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Inhalation of high concentrations of sulfur dioxide (SO 2 ) affects the lungs and can be immediately dangerous to life. We examined the development of acute and long-term effects after exposure of SO 2 in Sprague-Dawley rats, in particular inflammatory responses, airway hyperresponsiveness (AHR) and lung fibrosis. Animals were subjected to a single exposure of 2200ppm SO 2 during 10min and treated with a single dose of the anti-inflammatory corticosteroid dexamethasone 1h following exposure. Exposed rats showed labored breathing, decreased body-weight and an acute inflammation with neutrophil and macrophage airway infiltrates 5h post exposure. The acute effects were characterized by bronchial damage restricted to the larger bronchi with widespread injured mucosal epithelial lining. Rats displayed hyperreactive airways 24h after exposure as indicated by increased methacholine-induced respiratory resistance. The inflammatory infiltrates remained in lung tissue for at least 14 days but at the late time-point the dominating granulocyte types had changed from neutrophils to eosinophils. Analysis of immunoregulatory and pro-inflammatory cytokines in serum and airways implicated mixed macrophage phenotypes (M1/M2) and T helper cell activation of both T H 1 and T H 2 subtypes. Increased expression of the pro-fibrotic cytokine TGFβ1 was detected in airways 24h post exposure and remained increased at the late time-points (14 and 28 days). The histopathology analysis confirmed a significant collagen deposition 14 days post exposure. Treatment with dexamethasone significantly counteracted the acute inflammatory response but was insufficient for complete protection against SO 2 -induced adverse effects, i.e. treatment only provided partial protection against AHR and the long-term fibrosis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Chlorine (Cl2) is an industrial gas that is highly toxic and irritating when inhaled causing tissue damage and an acute inflammatory response in the airways followed by a long-term airway dysfunction. The aim of this study was to evaluate whether early anti-inflammatory treatment can protect against the delayed symptoms in Cl2-exposed mice. BALB/c mice were exposed by nose-only inhalation using 200ppm Cl2 during 15min. Assessment of airway hyperresponsiveness (AHR), inflammatory cell counts in bronchoalveolar lavage, occurrence of lung edema and lung fibrosis were analyzed 24h or 14days post-exposure. A single dose of the corticosteroid dexamethasone (10 or 100mg/kg) was administered intraperitoneally 1, 3, 6, or 12h following Cl2 exposure. High-dose of dexamethasone reduced the acute inflammation if administered within 6h after exposure but treated animals still displayed a significant lung injury. The effect of dexamethasone administered within 1h was dose-dependent; high-dose significantly reduced acute airway inflammation (100mg/kg) but not treatment with the relatively low-dose (10mg/kg). Both doses reduced AHR 14days later, while lung fibrosis measured as collagen deposition was not significantly reduced. The results point out that the acute inflammation in the lungs due to Cl2 exposure only partly is associated with the long-term AHR. We hypothesize that additional pathogenic mechanisms apart from the inflammatory reactions contribute to the development of long-term airway dysfunction. By using this mouse model, we have validated early administration of corticosteroids in terms of efficacy to prevent acute lung injury and delayed symptoms induced by Cl2 exposure., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chlorine is highly irritating when inhaled, and is a common toxic industrial gas causing tissue damage in the airways followed by an acute inflammatory response. In this study, we investigated mechanisms by which chlorine exposure may cause reactive airways dysfunction syndrome (RADS) and we examined the dose-dependency of the development of symptoms. Mice were exposed to 50 or 200 ppm Cl(2) during a single 15 min exposure in a nose-only container. The experiment terminated 2, 6, 12, 24, 48, 72 h and 7, 14, 28 and 90 days post exposure. Inflammatory cell counts in bronchoalveolar lavage (BAL), secretion of inflammatory mediators in BAL, occurrence of lung edema and histopathological changes in lung tissue was analyzed at each time-point. Airway hyperresponsiveness (AHR) was studied after 24 and 48 h and 7, 14, 28 and 90 days. The results showed a marked acute response at 6h (50 ppm) and 12h (200 ppm) post exposure as indicated by induced lung edema, increased airway reactivity in both central and peripheral airways, and an airway inflammation dominated by macrophages and neutrophils. The inflammatory response declined rapidly in airways, being normalized after 48 h, but inflammatory cells were sustained in lung tissue for at least seven days. In addition, a sustained AHR was observed for at least 28 days. In summary, this mouse model of chlorine exposure shows delayed symptoms of hyperreactive airways similar to human RADS. We conclude that the model can be used for studies aimed at improved understanding of adverse long-term responses following inhalation of chlorine., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Context: Exposure to toxic alkylating mustard agents causes both acute and long-term effects to the lungs as indicated by increased number of inflammatory cells in airways, lung edema and lung tissue fibrosis. We have previously demonstrated that treatment with the corticosteroid dexamethasone 1 h after lung exposure to the nitrogen mustard analog melphalan protects mice from acute and sub-acute inflammatory responses, as well as from lung tissue fibrosis., Objective: In order to address the importance of early anti-inflammatory treatment, we investigated the therapeutic effect of dexamethasone administered 1, 2 or 6 h following exposure to melphalan., Methods: C57BL/6 mice were exposed to melphalan and treated with dexamethasone 1, 2 or 6 h after exposure. Twenty hours or 14 days post exposure mice were subjected to analysis of respiratory mechanics where the effects of incremental doses of methacholine on central and peripheral lung components were measured. We also determined the amount of inflammatory cells in the bronchoalveolar lavage fluid and measured the amount of collagen content in the lungs., Results: Melphalan exposure increased airway hyperresponsiveness in both central and peripheral airways and induced an airway inflammation dominated by infiltration of macrophages and neutrophils. Dexamethasone given 1 h after exposure to melphalan provided better protection against airway inflammation than administration 2 or 6 h after exposure. Collagen deposition 14 days after exposure was decreased due to dexamethasone treatment., Conclusion: Early treatment with dexamethasone is important in order to reduce the airway hyperresponsiveness and inflammation caused by toxic alkylating mustards such as melphalan., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

It is widely established that mast cells (MCs) have a harmful role in asthma, for example by secreting various proinflammatory substances stored within their secretory granule. However, in this study, we show that one of the substances stored within MC granule, chymase, in fact has a protective role in allergic airway inflammation, indicating that MCs may possess both harmful and protective activities in connection with this type of disease. Wild-type (WT) mice and mice lacking mouse MC protease 4 (mMCP-4), a chymase that is functionally homologous to human chymase, were sensitized and challenged with OVA, followed by the assessment of airway physiology and inflammatory parameters. Our results show that the airway hyperresponsiveness was significantly higher in mMCP-4(-/-) as compared with WT mice. Moreover, the degree of lung tissue inflammation was markedly higher in mice lacking mMCP-4 than in WT controls. Histological analysis revealed that OVA sensitization/challenge resulted in a marked increased in the thickness of the smooth muscle cell (SMC) layer and, notably, that the degree of SMC layer thickening was more pronounced in mMCP-4(-/-) animals than in WT controls, thus indicating that chymase may have an effect on airway SMCs. In support of this, mMCP-4-positive MCs were located in the close vicinity of the SMC layer, mainly in the upper airways, and mMCP-4 was shown to be the major chymase expressed in these MCs. Taken together, our results indicate that chymase present in the upper airways protects against allergic airway responses, possibly by regulating SMCs.

Context: Inhalation of sulfur dioxide (SO2) affects the lungs and exposure to high concentrations can be lethal. The early pulmonary response after inhaled SO2 involves tissue injury, acute neutrophilic lung inflammation and airway hyperresponsiveness (AHR). In rats, long-term pulmonary fibrosis is evident 14 days post-exposure as indicated by analysis of collagen deposition in lung tissue. Early treatment with a single dose of dexamethasone (DEX,10 mg/kg) significantly attenuates the acute inflammatory response in airways. However, this single DEX-treatment is not sufficient for complete protection against SO2-induced injuries. Methods: Female Sprague-Dawley rats exposed to SO2 (2200 ppm, nose-only exposure, 10 min) were given treatments (1, 5 and 23 h after SO2-exposure) with the anti-fibrotic and anti-inflammatory substance Pirfenidone (PFD, 200 mg/kg) or DEX (10 mg/kg) to evaluate whether the inflammatory response, AHR and lung fibrosis could be counteracted. Results: Both treatment approaches significantly reduced the total leukocyte response in bronchoalveolar lavage fluid and suppressed pulmonary edema. In contrast to DEX-treatment, PFD-treatment reduced the methacholine-induced AHR to almost control levels and partially suppressed the acute mucosal damage whereas multiple DEX-treatment was the only treatment that reduced collagen formation in lung tissue. Conclusions: To enable an accurate extrapolation of animal derived data to humans, a detailed understanding of the underlying mechanisms of the injury, and potential treatment options, is needed. The findings of the present study suggest that treatments with the capability to reduce both AHR, the inflammatory response, and fibrosis are needed to achieve a comprehensive mitigation of the acute lung injury caused by SO2. [ABSTRACT FROM AUTHOR]

The acute pulmonary responses after exposure to sulfur and nitrogen mustards are well documented whereas the late pulmonary effects are not. With a novel focus on the immune system this paper investigate whether late phase pulmonary effects developed in rats exposed to the nitrogen mustard melphalan are linked to the acute responses and whether the reactions are genetically regulated. The DA rat strain was used to establish a lung exposure model. Five other inbred rat strains (PVG, PVG.1AV1, LEW, WF and F344) were compared within the model at selected time points. All rat strains displayed a biphasic pattern of leukocyte infiltration in the lungs, dominated by neutrophils 2 days after exposure and a second peak dominated by macrophages 29 days after exposure. The number of macrophages was higher in the DA rat compared with the other strains. The infiltration of lymphocytes in the lungs varied in both time of appearance and magnitude between strains. The quantity of collagen deposition in the lungs varied between strains at day 90; LEW and WF displayed high collagen content which coincided with an increased level of cytotoxic T cells. LEW further displayed an increased number of T helper cells and natural killer (NK) T cells in the lungs. The results in this study suggest there is a link between the development of lung fibrosis and high cytotoxic cell responses and that there is a genetic influence, as there are variations in acute and late adverse reactions between rat strains in both timing and magnitude. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Context: Titanium dioxide (TiO2) nanoparticles (NPs) are regarded as relatively non-toxic in concentrations occurring in occupational environments. Nevertheless, it is conceivable that adverse health effects may develop in sensitive populations such as individuals with respiratory diseases. Objective: We investigated whether single or repeated exposure to TiO2 could aggravate inflammatory responses in naïve mice and mice with ovalbumin (OVA)-induced airway inflammation. Methods: Exposure to aerosolized TiO2 was performed during OVA sensitization, before, or during the OVA challenge period. The effects on respiratory physiology, inflammatory cells in bronchoalveolar lavage (BAL) and inflammatory mediators in BAL and serum were assessed 24 h after the last OVA challenge or TiO2 exposure. Results: A single exposure of TiO2 had a marked effect on responses in peripheral airways and increasing infiltration of neutrophils in airways of naïve animals. Marked aggravation of airway responses was also observed in animals with allergic disease provided that the single dose TiO2 was given before allergen challenge. Repeated exposures to TiO2 during sensitization diminished the OVA-induced airway eosinophilia and airway hyperresponsiveness but concomitant exposure to TiO2 during the OVA challenge period resulted in neutrophilic airway inflammation and a decline in general health condition as indicated by the loss of body weight. Conclusion: We conclude that inhalation of TiO2 may aggravate respiratory diseases and that the adverse health effects are highly dependent on dose and timing of exposure. Our data imply that inhalation of NPs may increase the risk for individuals with allergic airway disease to develop symptoms of severe asthma. [ABSTRACT FROM AUTHOR]

We have compared the cellular uptake and responses of five preparations of nanocrystalline titanium dioxide (TiO2) between normal human bronchial epithelial (NHBE) cells and epithelial cell lines (A549 and BEAS-2B). The P25 nanoparticles, containing both anatase and rutile modifications, induced reactive oxygen species (ROS) and secretion of the neutrophil chemoattractant IL-8 in all three cell types used. Pure anatase and rutile particles provoked differential IL-8 response in A549 and no response in BEAS-2B cells despite similar formation of ROS. The pure TiO2 modifications also provoked release of the inflammatory mediators: IL-6, G-CSF and VEGF, in NHBE cells but not in the two cell lines. We conclude that the responsiveness of lung epithelial cells is strongly dependent on both the physicochemical properties of TiO2 nanoparticles and the type of responder cells. The differential pro-inflammatory responsiveness of primary lung epithelial cells compared with immortalized cell lines should be considered in the assessment of adverse reactions to inhaled nanoparticles. [ABSTRACT FROM AUTHOR]

Nanomaterial of titanium dioxide (TiO2) is manufactured in large-scale production plants, resulting in risks for accidental high exposures of humans. Inhalation of metal oxide nanoparticles in high doses may lead to both acute and long-standing adverse effects. By using the Dark Agouti (DA) rat, a strain disposed to develop chronic inflammation following exposure to immunoactivating adjuvants, we investigated local and systemic inflammatory responses after lung exposure of nanosized TiO2 particles up to 90 days after intratracheal instillation. TiO2 induced a transient response of proinflammatory and T-cell-activating cytokines (interleukin [IL]-1αα, IL-1ββ, IL-6, cytokine-induced neutrophil chemoattractant [CINC]-1, granulocyte--macrophage colony-stimulating factor [GM-CSF], and IL-2) in airways 1--2 days after exposure, accompanied by an influx of eosinophils and neutrophils. Neutrophil numbers remained elevated for 30 days, whereas the eosinophils declined to baseline levels at Day 8, simultaneously with an increase of dendritic cells and natural killer (NK) cells. The innate immune activation was followed by a lymphocyte expansion that persisted throughout the 90-day study. Lymphocytes recruited to the lungs were predominantly CD4++ helper T-cells, but we also demonstrated presence of CD8++ T-cells, B-cells, and CD25++ T-cells. In serum, we detected both an early cytokine expression at Days 1--2 (IL-2, IL-4, IL-6, CINC-1, IL-10, and interferon-gamma [IFN-γγ] and a second response at Day 16 of tumor necrosis factor-alpha (TNF-αα), indicating systemic late-phase effects in addition to the local response in airways. In summary, these data demonstrate a dynamic response to TiO2 nanoparticles in the lungs of DA rats, beginning with an innate immune activation of eosinophils, neutrophils, dendritic cells, and NK cells, followed by a long-lasting activation of lymphocytes involved in adaptive immunity. The results have implications for the assessment of risks for adverse and persistent immune stimulation following nanoparticle exposures in sensitive populations. [ABSTRACT FROM AUTHOR]

Summary Allergic airway inflammation induced in mice is T-cell dependent and recruitment of eosinophils to airspaces requires both αβ and γδ T cells. From previous studies it is evident that αβ T cells are essential for the allergic T helper type 2 (Th2)-like response, while the mechanistic contribution of γδ T cells is still unclear. In this study, we have investigated the role of γδ T cells in allergic airway eosinophilia induced by ovalbumin hypersensitivity. By comparing the responsiveness to sensitizing allergen of wild-type mice with that of T-cell receptor γδ knockout mice (TCRγδ KO) we demonstrated that mice lacking γδ T cells are defective in the systemic ovalbumin-specific immunoglobulin E (IgE) response. Furthermore, after aerosol challenge with allergen, γδ T-cell deficient mice exhibited a significantly decreased migration of B cells and natural killer cells to airways and reduced levels of allergen-specific IgG and IgA in bronchoalveolar lavage fluid. The role for B cells in the airway inflammation was indicated by the impaired ability of mice lacking functional B cells to evoke an eosinophilic response. The diminished eosinophilia in TCRγδ KO mice could not be explained by a defective Th2 activation since these mice displayed a normal IgG response in serum and an unaffected IG2b /IgG1 ratio in airways. Analysis of immunoregulatory cytokines in isolated lung tissue, thoracic lymph nodes and spleen further supported the notion that these mice are able to evoke a sufficient activation of T helper cells and that γδ T cells are not required for maintaining the Th2 profile. These results indicate that γδ T cells contribute to allergic airway inflammation by pathways separate from classical Th2 immune activation. [ABSTRACT FROM AUTHOR]

Abstract: Low-dose exposure of alkylating mustard gas causes long-term respiratory complications characterized by bronchitis and lung fibrosis. In this study, we utilized a mouse model for lung exposure of the nitrogen mustard melphalan, in order to define early and late events in the pathogenesis such as expression of pro-inflammatory cytokines, recruitment of inflammatory cells to airways and late-phase fibrosis. We investigated the roles of different T lymphocyte subsets on the inflammatory response by using knockout mice lacking either the genes expressing T cell receptor (TCR)αβ or TCRγδ, and compared the responsiveness with that of wild type mice and double knockout mice completely deficient in T cells. Exposure to melphalan induced an early burst of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and IL-23 in airways, followed by extensive infiltration of neutrophils in the lung tissue and airways within 24h. The acute phase was followed by a sustained lymphocytic response that persisted for at least 14 days with resulting lung fibrosis. Engagement of T lymphocytes, particularly the γδ T cell subset, was crucial both for the acute cytokine and neutrophil response and for the late-phase lung fibrosis as indicated by the lack of response in γδ T cell deficient mice. Our data demonstrate that T lymphocytes play a prominent role in the pathogenesis of long-term lung injuries caused by strong alkylating agents. [Copyright &y& Elsevier]

Inhalation of chlorine (Cl 2 ) may cause oxidative acute lung injury (ALI) characterized by pulmonary edema, pneumonitis, and hyperreactive airways. The aim of the study was to identify possible biomarkers for Cl 2 -induced ALI. Female BALB/c mice were exposed to Cl 2 for 15 min using two protocols 1) concentration-dependent response (25–200 ppm) and 2) time-kinetics (2h–14 days post-exposure). Exposure to 50–200 ppm Cl 2 caused a concentration-dependent inflammatory response with increased expression of IL-1β, IL-6 and CXCL1/KC in bronchoalveolar lavage fluid 2–6 h after exposure which was followed by increased lung permeability and a neutrophilic inflammation 12–24 h post-exposure. The early inflammatory cytokine response was associated with a clear but transient increase of 8-isoprostane, a biomarker for oxidative stress, with its maximum at 2 h after exposure. An increase of 8-isoprostane could also be detected in serum 2 h after exposure to 200 ppm Cl 2 , which was followed by increased levels of IL-6 and CXCL1/KC and signs of increased fibrinogen and PAI-1. Melphalan, a non-oxidizing mustard gas analog, did not increase the 8-isoprostane levels, indicating that 8-isoprostane is induced in airways through direct oxidation by Cl 2 . We conclude that 8-isoprostane represents an early biomarker for oxidative stress in airways and in the blood circulation following Cl 2 -exposure. [ABSTRACT FROM AUTHOR]

We investigated acute and delayed respiratory changes after inhalation exposure to chlorine (Cl 2 ) with the aim to understand the pathogenesis of the long-term sequelae of Cl 2 -induced lung-injury. In a rat model of nose-only exposure we analyzed changes in airway hyperresponsiveness (AHR), inflammatory responses in airways, expression of pro-inflammatory markers and development of lung fibrosis during a time-course from 5 h up to 90 days after a single inhalation of Cl 2 . A single dose of dexamethasone (10 mg/kg) was administered 1 h following Cl 2 -exposure. A 15-min inhalation of 200 ppm Cl 2 was non-lethal in Sprague-Dawley rats. At 24 h post exposure, Cl 2 -exposed rats displayed elevated numbers of leukocytes with an increase of neutrophils and eosinophils in bronchoalveolar lavage (BAL) and edema was shown both in lung tissue and the heart. At 24 h, the inflammasome-associated cytokines IL-1β and IL-18 were detected in BAL. Concomitant with the acute inflammation a significant AHR was detected. At the later time-points, a delayed inflammatory response was observed together with signs of lung fibrosis as indicated by increased pulmonary macrophages, elevated TGF-β expression in BAL and collagen deposition around airways. Dexamethasone reduced the numbers of neutrophils in BAL at 24 h but did not influence the AHR. Inhalation of Cl 2 in rats leads to acute respiratory and cardiac changes as well as pulmonary inflammation involving induction of TGF-β1. The acute inflammatory response was followed by sustained macrophage response and lack of tissue repair. It was also found that pathways apart from the acute inflammatory response contribute to the Cl 2 -induced respiratory dysfunction. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the inflammatory and immunological responses in airways and lung-draining lymph nodes (LDLNs), following lung exposure to iron oxide (hematite) nanoparticles (NPs). The responses to the hematite NPs were evaluated in both healthy non-sensitized mice, and in sensitized mice with an established allergic airway disease. The mice were exposed intratracheally to either hematite NPs or to vehicle (PBS) and the cellular responses were evaluated on days 1, 2, and 7, post-exposure. Exposure to hematite NPs increased the numbers of neutrophils, eosinophils, and lymphocytes in the airways of non-sensitized mice on days 1 and 2 post-exposure; at these time points the number of lymphocytes was also elevated in the LDLNs. In contrast, exposing sensitized mice to hematite NPs induced a rapid and unspecific cellular reduction in the alveolar space on day 1 post-exposure; a similar decrease of lymphocytes was also observed in the LDLN. The results indicate that cells in the airways and in the LDLN of individuals with established airway inflammation undergo cell death when exposed to hematite NPs. A possible explanation for this toxic response is the extensive generation of reactive oxygen species (ROS) in the pro-oxidative environment of inflamed airways. This study demonstrates how sensitized and non-sensitized mice respond differently to hematite NP exposure, and it highlights the importance of including individuals with respiratory disorders when evaluating health effects of inhaled nanomaterials. [ABSTRACT FROM AUTHOR]

There is a compelling need to understand and assess the toxicity of industrially produced nanoparticles (NPs). In order to appreciate the long-term effects of NPs, sensitive human-based screening tests that comprehensively map the NP properties are needed to detect possible toxic mechanisms. Animal models can only be used in a limited number of test applications and are subject to ethical concerns, and the interpretation of experiments in animals is also distorted by the species differences. Here, we present a novel easy-to-perform highly sensitive whole-blood model using fresh non-anticoagulated human blood, which most justly reflects complex biological cross talks in a human system. As a demonstrator of the tests versatility, we evaluated the toxicity of TiO 2 NPs that are widely used in various applications and otherwise considered to have relatively low toxic properties. We show that TiO 2 NPs at very low concentrations (50 ng/mL) induce strong activation of the contact system, which in this model elicits thromboinflammation. These data are in line with the finding of components of the contact system in the protein corona of the TiO 2 NPs after exposure to blood. The contact system activation may lead to both thrombotic reactions and generation of bradykinin, thereby representing fuel for chronic inflammation in vivo and potentially long-term risk of autoimmunity, arteriosclerosis and cancer. These results support the notion that this novel whole-blood model represents an important contribution to testing of NP toxicity. [ABSTRACT FROM AUTHOR]

Abstract: In this study aerosol samples collected in an Asian mega-city (Kabul, Afghanistan) were compared to PM samples collected in a European location with traffic (Umeå, Sweden) and a reference urban dust material (SRM 1649b). The toxicity of each sample towards normal human bronchial epithelial (NHBE) cells and a human bronchial epithelial cell line (BEAS-2B) was tested along with their ability to induce reactive oxygen species (ROS) formation and inflammatory responses. The extracts’ morphology and elemental composition was studied by SEM-EDXRF, and filter samples were analyzed for metals and organic compounds. The PM from Kabul contained a larger fraction of fine particles, 19 times more polyaromatic hydrocarbons (PAH) and 37 times more oxygenated PAH (oxy-PAH) compared to samples from Umeå. The PM-samples from Kabul and the reference material (SRM 1649b) induced significantly stronger oxidative stress responses than the samples from Umeå. Furthermore, samples collected in Kabul induced significantly higher secretion of the cytokines IL-6, IL-8 and GM-CSF while SRM1649b induced a cytokine pattern more similar to samples collected in Umeå. Several properties of the particles could potentially explain these differences, including differences in their size distribution and contents of PAH and oxy-PAH, possibly in combination with their relative transition metal contents. [Copyright &y& Elsevier]