the concentration of 0 or 2 mg/m3 for 6 months (20 hr/day, 7 days/week) in the first series of experiments, and at 0, 1, 2, or 20 mg/m3 of FA for 3 months in the subsequent experiments. Particle-laden AMs accumulated dose- and time-dependently in alveoli. In the lungs of 1 and 2 mg/m3-exposed groups. AMs first appeared in a cluster in alveoli at the alveolar-bronchiole junctions proximal to the lobar bronchus. Agglomerated AMs in these regions were generally larger in size and ingested more particles than those in the peripheral regions. These results indicate that the accumulation of AMs is closely related to the amount of particles deposited in alveoli and that ingested by AMs. Histochemical analysis revealed that AMs with small amount of particles showed the positive activity of acid phosphatase. On the other hand, heavily particle-laden AMs showed no such activity. Scanning electron microscopic observations revealed the time-related formation of small blebs and loss of surface features on the cell surface of AMs. These results suggest that the accumulation of particle-laden AMs might be caused by the decrease and/or loss of their activities, especially their mobility during migration toward terminal bronchioles from alveoli, due to the increase of ingested particles in parallel with the prolongation of exposure time.

Translocation of particle-laden alveolar macrophages and intra-alveolar granuloma formation in rats exposed to Ludox colloidal amorphous silica by inhalation

Rats were exposed to Ludox colloidal amorphous silica at concentrations of 0, 10, 50 and 150 mg/m 3 for 6 h/day, 5 days/week for 4 weeks. The rats were killed after 4 weeks exposure and at 10 days or 3 months post-exposure (PE). Dose-related pulmonary lesions were observed at 50 and 150 mg/m 3, but not at 10 mg/m 3. Inhaled particles were mostly phagocytized by alveolar macrophages (AMs) in the alveolar duct region and a few free particles were found in Type I pneumocytes in the alveoli. Particle-laden AMs directly penetrated into the bronchiolar interstitium from alveoli and accumulated in bronchus-associated lymphoid tissue (BALT), peribronchiolar, or perivascular interstitium. The particle-laden AMs in the interstitium further migrated into the peribronchial or perivascular lymphatics and accumulated in the tracheo-bronchial lymph nodes (TBLN). Some particle-laden AMs in the BALT transmigrated directly into bronchial lumen through the epithelium. The transmigrated particle laden-AMs in the TBLN were indistinguishable from those seen in the alveoli. They were characterized by slender cytoplasmic processes, phagosomes, myelin figures (alveolar surfactant), cholesterol clefts and lipid droplets. Many migrated particle-laden AMs were necrotic and released particles in the TBLN. The released particles were phagocytized by histiocytes and formed histiocytic granulomas. Silicotic granulomas were initially formed in alveoli with particle-laden AMs and proliferating epithelioid cells. Subsequently, the granulomas were incorporated into the interstitium. However, perivascular silicotic granulomas were developed with accumulation of transmigrated particle-laden AMs and minimal collagenized fibers at 3 months PE. There was no alveolar lipo-proteinosis as seen in crystalline silica exposure.

Lung free cells following short-term inhalation of coal fly ash particles in golden hamsters.

Male golden hamsters were exposed to coal fly ash (FA) at the concentration of 2 or 5 mg/m3 for 10 days (20 hr/day, Experiment 1) or 5 mg/m3 for 4 weeks (10 hr/day, 7 days/week) and maintained in clean air condition for subsequent 4 weeks (Experiment 2) to investigate the effect on the induced changes of lung free cells. Free cells were collected by lung lavage in Exp. 1 and histologically examined in lung sections in Exp.2. In Exp. 1 the number of alveolar macrophages (AMs) increased in a dose-dependent manner. In Exp. 2 AMs increased till at 2 weeks of exposure, while the subsequent increase did not occur. The ratio of particle-laden AMs increased in a dose- and time-dependent manner in each exposed group. Particle number ingested in AMs tended to increase with exposure time. In the recovery period in Exp. 2 the ratio of particle-laden AMs reduced from about 80 to 20% at the 1st week after the cessation of exposure and remained at the similar level to the termination of the experiment. On the other hand decrease in the number of total AMs was slight in the recovery period. These results suggest that the pool size of AMs is mainly dependent on the inhaled dust concentrations and some of particle-laden AMs remain in alveoli for a long time. The number of polymorphonuclear leukocytes (PMNs) also increased after inhalation of FA particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary inflammation and fibrosis following subacute inhalational exposure to silica: determinants of progression

To evaluate components of the pulmonary cellular response to inhaled silica that might be determinants of progression to fibrosis, we developed a model of the early stages of chronic human silicosis. Groups of mice were subacutely exposed either to alpha-quartz or to nonfibrogenic titanium dioxide dust as a control. Induction of lesions by inhaled silica was dependent upon the size distribution and dose of the particles. A novel observation was that low intensity exposure to silica evoked reversible inflammatory lesions that were characterized by focal aggregation of particle-laden alveolar macrophages near terminal airways. In contrast, higher intensity exposure elicited progressive pulmonary inflammation, including a significant perivascular influx of T-lymphocytes early in the response. The airspace inflammatory lesions exhibited a statistically significant decline in numerical density over time. Meanwhile, deposition of collagen was observed at perivascular locations, which were anatomically distinct from the initial foci of inflammation, and the numerical density of fibrotic lesions increased significantly with time. We speculate that this pattern of response might be related to alveolar clearance mechanisms being overwhelmed, followed by translocation and sequestration of particles in the interstitium, subsequently leading to T-lymphocyte recruitment and ultimately to the development of fibrosis.

The pulmonary response and clearance of Ludox colloidal silica after a 4-week inhalation exposure in rats

Rats were exposed to Ludox colloidal silica (CS) at concentrations of 0, 10, 50, and 150 mg/m 3 for 6 hr/day, 5 days/week for 4 weeks. Rats were killed after 4 weeks of exposure and 10 days or 3 months postexposure (PE). The exposure concentration of 10 mg/m 3 Ludox CS is considered to be the no-effect concentration. There were no exposure-related clinical signs in any group. After 4 weeks exposure, lung weights were increased significantly in rats exposed to 50 and 150 mg/m 3 Ludox CS, but lung weights were similar to those of controls at 3 months PE. After 4 weeks exposure to 50 mg/m 3 Ludox CS, a slight alveolar macrophage response, polymorphonuclear leukocytic infiltration, and Type II pneumocyte hyperplasia in alveolar duct regions were present. After 3 months PE, these pulmonary lesions had almost disappeared with removal of most dust-laden alveolar macrophages (AMs). The pulmonary response to 150 mg/m 3 Ludox CS was similar in character but increased in magnitude from that seen at 50 mg/m 3. At 3 months PE, most particleladen AMs had disappeared and the remaining AMs were aggregated and sharply demarcated. A few aggregates of particleladen AMs appeared to transform into silicotic nodules comprising macrophages, epithelioid cells, and lymphocytic infiltration in some animals. Some silicotic nodules showed reticular fiber networks with minute collagen fiber deposition. Tracheobronchial lymph nodes were enlarged with aggregates of particleladen AMs and hyperplastic histiocytic cells. Lung-deposited Ludox cleared rapidly from the lungs with half-times of approximately 40 and 50 days for the 50 and 150 mg/m 3 groups, respectively.

Migratory Behaviors of Alveolar Macrophages During the Alveolar Clearance of Light to Heavy Burdens of Particles

We investigated the unstimulated and stimulated migratory activities of lavaged alveolar macrophages (AMs) in vitro over the course of alveolar clearance of three different mass lung burdens of microspheres. Our intent was to uncover potentially important relationships between the migratory behaviors of the AM and the retention kinetics of particles. Groups of adult, male Fischer-344 rats were intratracheally instilled with approximately 86 micrograms (low burden, LB), approximately 1 mg (medium burden, MB), or approximately 3.7 mg (high burden, HB) of polystyrene microspheres (2.13 microns diameter), or with carrier vehicle (phosphate buffered saline, PBS) alone. The lung retention kinetics of the particles were determined over an approximately 170 day period. On days 14, approximately 57, and approximately 85, lavaged AMs were assessed for their abilities to migrate through 5-microns pore membranes in response to inactivated rat serum (unstimulated condition) and yeast-activated rat serum (stimulated condition). The retention characteristics of the three burdens could be satisfactorily described by two-component, negative exponential equations. The kinetics of retention of the LB and MB were similar, although some evidence indicated the MB slightly retarded the lung clearance process. Deposition of the HB resulted in more marked prolongations of both the early, more rapid, and the slower, longer term components of alveolar clearance. The unstimulated migration indices of AMs from the particle-instilled lungs were generally not significantly different from those of AMs from PBS-instilled lungs except for a significant increase in the migration indices of LB AMs at the last assay time. The stimulated migration indices of MB and HB AMs were significantly decreased on assay days 14 and approximately 57. On day approximately 85, however, the migration indices of LB, MB, and HB AMs were all increased above the PBS AMs. Comparisons of the frequency distributions of particles in the unstimulated and stimulated AM that migrated to those in corresponding parent AM populations consistently indicated a preferential migration of particle-free AMs and of AMs with lesser loads of microspheres. The overall results of this study suggest that the unstimulated and stimulated migratory activities of particle-laden AMs are depressed in vitro. Our results also suggest that the migratory activities of generally particle-free AMs may be enhanced over a prolonged period of time following the deposition of particles in the lung.

Subchronic Inhalation Study of Toner in Rats

AbstractA subchronic inhalation study of a special test toner was conducted by exposure of groups of F-344 SPF (specific pathogen free) rats for 6 hlday, 5 dayslwk for 13 wk. The test material was a special 9000 type xerographic toner, enriched in respirable size particles compared to commercial toner, such that it was about 35% respirable according to the ACGIH criteria. The nominal aerosol exposure concentrations were 0, 7.0, 4.0, 76.0, and 64.0 mg/m3 Body weight, clinical chemistry values, food consumption, and organ weights were normal except for a 40% increase in lung weight for the highest exposure group. Histopathological examination of the lungs indicated an exposure-related accumulation of particle-laden alveolar macrophages. A very slight degree of septal thickening of the alveolar structure was noted in the highest exposure group. Clearance results for the test material and a superimposed spike of 59Fe2O3 were essentially unchanged at exposure concentrations of 0, 1, and 4 mg/m3. At 76 mg/m3, s...

Pulmonary deposition and effects of inhaled silica particles after short-term exposures in the rat.

The present study was designed to evaluate the pulmonary deposition and the effects of inhaled silica particles in the rat model. Wistar (W/M strain) rats were exposed to silica aerosols generated from a fluidized bed dust generator for 1 hr a day, intermittently for 6 days, using a "nose-only" inhalation chamber. After the cessation of the exposures, analysis of lavaged bronchoalveolar cells (BAC) and histological examinations of lungs and tracheobronchial lymph nodes (TBLN) were performed during a period of 6 months. Total cell yields and the proportions of alveolar macrophages (AM) in BAC were not altered, whereas the proportions of lymphocytes in BAC were significantly increased in the exposed animals. Although the proportions of silica-laden AM in BAC were gradually decreased during the 6 months, particle-laden AM were predominantly and persistently observed in the alveoli under light microscopy. Silica particles were also identified in macrophages of granulomatous nodules in pulmonary peribronchial lymphoid tissues(PBLT) and TBLN, indicating the translocation of particles via the lymph. Associated with pulmonary particle deposition, some characteristic histopathological features were evident, including thickening of alveolar duct bifurcations and lymphocyte infiltrations both in the alveolar sacs and around the interstitial blood vessels. At later months after the exposures, the alveolar interstitium was thickened with the increase of fibroblasts and collagen. These results implicate that short-term exposures of silica particles in the rat can evoke histopathologic changes in the lungs and lymphatic tissues, associated with their deposition and translocation.