Cellular and biochemical response of the human lung after intrapulmonary instillation of ferric oxide particles

Bronchoalveolar lavage (BAL) was used to sample lung cells and biochemical components in the lung air spaces at various times from 1 to 91 d after intrapulmonary instillation of 2.6 microm-diameter iron oxide particles in human subjects. The instillation of particles induced transient acute inflammation during the first day post instillation (PI), characterized by increased numbers of neutrophils and alveolar macrophages as well as increased amounts of protein, lactate dehydrogenase, and interleukin-8 in BAL fluids. This response was subclinical and was resolved within 4 d PI. A similar dose-dependent response was seen in rats 1 d after intratracheal instillation of the same particles. The particles contained small amounts of soluble iron (240 ng/mg) and possessed the capacity to catalyze oxidant generation in vitro. Our findings indicate that the acute inflammation after particle exposure may, at least partially, be the result of oxidant generation catalyzed by the presence of residual amounts of ferric ion, ferric hydroxides, or oxyhydroxides associated with the particles. These findings may have relevance to the acute health effects associated with increased levels of ambient particulate air pollutants.     

Biochemical and morphologic changes in rabbit lung following endotracheal instillation of zymosan particles

In order to establish a model of lung disease in which the usefulness of potential antiinflammatory compounds can be evaluated, we have analyzed the biochemical and cellular responses of rabbits to zymosan deposition in their lungs. A suspension of zymosan particles was instilled into the lungs of rabbits using an intratracheal catheter. Because the influx of leukocytes and the transudation of plasma into affected lungs was expected to contribute to the total cellular enzyme and protein levels, lungs were homogenized and assayed after various time intervals for six cellular enzymes and for protein content. After one day, alkaline phosphatase and neutral protease levels were elevated by 90% and 50%, respectively, above normal values. After two and three days, all of the pulmonary enzymes assayed displayed maximal two- to fourfold increases in their levels of activity. After seven days, only the alkaline phosphatase and neutral protease levels remained slightly elevated by 50% and 30%, respectively. Histologic analysis revealed focal and diffuse intraalveolar, interstitial, peribronchiolar, and perivascular accumulations of macrophages, granulocytes, and lymphocytes. Severe pulmonary edema, evident microscopically after one to three days, correlated well with 100% increases in both the wet weight and protein content of the lungs. In control experiments, the intratracheal infusion of saline solution minus zymosan particles resulted in a variety of enzymatic changes in the lungs after three days, which could be distinguished both enzymatically and histologically from those following zymosan deposition; histopathologic analysis revealed a pattern of intravascular congestion with erythrocytes, edematous thickening of alveolar septa, and focal intraalveolar hemorrhages, but with no inflammatory infiltration. In summary, this study demonstrates the time course of an experimental model for acute and chronic lung inflammation, the extent of which may be quantitatively evaluated using cellular enzymatic markers.     

Pathways of cellular efflux and particulate clearance after carbon instillation to the lung

The pulmonary response to the deposition of carbon particles was investigated to determine the routes of cellular efflux and the mechanisms of particulate clearance. At intervals to 6 months after the intratracheal instillation of 4 mg carbon, the lungs of mice were fixed in situ by perfusion without lavage. Within a few hours, migration of granulocytes into the bronchioles was observed; large numbers of PMNs were not seen in alveoli until 24 h. Associated with the PMN efflux there was transient oedema with no evidence of pulmonary cell necrosis. The number of free macrophages increased in response to the carbon and mononuclear cell migration into alveoli and bronchioles was also observed. The bronchiolar component of the cellular efflux indicates that PMNs and macrophages recovered from the lung by lavage are not entirely of alveolar origin. Whereas most particles, either free or in phagocytic cells, were cleared by the bronchial tree, some transepithelial passage of free particles to the interstitium was observed. Some carbon was found in hilar lymph nodes but overall lymphatic clearance was low.     

Apoptosis-dependent acute lung injury and repair after intratracheal instillation of noradrenaline in rats

Earlier work in this laboratory showed that noradrenaline (NA) induces apoptosis in primary cultures of alveolar epithelial cells (AECs). Apoptosis of alveolar epithelial cells may promote the collapse of lung barrier function. On this basis we hypothesized that exogenous NA, administered by intratracheal (I.T.) instillation, might induce AEC apoptosis in vivo followed by acute lung injury. Delivery of NA (10 microM) I.T. into male Wistar rats increased labelling of both fragmented DNA, measured by in situ end labelling (ISEL), and the active form of caspase 3 (anti-Casp3) 6 and 20 h after administration (P < 0.05), but instillation of the vehicle alone (PBS) had no effect. Both ISEL and anti-Casp3 labelling were attenuated by concurrent I.T. delivery of the broad-spectrum caspase inhibitor ZVADfmk. After 6 h, most ISEL- and Casp3-positive cells were located in the surfaces of alveolar walls, but after 20 h more were found in alveolar spaces (P < 0.05). Instillation of NA also increased the bronchoalveolar lavage (BAL) content of fluorescent albumin (BODIPY-alb), which had previously been injected intravenously; the increase was reversed by concurrent ZVADfmk administration. These data suggest that NA-induced apoptosis of AECs in vivo is sufficient to invoke transient collapse of AEC barrier function that is rapidly repaired.     

Intraluminal fibrosis induced unilaterally by lobar instillation of CdCl2 into the rat lung.

Lung injury induced by intratracheal instillation of cadmium chloride (CdCl2) into the rat lung may serve as a model of human interstitial lung disease. In this study, CdCl2 solutions were instilled through a lobar bronchus into the left lung of the rat. Two doses (400 micrograms or 50 micrograms of CdCl2, each in 400 microliters of neutral saline) were used and the morphologic changes occurring during the first 7 days after a single exposure were documented by light and electron microscopy. With the higher dose, inflammatory cells appeared in the alveolar interstitium 1 day after CdCl2 administration. Edema and thickening of the alveolar walls were evident, as were damaged type I epithelial cells and denuded basement membranes. Fibrin was found in the air spaces. Within 2 days, inflammatory cells were seen in large numbers and fibroblasts were observed passing through gaps in the alveolar basement membranes into the air spaces. By 4 and 7 days after CdCl2, various forms of intraluminal fibrosis, including intrabronchiolar budding, mural incorporation, and obliterative changes, were observed. The contralateral lungs had normal-appearing architecture for all the time points investigated. In the lower dose exposure, gradients of alveolar damage were observed in which normal lung, interstitial fibrosis, and/or intraluminal fibrosis were seen within treated lungs. In the mildly damaged regions, interstitial fibrosis predominated, while in the more severely damaged regions, mural incorporation of the convoluted basement membranes was observed. The pulmonary fibrosis that developed appeared to be similar to some human interstitial lung diseases and may offer a system in which to study the regulation of collagen deposition and fibrosis development in these pathologic conditions.     

Morphological changes in rat tracheal mucosa immediately after antigen challenge

We have investigated the morphological effects of an intratracheal challenge with 50 micrograms ovalbumin (OA) on sensitized rat tracheas, in vivo. Female Sprague-Dawley rats were primed ten days before challenge with a single i.t. injection of 100 micrograms OA plus Bordetella pertussis (OA-BP). Two additional groups of animals served as controls: primed animals challenged with saline only and non-primed but OA-challenged animals. Sacrifices--and subsequent morphological studies--were performed prior to and 5, 15 and 60 min after challenge. At each time, the total numbers of epithelial nuclei, subepithelial mast cells (SEMC) and intraepithelial mast cells (IEMC) were scored in six non-adjacent cross sections per trachea. We found that: 1) priming with OA-BP alone did not induce any change in the tracheal mucosa with respect to morphological structure and mast cell counts; 2) no morphological change nor significant modification of the cell counts occurred at any time in tracheas from either of the control groups; 3) in contrast, a luminal heterogeneous exudate and a subepithelial oedema developed in 9 of the 15 tracheas of primed animals within 60 min of OA challenge. In those nine tracheas, the scores of intraepithelial nuclei, of IEMC and of SEMC were found to decrease significantly 15 min after challenge as compared with starting values (p less than 0.05 for each score). The decrease in the number of mucosal mast cells is probably related to the damages of the epithelial cells and to the difficulty with which depleted mast cells can be seen by toluidine blue staining.     

Temporal pattern of accelerated lung growth after tracheal occlusion in the fetal rabbit.

Tracheal occlusion in utero is a potent stimulus of fetal lung growth. We describe the early growth mechanics of fetal lungs and type II pneumocytes after tracheal ligation (TL). Fetal rabbits underwent TL at 24 days gestational age (DGA; late pseudoglandular stage; term = 31 to 33 days) and were sacrificed at time intervals ranging from 1 to 5 days after TL. Lung growth was measured by stereological volumetry and bromodeoxyuridine (BrdU) pulse labeling. Pneumocyte II population kinetics were analyzed using a combination of anti-surfactant protein A and BrdU immunohistochemistry and computer-assisted morphometry. Nonoperated littermates served as controls. TL resulted in dramatically enhanced lung growth (lung weight/body weight was 5.00 +/- 0.81% in TL versus 2.52 +/- 0.13% in controls at 29 DGA; P < 0.001, unpaired Student's t-test). Post-TL lung growth was characterized by a 3-day lag-phase typified by relative stagnation of growth, followed by distension of airspaces, increased cell proliferation, and accelerated architectural and cellular maturation by postligation days 4 and 5. During the proliferation phase, the replicative activity of type II cells was markedly increased (type II cell BrdU labeling index was 10.0 +/- 4.1% in TL versus 1.1 +/- 0.3% for controls at 29 DGA; P < 0.02), but their numerical density decreased (3.0 +/- 0.5 x 10(-3)/microm2 in TL versus 4.5 +/- 0.3 x 10(-3)/microm2 in controls at 29 DGA; P < 0.02), suggesting accelerated terminal differentiation to type I cells. In conclusion, post-TL lung development is characterized by a well defined temporal pattern of lung growth and maturation. The rabbit model lends itself well to study the regulatory mechanisms underlying accelerated fetal lung growth after TL.     

Effects of components derived from diesel exhaust particles on lung physiology related to antigen

Our previous study has shown that diesel exhaust particles (DEP), main constituents in ambient particulate matters (PM), enhance airway hyperresponsivness in a murine model of allergic asthma (Takano et al., 1998). However, it remains unknown which components in DEP are responsible for the enhancement. The present study investigated the effects of repeated pulmonary exposure to DEP components (extracted organic chemicals in DEP; DEP-OC, carbonaceous nuclei of DEP after extraction; washed DEP) on lung physiology in the presence or absence of antigen. ICR mice were divided into six experimental groups. Vehicle, DEP components, ovalbumin (OVA), or DEP components plus OVA was administered intratrachally for 6 weeks. Twenty-four hr after the last instillation, cholinergic lung reactivity was examined. DEP components alone did not induce any facilitation of lung function as compared to vehicle alone. The values of total respiratory system resistance (R), elastance (E), Newtonian resistance (R(n)), tissue damping (G), and tissue elastance (H) were higher and the value of compliance (C) was lower in the OVA or the DEP component + OVA groups than in the vehicle group. In particular, the hyperreactivity was most prominent in the washed DEP + OVA group. The values in the DEP-OC + OVA group were not significantly different from those in the OVA group. These data suggest that carboneous component in DEP, rather than organic chemical one, can be attributable to the enhancement of lung hyperresponsiveness in allergic asthma.     

Evaluation of the release of mutagens and 1-nitropyrene from diesel particles in the presence of lung macrophages in culture

The release and recovery of mutagenic activity and 1-nitropyrene from diesel particles phagocytized and cultured with lung macrophages were studied. The Ames Salmonella typhimurium plate incorporation assay was used to measure mutagenic activity. Quantitative analysis of 1-nitropyrene was performed with liquid chromatography/fluorescence analysis. The cytotoxicity and phagocytosis of diesel particles with and without fetal calf serum were evaluated to select exposure concentrations that resulted in minimal toxicity and maximal engulfment of particles by the macrophages. The diesel-particle exposure concentrations for the mutagenicity studies were 200 micrograms/ml in the absence of serum and 375 micrograms/ml in the presence of serum. Engulfment and incubation of diesel particles with lung macrophages resulted in the loss of considerable mutagenic activity (97-98%) and significantly less 1-nitropyrene (10-25%). These studies suggest that lung macrophages have the capability to metabolize mutagenic nitroaromatics found in diesel particles.     

Expansion pattern of terminal air-spaces in the premature rabbit lung after tracheal deposition of surfactant

Summary The expansion pattern of the lungs of full-term and premature newborn rabbits was evaluated by direct observation of the lung surface during an inflation-deflation cycle, with particular reference to the effect of tracheal deposition of surfactant before the first breath. In non-treated premature fetuses, aeration of alveoli was initiated at an endotracheal pressure of 35 cm H₂O; the mean corresponding opening pressure was 25 cm H₂O in both full-term and surfactant-treated premature animals. The process of alveolar aeration was asynchronous in all groups but at maximal inflation pressure (35 cm H₂O) the expansion pattern was uniform in full-term and surfactant-treated premature fetuses; these animals also developed residual volume. In non-treated premature animals aeration was patchy, even at maximal inflation, and the lungs emptied at the end of deflation.This work was supported by grants from The Swedish Medical Research Council (Project No. 12X-3352)     

Bioassay by intratracheal instillation for detection of lung toxicity due to fine particles in F344 male rats.

We have established and documented an in vivo bioassay for detection of hazards with intratracheally instilled fine particles, which can be used for risk assessment of toxicity of materials inhaled into deep lung tissue of humans (Yokohira et al. Establishment of a bioassay system for detection of lung toxicity due to fine particle instillation: sequential histopathological changes with acute and subacute lung damage due to intratracheal instillation of quartz in F344 male rats. J Toxicol Pathol 2005;18:13-8). For validation we here examined toxicity of fine particles from quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black with this bioassay. A total of 108, 10-week-old F344/DuCrj male rats were randomly divided into 8 groups. Groups 1 to 5 underwent intratracheal instillation of the 5 test particles (4 mg/rat) suspended in 0.2 ml vehicle (saline or 10% propylene glycol and 1% sodium carboxymethyl cellulose in saline: PG-CMC) with a specially designed aerolizer, and subgroups of 7 rats were killed on Days 1 and 28 thereafter. Groups 6 and 7 similarly were exposed to saline and PG-CMC, respectively, as vehicle controls, while group 8 was maintained untreated. Using histopathological changes and immunohistochemically assessed bromodeoxyuridine (BrdU) labeling indices, inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-3 (MMP-3) levels as end points, the quartz treated group exhibited high toxicity, while the values for the other particle-treated groups pointed to only slight effects. Although additional efforts are needed to establish advantages and disadvantages with our bioassay, models featuring intratracheal instillation clearly can be useful for detection of acute or subacute lung toxicity due to inhaled fine particles by using histopathological scoring and markers like BrdU and iNOS for screening purposes in short-term studies.     

Ultrastructural changes of the air–blood barrier in mice after intratracheal instillation of lipopolysaccharide and ultrafine carbon black particles

Epidemiological studies have indicated associations between exposure to increased concentrations of ambient ultrafine particles and adverse health effects especially in susceptible individuals. To ellucidate the mechanisms underlying the findings from epidemiological studies, mice pretreated with lipopolysaccharide (LPS) (acute lung injury model) were intratracheally instilled with ultrafine carbon black particles (UFCB), and the air–blood barrier was observed to examine the translocation pathway of UFCB from the lung into the systemic circulation. In addition, lung toxicity induced by the intratracheal instillation of LPS and UFCB was studied with the use of electron microscope. LPS treatment induced acute inflammatory changes with increased number of activated macrophages and neutrophils in the degenerated alveolar walls. UFCB were demonstrated on or in the denuded basement membrane in the air–blood barrier; these findings were associated with edematous changes and fragmentation of the cytoplasms of alveolar epithelial cell type 1, and the damages of alveolar epithelial cell type 1 were frequently observed in the close vicinity of the clumps of UFCB. These findings suggest that translocation of the exposed ultrafine particles may be enhanced in the lung tissues with acute inflammatory changes.     

Uptake of marker particles by in vitro ventilated and perfused rat lung

Colloidal gold marker was present in the capillary lumen and at various stages of pinocytosis and passage through the endothelial cells. It was occasionally located in the peri-endothelial space, close to the basement membrane. The uptake of marker by other cells was not observed. These results suggest the existence of two barriers, the biological (pinocytotic and hydrolytic capacities of the endothelial cells) and mechanical (permeability of the basement membrane).     

Acute and subacute pulmonary toxicity of low dose of ultrafine colloidal silica particles in mice after intratracheal instillation

To study the acute and subacute lung toxicity of low dose of ultrafine colloidal silica particles (UFCSs), mice were intratracheally instilled with 0, 0.3, 3, 10, 30 or 100 microg of UFCSs. Cellular and biochemical parameters in bronchoalveolar lavage fluid (BALF), histological alteration and the body weight were determined at 3 days after instillation. Exposure to 30 or 100 microg of UFCSs produced moderate to severe pulmonary inflammation and tissue injury. To investigate the time response, mice were instilled with 30 microg of UFCSs and sacrificed at intervals from 1 to 30 days post-exposure. UFCSs induced moderate pulmonary inflammation and injury on BALF indices at acute period; however, these changes gradually regressed until recovery during the experiment. Concomitant histopathological and laminin immunohistochemical findings generally correlated to BALF data. TUNEL analyses in UFCSs-treated animals showed a significant increase of the apoptotic index in lung parenchyma at all observation times. 8-OHdG expression occurred in lung epithelial cells and activated macrophages, which correlated to lung lesions in UFCSs-treated mice. These findings suggest that instillation of a small dose of UFCSs causes transient acute moderate lung inflammation and tissue damage. Oxidative stress and apoptosis may underlie the lung tissue injury induction.     

Mesothelial cell proliferation after instillation of long or short asbestos fibers into mouse lung.

The relationship of asbestos deposition in the lung to subsequent cell proliferation at the pleural surface is not clear. The present study examines DNA synthesis by various pulmonary cells, particularly those at the pleura after intratracheal injection of 0.1 mg crocidolite to mice using: 1) long fibers (> 20 mu), which are deposited in bronchiolar regions and induce fibrosis; 2) short fibers (< 1 mu), which reach alveoli but do not induce fibrosis. Mice also received 2 microCi/g tritiated thymidine 1 hour before death at intervals to 16 weeks. Short fibers induced only a small increase in labeling of bronchiolar epithelial and interstitial cells, which subsided by 5 days, when a small increase in labeled mesothelial and subpleural cells was seen. In contrast, long fibers damaged the bronchiolar epithelium and became incorporated into connective tissue. During regeneration, 12% of cells were labeled at 3 days and labeling was greater than controls to 4 weeks. Increased peribronchiolar labeling of fibroblasts and interstitial macrophages was seen around long fibers, and increased DNA synthesis by mesothelial and subpleural cells was found. Up to 2% of mesothelial cells were labeled 1 week after long fibers compared to near zero in controls. No long fibers were found at the pleura. Activation of interstitial macrophages in response to long crocidolite fibers is associated with fibroblast proliferation. It is now suggested that mesothelial cells may also be stimulated by cytokines from activated interstitial macrophages that diffuse across the interstitium, without requiring actual fiber translocation to the pleura.     

Reduction of IL-12 p40 production in activated monocytes after exposure to diesel exhaust particles

BACKGROUND: A reduction of IL-12 production by lung macrophages may partly explain the presumed adjuvant effect of diesel exhaust particles (DEP) in allergy and asthma. IL-12 stimulates T helper type 1 (Th1) lymphocytes, which inhibit Th2 cells via Th1-specific cytokines. The aim of this study was to investigate the influence of DEP on the production of IL-12 p40 in lipopolysaccharide (LPS)-activated monocytes. METHODS: The human monocytic cell line Mono-Mac-6 was stimulated with LPS (200 ng/ml) and grown with DEP (0-200 microg/ml) for 0, 6 or 24 h. IL-12 p40 and the pro-inflammatory cytokine TNF were analysed in the cell supernatants by ELISA and a cell assay, respectively. RESULTS: Levels of IL-12 p40 correlated inversely with the DEP exposure concentrations, whereas TNF increased in parallel to the DEP concentrations. At a DEP concentration of 200 microg/ml, the amount of IL-12 p40 was 35% of that observed without DEP. The corresponding TNF value was 230% of the control. Reduced viability, binding of cytokines to DEP or endotoxin in the DEP samples cannot fully explain the changes in the concentrations of these two cytokines. CONCLUSION: DEP seem to inhibit the production of IL-12 p40 and stimulate that of TNF in activated monocytes. This may partly explain the presumed adjuvant effect of DEP in atopy; by altering the Th1/Th2 balance via down-regulation of IL-12, the Th2 response characteristic of allergy and asthma may be favoured.     

Accumulation of iron by primary rat hepatocytes in long-term culture: changes in nuclear shape mediated by non-transferrin-bound forms of iron.

We have previously shown that hepatocytes in long-term dimethylsulfoxide (DMSO) culture, fed a chemically defined medium, are highly differentiated and an excellent in vitro model of adult liver. Hepatocytes in long-term DMSO culture can be iron loaded by exposure to non-transferrin-bound iron (NTBI) in the form of ferrous sulfate (FeSO4), ferric nitrilotriacetate, or trimethylhexanoyl (TMH)-ferrocene. Holotransferrin, at equivalent times and concentrations, was unable to load hepatocytes. Of the iron compounds tested, TMH-ferrocene most accurately simulated the morphological features of iron-loaded hepatocytes in vivo. When exposed to 25 micromol/L TMH-ferrocene, hepatocytes loaded increasing amounts of iron for 2 months before the cells died. When exposed to lower concentrations of TMH-ferrocene (as low as 2.5 micromol/L), hepatocytes continuously loaded iron and remained viable for more than 2 months. The cellular deposition of iron was different in hepatocytes exposed to TMH-ferrocene compared with those exposed to FeSO4; exposure to TMH-ferrocene resulted in the presence of more ferritin cores within lysosomes than were seen with FeSO4. When the concentration of TMH-ferrocene was increased, a greater number of ferritin cores were observed within the lysosome, and total cellular ferritin, as assessed by Western blot, increased. The formation of hemosiderin was also observed. Furthermore, nuclear shape was distorted in iron-loaded hepatocytes. The extent of deviation from circularity in the nucleus correlated with increasing concentrations of TMH-ferrocene and was greater in hepatocytes exposed to FeSO4 than an equivalent concentration of TMH-ferrocene. The deviation from circularity was smallest in hepatocytes that contained well formed ferritin cores and increased in hepatocytes that contained greater amounts of hemosiderin. Furthermore, in hepatocytes treated with FeSO4, a large amount of cell-associated iron was detected but without a significant increase in the total amount of ferritin. The deviation from circularity was the largest in FeSO4-treated hepatocytes, indicating that iron not properly incorporated into ferritin caused more cellular damage. We conclude that iron-loaded hepatocytes in long-term DMSO culture represent a flexible system for studying the effects of chronic iron loading on hepatocytes.     

气管滴注红细胞诱导大鼠肺损伤

目的:研究红细胞滴注对肺组织结构和机能代谢的影响。方法:健康雄性大鼠48只随机分为2组(n=24):生理盐水组和红细胞滴注组。分别在气管滴注0.5mL生理盐水或0.5mL1×1010cells/L红细胞2d、7d、14d后,随机选择8只大鼠,右肺肺泡灌洗后进行总细胞计数、分类计数;肺泡灌洗上清测定总蛋白、乳酸脱氢酶含量;左肺进行常规病理学检查和普鲁氏蓝染色。结果:滴注红细胞2d后,肺大体形态学和病理学观察结果提示滴注红细胞可以引起肺组织水肿、肺间隔增宽;并且有炎症细胞浸润;肺泡灌洗液中细胞数量增加,以巨噬细胞为主;肺泡灌洗上清中总蛋白、乳酸脱氢酶含量也增加。这些病理变化在第7d开始减轻,在14d基本恢复正常。气管滴注生理盐水对肺组织无明显影响。结论:滴注红细胞可以引起肺组织损伤,导致异常的结构和机能代谢的变化。