COPD (chronic obstructive pulmonary disease) is caused by exposure to toxic gases and particles, most often CS (cigarette smoke), leading to emphysema, chronic bronchitis, mucus production and a subsequent decline in lung function. The disease pathogenesis is related to an abnormal CS-induced inflammatory response of the lungs. Similar to active (mainstream) smoking, second hand (sidestream) smoke exposure severely affects respiratory health. These processes can be studied in vivo in models of CS exposure of mice. We compared the acute inflammatory response of female C57BL/6 mice exposed to two concentrations [250 and 500 mg/m3 TPM (total particulate matter)] of sidestream and mainstream CS for 3 days and interpreted the biological effects based on physico-chemical differences in the gas and particulate phase composition of CS. BAL (bronchoalveolar lavage fluid) was obtained to perform differential cell counts and to measure cytokine release. Lung tissue was used to determine mRNA and protein expression of proinflammatory genes and to assess tissue inflammation. A strong acute inflammatory response characterized by neutrophilic influx, increased cytokine secretion [KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factor α), MIP-2 (macrophage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein-1)], pro-inflammatory gene expression [KC, MIP-2 and MMP12 (matrix metalloproteinase 12)] and up-regulated GM-CSF (granulocyte macrophage colony-stimulating factor) production was observed in the mainstream model. After sidestream exposure there was a dampened inflammatory reaction consisting only of macrophages and diminished GM-CSF levels, most likely caused by elevated CO concentrations. These results demonstrate that the composition of CS determines the dynamics of inflammatory cell recruitment in COPD mouse models. Different initial inflammatory processes might contribute to COPD pathogenesis in significantly varying ways, thereby determining the outcome of the studies.
chronic obstructive pulmonary disease (COPD); inflammation; mainstream; neutrophil; sidestream; smoke; BAL, bronchoalveolar lavage; CC, carbonyl compounds; CMD, count median diameter; CO-Hb, carboxyhaemoglobin; COPD, chronic obstructive pulmonary disease; CS, cigarette smoke; DNPH, 2,4-dinitrophenylhydrazine; GC-SIM-MS, gas-chromatography with selective ion monitoring MS; GM-CSF, granulocyte macrophage colony-stimulating factor; HO, haem oxygenase; H&E, haematoxylin and eosin; HPRT-1, hypoxanthine–guanine phosphoribosyltransferase; IFNβ, interferon β; IL-1β, interleukin 1β; KC, keratinocyte chemoattractant; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; MIP, macrophage inflammatory protein; MMD, mass median diameter; MMP12, matrix metalloproteinase 12; NE, neutrophil elastase; NF-κB, nuclear factor κB; PAH, polycyclic aromatic hydrocarbons; PM, particulate matter; TNF-α, tumour necrosis factor α; TPM, total particulate matter
Involuntary inhalation of tobacco smoke has been shown to aggravate the allergic response. Antibodies to fungal antigens such as Aspergillus fumigatus (Af) cause an allergic lung disease in humans. This study was carried out to determine the effect of environmental tobacco smoke (ETS) on a murine model of allergic bronchopulmonary aspergillosis (ABPA). BALB/c mice were exposed to aged and diluted sidestream cigarette smoke to simulate 'second-hand smoke'. The concentration was consistent with that achieved in enclosed public areas or households where multiple people smoke. During exposure, mice were sensitized to Af antigen intranasally. Mice that were sensitized to Af antigen and exposed to ETS developed significantly greater airway hyperreactivity than did mice similarly sensitized to Af but housed in ambient air. The effective concentration of aerosolized acetylcholine needed to double pulmonary flow resistance was significantly lower in Af + ETS mice compared to the Af + AIR mice. Immunological data that supports this exacerbation of airway hyperresponsiveness being mediated by an enhanced type 1 hypersensitivity response include: eosinophilia in peripheral blood and lung sections. All Af sensitized mice produced elevated levels of IL4, IL5 and IL10 but no IFN-γ indicating a polarized Th2 response. Thus, ETS can cause exacerbation of asthma in ABPA as demonstrated by functional airway hyperresponsiveness and elevated levels of blood eosinophilia.
Rationale: Adipose-derived stem cells express multiple growth factors that inhibit endothelial cell apoptosis, and demonstrate substantial pulmonary trapping after intravascular delivery.
Objectives: We hypothesized that adipose stem cells would ameliorate chronic lung injury associated with endothelial cell apoptosis, such as that occurring in emphysema.
Methods: Therapeutic effects of systemically delivered human or mouse adult adipose stem cells were evaluated in murine models of emphysema induced by chronic exposure to cigarette smoke or by inhibition of vascular endothelial growth factor receptors.
Measurements and Main Results: Adipose stem cells were detectable in the parenchyma and large airways of lungs up to 21 days after injection. Adipose stem cell treatment was associated with reduced inflammatory infiltration in response to cigarette smoke exposure, and markedly decreased lung cell death and airspace enlargement in both models of emphysema. Remarkably, therapeutic results of adipose stem cells extended beyond lung protection by rescuing the suppressive effects of cigarette smoke on bone marrow hematopoietic progenitor cell function, and by restoring weight loss sustained by mice during cigarette smoke exposure. Pulmonary vascular protective effects of adipose stem cells were recapitulated by application of cell-free conditioned medium, which improved lung endothelial cell repair and recovery in a wound injury repair model and antagonized effects of cigarette smoke in vitro.
Conclusions: These results suggest a useful therapeutic effect of adipose stem cells on both lung and systemic injury induced by cigarette smoke, and implicate a lung vascular protective function of adipose stem cell derived paracrine factors.
pulmonary disease, chronic obstructive; endothelium; cell death; regenerative medicine; human
Exposure to environmental tobacco smoke (ETS) is known to contribute to and exacerbate inflammatory diseases of the lung such as chronic obstructive pulmonary disease (COPD) and asthma. The effect of ETS on angiogenesis and leukocyte recruitment, both of which promote lung inflammation, was investigated using lung tissue from mice exposed to aged and diluted sidestream cigarette smoke or fresh air for 12 weeks and transplanted into dorsal skin-fold chambers in nude mice. Lung tissue from mice exposed to cigarette smoke for 12 weeks exhibited significantly increased vascular density (angiogenesis) associated with selectin-mediated increased intravascular leukocyte rolling and adhesion compared to fresh air–exposed lung tissue by intravital microscopy. Further, neutrophils from nicotine-exposed mice displayed significantly increased rolling and adhesion compared to control neutrophils in microvessels of nicotine-exposed lungs versus control lung microvessels, suggesting that nicotine in cigarette smoke can augment leukocyte-endothelial interactions. ETS-induced angiogenesis and leukocyte trafficking may play a key role in airway recruitment of inflammatory cells in ETS-associated disorders such as COPD bronchitis or asthma.
environmental tobacco smoke; leukocyte trafficking; lung angiogenesis; nicotine
Rationale: Matrix metalloprotease (MMP)-9 is an elastolytic endopeptidase produced by activated macrophages that may be involved in the development of human pulmonary emphysema and could be inhibited with existing compounds. Mouse models have demonstrated that excess MMP-9 production can result in permanent alveolar destruction.
Objectives: To determine if MMP-9 causes cigarette smoke–induced emphysema using MMP-9 knockout mice and human samples.
Methods: Mouse lungs were analyzed for inflammation and airspace enlargement using a mainstream smoke-exposure model. Human macrophage mRNA was isolated from subjects with emphysema by laser capture microdissection. Human blood monocyte mRNA was isolated from subjects with greater than 30 pack-year smoking history. Human gene expression was determined by quantitative polymerase chain reaction and compared with emphysema severity determined by automated computed tomography analysis. Plasma Clara cell secretory protein and surfactant protein-D were quantified to measure ongoing lung injury.
Measurements and Main Results: Mice deficient in MMP-9 develop the same degree of cigarette smoke–induced inflammation and airspace enlargement as strain-matched controls. Macrophages are the predominant source of MMP-9 production in human emphysema specimens and similar quantities of macrophage MMP-9 mRNA is present in areas of lung with and without emphysema. Circulating monocytes produce more MMP-9 in individuals with advanced emphysema severity despite no correlation of MMP-9 with markers of ongoing lung damage.
Conclusions: These results suggest that MMP-9 in humans who smoke is similar to smoke-exposed mice, where MMP-9 is present in emphysematous lung but not correlated with the emphysema. To the degree that the mechanisms of emphysema in humans who smoke resemble the mouse model, these data suggest specific inhibition of MMP-9 is unlikely to be an effective therapy for cigarette smoke–induced emphysema.
Clinical trial registered with www.clinicaltrials.gov (NCT 00757120).
pulmonary disease, chronic obstructive; laser capture microdissection; mice, knockout
Tobacco smoke exhaled from smokers is a key component of secondhand smoke, contributing to lung alveolar wall destruction seen in chronic lung diseases. Although mainstream and sidestream tobacco smoke are cyto-toxic to lung cells, it is unclear whether exhaled smoke induces lung cell injury or even death. We sought to establish an in vitro model to examine the effects of exhaled smoke on lung cells. Phosphate-buffered saline-conditioned cigarette smoke (CCS) derived from a blow-by system was used to mimic exhaled tobacco smoke exposure. Exposure of medium to CCS leads to dose-dependent increases in nicotine/cotinine levels. Scanning spectrophotometric analysis of the CCS-exposed medium reveals an absorption peak at 290 nm wavelength. The OD values at 290 nm are correlated with nicotine levels in the exposed medium, indicating that a simple measurement of OD at 290 nm can be used to monitor CCS exposure. Tobacco smoke contacts the microvascular endothelium located at lung alveoli, before it enters the blood stream. Hence, human lung microvascular endothelial cells (hMVEC) were exposed to CCS and assessed for cell injury and death. Exposure of hMVEC to CCS equivalent to burning 12-16 cigarettes leads to increased LDH release from the cells into the medium. This suggests that CCS can induce lung cell injury. CCS at a low level increases cell growth, whereas the high level of CCS decreases cell viability. In addition, CCS exposure induces cell detachment and morphological changes. Our results demonstrate that exposure of buffer-conditioned mainstream cigarette smoke leads to increased nicotine/cotinine levels and cell injury/death, which may contribute to the pathophysiology of passive smoking-associated lung diseases.
Cellular model; secondhand smoke; chronic lung diseases; lung microvascular endothelial cells; passive smoking-associated lung diseases
Chronic obstructive pulmonary disease (COPD) is characterized by the presence of airflow obstruction and lung destruction with airspace enlargement. In addition to cigarette smoking, respiratory pathogens play a role in pathogenesis, but specific organisms are not always identified. Recent reports demonstrate associations between the detection of Pneumocystis jirovecii DNA in lung specimens or respiratory secretions and the presence of emphysema in COPD patients. Additionally, human immunodeficiency virus-infected individuals who smoke cigarettes develop early emphysema, but a role for P. jirovecii in pathogenesis remains speculative. We developed a new experimental model using immunocompetent mice to test the interaction of cigarette smoke exposure and environmentally acquired Pneumocystis murina infection in vivo. We hypothesized that cigarette smoke and P. murina would interact to cause increases in total lung capacity, airspace enlargement, and pulmonary inflammation. We found that exposure to cigarette smoke significantly increases the lung organism burden of P. murina. Pulmonary infection with P. murina, combined with cigarette smoke exposure, results in changes in pulmonary function and airspace enlargement characteristic of pulmonary emphysema. P. murina and cigarette smoke exposure interact to cause increased lung inflammatory cell accumulation. These findings establish a novel animal model system to explore the role of Pneumocystis species in the pathogenesis of COPD.
Approximately 15% of cases of COPD occur in non-smokers. Among the potential risk factors for COPD in non-smokers is second hand smoke (SHS) exposure. However, the Surgeon General reported in 2006 that the evidence linking second hand smoke and COPD is insufficient to infer a causal relationship, largely because current evidence does not establish a biological link.
The goal of this study was to determine whether SHS exposure can induce alveolar macrophage recruitment and expression of activation markers that we have previously demonstrated in human smokers and in mouse models of emphysema. To achieve these goals, we studied mice exposed to an ambient mixture of predominantly [89%] sidestream smoke at increasing doses over 3 months.
We found that second hand smoke exposure induced a dose-dependent increase in alveolar macrophage recruitment (mean ± sd; 224,511 ± 52,330 vs 166,152 ± 47,989 macrophages/ml of bronchoalveolar lavage in smoke-exposed vs air-exposed controls at 3 months, p=0.003). We also found increased expression of several markers of alveolar macrophage activation (PLA2g7, dkfzp434l142, Trem-2, and pirin, all p<0.01 at 3 months) and increased lavage levels of two inflammatory mediators associated with COPD (CCL2 [MCP-1], 58 ± 12 vs. 43 ± 22 pg/ml, p=0.03; and TNFα, 138 ± 43 vs 88 ± 78 pg/ml, p=0.04 at 3 months).
These findings indicate that second smoke exposure can cause macrophage recruitment and activation, providing a biological link between second hand smoke exposure and the development of inflammatory processes linked to COPD.
macrophage; tobacco; cigarette smoke; inflammation; cytokine
Chronic Obstructive Pulmonary Disease (COPD) is one of the foremost causes of death worldwide. It is primarily caused by tobacco smoke, making it an easily preventable disease, but facilitated by genetic α-1 antitrypsin deficiency. In addition to active smokers, health problems also occur in people involuntarily exposed to second hand smoke (SHS). Currently, the relationship between SHS and COPD is not well established. Knowledge of pathogenic mechanisms is limited, thereby halting the advancement of new treatments for this socially and economically detrimental disease. Here, we attempt to summarize tobacco smoke studies undertaken in animal models, applying both mainstream (direct, nose only) and side stream (indirect, whole body) smoke exposures. This overview of 155 studies compares cellular and molecular mechanisms as well as proteolytic, inflammatory, and vasoreactive responses underlying COPD development. This is a difficult task, as listing of exposure parameters is limited for most experiments. We show that both mainstream and SHS studies largely present similar inflammatory cell populations dominated by macrophages as well as elevated chemokine/cytokine levels, such as TNF-α. Additionally, SHS, like mainstream smoke, has been shown to cause vascular remodeling and neutrophil elastase-mediated proteolytic matrix breakdown with failure to repair. Disease mechanisms and therapeutic interventions appear to coincide in both exposure scenarios. One of the more widely applied interventions, the anti-oxidant therapy, is successful for both mainstream and SHS. The comparison of direct with indirect smoke exposure studies in this review emphasizes that, even though there are many overlapping pathways, it is not conclusive that SHS is using exactly the same mechanisms as direct smoke in COPD pathogenesis, but should be considered a preventable health risk. Some characteristics and therapeutic alternatives uniquely exist in SHS-related COPD.
second hand cigarette smoke; COPD; emphysema; inflammation; animal model; chamber; pulmonary hypertension; matrix degradation
Exposure to environmental tobacco smoke (ETS), which is largely composed of the sidestream cigarette smoke, has been implicated in increased incidence of cancer among nonsmokers. The present study was conducted to compare the potential of mainstream and sidestream cigarette smoke to induce DNA adducts in mice. Groups of female C57Bl and DBA mice were exposed twice daily for 65-70 weeks to mainstream or sidestream smoke from the University of Kentucky reference cigarettes (2R1) in a nose-only exposure system. Animals received a total particulate matter dose of about 16 and 6 mg/kg body weight/exposure and exhibited blood carboxyhemoglobin levels of about 16 and 34%, for mainstream and sidestream smoke-exposed groups, respectively. Pulmonary aryl hydrocarbon hydroxylase (AHH) activity was induced by about 2- to 3-fold in both mainstream and sidestream groups of C57Bl and in mainstream smoke-exposed group of DBA mice, but not in sidestream smoke-exposed DBA mice. An analysis of total DNA adduct levels by the 32P-postlabeling assay showed a significant (12- to 25-fold) increase in the magnitude of preexisting lung DNA adducts in both mainstream and sidestream smoke-exposed C57Bl and DBA mice. Smoke exposures did not affect the total preexisting DNA adducts in liver of either strain. It is concluded that both mainstream and sidestream smoke are capable of enhancing preexisting DNA adducts in the lungs of chronically smoke-exposed mice.
Chronic obstructive pulmonary disease (COPD) is a progressive condition characterized by chronic airway inflammation and airspace remodeling, leading to airflow limitation that is not completely reversible. Smoking is the leading risk factor for compromised lung function stemming from COPD pathogenesis. First- and second-hand cigarette smoke contain thousands of constituents, including several carcinogens and cytotoxic chemicals that orchestrate chronic lung inflammation and destructive alveolar remodeling. Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors primarily expressed by diverse lung cells. RAGE expression increases following cigarette smoke exposure and expression is elevated in the lungs of patients with COPD. RAGE is responsible in part for inducing pro-inflammatory signaling pathways that culminate in expression and secretion of several cytokines, chemokines, enzymes, and other mediators. In the current review, new transgenic mouse models that conditionally over-express RAGE in pulmonary epithelium are discussed. When RAGE is over-expressed throughout embryogenesis, apoptosis in the peripheral lung causes severe lung hypoplasia. Interestingly, apoptosis in RAGE transgenic mice occurs via conserved apoptotic pathways also known to function in advanced stages of COPD. RAGE over-expression in the adult lung models features of COPD including pronounced inflammation and loss of parenchymal tissue. Understanding the biological contributions of RAGE during cigarette smoke-induced inflammation may provide critically important insight into the pathology of COPD.
RAGE; COPD; tobacco; mouse model
Atherosclerosis is the leading cause of death in western societies and cigarette smoke is among the factors that strongly contribute to the development of this disease. The early events in atherogenesis are stimulated on the one hand by cytokines that chemoattract leukocytes and on the other hand by decrease in circulating molecules that protect endothelial cells (ECs) from injury. Here we focus our studies on the effects of "second-hand" smoke on atherogenesis.
To perform these studies, a smoking system that closely simulates exposure of humans to second-hand smoke was developed and a mouse model system transgenic for human apoB100 was used. These mice have moderate lipid levels that closely mimic human conditions that lead to atherosclerotic plaque formation.
"Second-hand" cigarette smoke decreases plasma high density lipoprotein levels in the blood and also decreases the ratios between high density lipoprotein and low density lipoprotein, high density lipoprotein and triglyceride, and high density lipoprotein and total cholesterol. This change in lipid profiles causes not only more lipid accumulation in the aorta but also lipid deposition in many of the smaller vessels of the heart and in hepatocytes. In addition, mice exposed to smoke have increased levels of Monocyte Chemoattractant Protein–1 in circulation and in the heart/aorta tissue, have increased macrophages in the arterial walls, and have decreased levels of adiponectin, an EC-protective protein. Also, cytokine arrays revealed that mice exposed to smoke do not undergo the switch from the pro-inflammatory cytokine profile (that develops when the mice are initially exposed to second-hand smoke) to the adaptive response. Furthermore, triglyceride levels increase significantly in the liver of smoke-exposed mice.
Long-term exposure to "second-hand" smoke creates a state of permanent inflammation and an imbalance in the lipid profile that leads to lipid accumulation in the liver and in the blood vessels of the heart and aorta. The former potentially can lead to non-alcoholic fatty liver disease and the latter to heart attacks.
Chronic obstructive pulmonary disease is associated with a chronic inflammatory response of the host to chronic exposure to inhaled toxic gases and particles. Although inflammatory cells of both the innate and adaptive immune system infiltrate the lungs in pulmonary emphysema and form lymphoid follicles around the small airways, the exact role of the acquired immune system in the pathogenesis of emphysema is not known.
In this study, wild type Balb/c mice and immunodeficient scid mice – which lack functional B- and T-cells – were exposed to mainstream cigarette smoke (CS) for 5 weeks or 6 months.
Subacute CS-exposure for 5 weeks significantly increased innate inflammatory cells (neutrophils, macrophages and dendritic cells) in the bronchoalveolar lavage (BAL) fluid of wild type mice and scid mice, which correlated with the CS-induced upregulation of the chemokines Monocyte Chemotactic Protein-1, Macrophage Inflammatory Protein-3α and KC (= mouse Interleukin-8). Chronic CS-exposure for 6 months significantly increased the number of neutrophils, macrophages, dendritic cells, CD4+ and CD8+ T-lymphocytes in BAL fluid and lungs of wild type mice compared to air-exposed littermates, and augmented the size and number of peribronchial lymphoid follicles. In contrast, neither B-lymphocytes, nor T-lymphocytes, nor lymphoid follicles could be discerned in the lungs of air- or CS-exposed scid mice. Importantly, chronic CS-exposure induced pulmonary emphysema in both wild type animals and scid mice, as evidenced by a significant increase in the mean linear intercept and the destructive index of CS-exposed versus air-exposed animals. The CS-induced emphysema was associated with increased mRNA expression of matrix metalloproteinase-12 in the lungs and increased protein levels of Tumor Necrosis Factor-α in the BAL fluid of CS-exposed Balb/c and scid mice compared to air-exposed littermates.
This study suggests that the adaptive immune system is not required per se to develop pulmonary emphysema in response to chronic CS-exposure, since emphysema can be induced in scid mice, which lack lymphoid follicles as well as functional B- and T-cells.
Increased numbers of macrophages are found in the lungs of smokers and those with chronic obstructive pulmonary disease. Experimental evidence shows the central role of macrophages in elaboration of inflammatory mediators such as TNF-a and the progression toward cigarette smoke-induced emphysema. We investigated the role of CX3CR1 in recruitment of mononuclear phagocytes, inflammatory cytokine responses, and tissue destruction in the lungs after cigarette smoke exposure. Using mice in which egfp is expressed at the locus of the cx3cr1 gene, we show that alveolar macrophages increased transmembrane ligand CX3CL1 expression and soluble CX3CL1 was detectable in the airspaces, but cx3cr1GFP/GFP and cx3cr1GFP/+ mice failed to show recruitment of CX3CR1+ cells into the airspaces with cigarette smoke. In contrast, cigarette smoke increased the accumulation of CX3CR1+CD11b+ mononuclear phagocytes that were spatially confined to the lung interstitium and heterogenous in their expression of CD11c, MHC class II, and autofluorescent property. Although an intact CX3CL1–CX3CR1 pathway amplified the percentage of CX3CR1+CD11b+ mononuclear phagocytes in the lungs, it was not essential for recruitment. Rather, functional CX3CR1 was required for a subset of tissue-bound mononuclear phagocytes to produce TNF-α and IL-6 in response to cigarette smoke, and the absence of functional CX3CR1 protected mice from developing tissue-destructive emphysema. Thus, CX3CR1+ “tissue resident” mononuclear phagocytes initiate an innate immune response to cigarette smoke by producing TNF-α and IL-6 and are capable of promoting emphysema.
Rationale: Arhgef1 is an intracellular protein, expressed by hematopoietic cells, that regulates signaling by both G protein–coupled receptors and RhoA, and, consequently, is required for appropriate migration and adhesion of diverse leukocyte populations.
Objectives: To evaluate a possible contribution for Arhgef1 in the development of airway inflammation and airway hyperreactivity.
Methods: Arhgef1-deficient (Arhgef1−/−) and wild-type (WT) mice were sensitized and airway challenged, followed by measurement of airway responsiveness to inhaled methacholine. Inflammation was assessed by several parameters that included flow cytometric analysis and histology. Arhgef1-deficient recipients were reconstituted with WT T lymphocytes before sensitization and challenge, and again measured for airway responsiveness and inflammation. Cytokine production in response to specific antigen was measured in cultures of isolated leukocytes from lung and spleen and compared with the levels generated in lung and spleen explant cultures.
Measurements and Main Results: Arhgef1−/− mice display significantly reduced airway hyperreactivity, Th2 cytokine production, and lung inflammation, despite intact systemic immunity. After airway challenge of Arhgef1−/− mice, antigen-specific T cells were present in mutant lungs, but were found to interact with CD11c+ cells at a significantly reduced frequency. Adoptive transfer of WT T cells into Arhgef1−/− mice restored airway hyperreactivity and inflammation.
Conclusions: These data demonstrate that T cells depend on Arhgef1 to promote lung inflammation. Moreover, a deficiency in Arhgef1 results in reduced T cell–CD11c+ antigen-presenting cell interaction, and likely underscores the inability of Arhgef1−/− mice to mount an adaptive immune response to airway challenge.
airway hyperreactivity; cytokines; lung inflammation; T cells
Secondhand smoke (SHS) has been associated with a variety of adverse health outcomes in nonsmokers, including emphysema (a chronic obstructive pulmonary disease). One way to detect SHS exposure is to measure the concentration of cotinine, the primary metabolite of nicotine, in bodily fluids. We have developed a method for cotinine analysis by combining micellar electrokinetic chromatography with enrichment techniques. We employed the method to measure cotinine concentrations in serum samples of mice exposed to tobacco smoke for 12 or 24 weeks and found that it was 3.1-fold or 4.8-fold higher than those exposed to room air for the same period. Further, we investigated the morphological changes in lungs of mice and observed tobacco smoke induced emphysema. Our results indicate that the method can be used to measure cotinine and there is an association between the serum cotinine concentration and tobacco smoke-induced emphysema in mice.
In utero exposure to second-hand smoke (SHS) is associated with exacerbated asthmatic responses in children. We tested the hypothesis that in utero SHS will aggravate the lung responses of young adult mice re-exposed to SHS. We exposed Balb/c mice in utero to SHS (S) or filtered air (AIR; A), and re-exposed the male offspring daily from 11–15 weeks of age to either SHS (AS and SS) or AIR (AA and SA). After the adult exposures, we analyzed samples of bronchoalveolar lavage fluid (BALF), examined the results of histopathology, and assessed pulmonary function and gene expression changes in lung samples. In SS mice, compared with the other three groups (AA, AS, and SA), we found decreases in breathing frequency and increases in airway hyperresponsiveness (AHR), as well as low but significantly elevated concentrations of BALF proinflammatory cytokines (IL-1b, IL-6, and keratinocyte-derived chemokine). Lung morphometric analyses revealed enlarged airspaces and arteries in SA and SS mice compared with their in utero AIR counterparts, as well as increased collagen deposition in AS and SS mice. Unique gene expression profiles were found for in utero, adult, and combined exposures, as well as for mice with elevated AHR responses. The profibrotic metalloprotease genes, Adamts9 and Mmp3, were up-regulated in the SS and AHR groups, suggesting a role for in utero SHS exposure on the adult development of chronic obstructive pulmonary disease. Our results indicate that in utero exposures to environmentally relevant concentrations of SHS alter lung structure more severely than do adult SHS exposures of longer duration. These in utero exposures also aggravate AHR and promote a profibrotic milieu in adult lungs.
second-hand smoke; in utero exposure; airway hyperresponsiveness; lung structure changes; gene expression
Cigarette smoke is the major risk factor associated with the development of chronic obstructive pulmonary disease (COPD). Recent studies propose a link between endoplasmic reticulum (ER) stress and emphysema, demonstrated by increased ER stress markers under smoking conditions. Here, we investigate whether cigarette smoke-induced ER stress is cell specific and correlates with acute and chronic cigarette smoke exposure.
Gene and protein expression changes in human primary lung cell cultures following cigarette smoke extract (CSE) exposure were monitored by qPCR and Western blot analysis. Mice and guinea pigs were exposed to cigarette smoke and ER stress markers examined in whole lung homogenates. Inflammatory cells from the bronchoalveolar lavage fluid of 10 days smoke exposed mice were also examined.
Cigarette smoke induced a trend increase in the ER stress response through an activating transcription factor 4 (ATF4) mediated induction of C/EBP homologous protein (CHOP) in primary small airway epithelial cells. Bronchial epithelial cells and macrophages responded similarly to CSE. Wild-type mice and guinea pigs exposed to acute levels of cigarette smoke exhibited increased levels of CHOP but not at significant levels. However, after long-term chronic cigarette smoke exposure, CHOP expression was reduced. Interestingly, inflammatory cells from smoke exposed mice had a significant increase in CHOP/ATF4 expression.
A trend increase in CHOP levels appear in multiple human lung cell types following acute cigarette smoke exposure in vitro. In vivo, inflammatory cells, predominately macrophages, demonstrate significant cigarette smoke-induced ER stress. Early induction of CHOP in cigarette smoke may play a pivotal role in early induction of lung disease, however in vivo long-term cigarette smoke exposure exhibited a reduction in the ER stress response.
COPD; ER stress; cigarette smoke; CHOP
Smoking cessation has been demonstrated to reduce the rate of loss of lung function and mortality among patients with mild to moderate chronic obstructive pulmonary disease (COPD). There is a paucity of evidence about the effects of smoking cessation on the risk of COPD exacerbations.
We sought to examine whether smoking status and the duration of abstinence from tobacco smoke is associated with a decreased risk of COPD exacerbations.
We assessed current smoking status and duration of smoking abstinence by self-report. Our primary outcome was either an inpatient or outpatient COPD exacerbation. We used Cox regression to estimate the risk of COPD exacerbation associated with smoking status and duration of smoking cessation.
We performed a cohort study of 23,971 veterans who were current and past smokers and had been seen in one of seven Department of Veterans Affairs (VA) primary care clinics throughout the US.
MEASUREMENTS AND MAIN RESULTS
In comparison to current smokers, ex-smokers had a significantly reduced risk of COPD exacerbation after adjusting for age, comorbidity, markers of COPD severity and socio-economic status (adjusted HR 0.78, 95% CI 0.75–0.87). The magnitude of the reduced risk was dependent on the duration of smoking abstinence (adjusted HR: quit <1 year, 1.04; 95% CI 0.87–1.26; 1–5 years 0.93, 95% CI 0.79–1.08; 5–10 years 0.84, 95% CI 0.70–1.00; ≥10 years 0.65, 95% CI 0.58–0.74; linear trend <0.001).
Smoking cessation is associated with a reduced risk of COPD exacerbations, and the described reduction is dependent upon the duration of abstinence.
chronic obstructive pulmonary disease; exacerbation; smoking cessation
Background: Exposure to secondhand smoke causes lung cancer; however, there are little data in the open literature on the in vivo toxicology of fresh sidestream cigarette smoke to guide the debate about smoke-free workplaces and public places.
Objective: To investigate the unpublished in vivo research on sidestream cigarette smoke done by Philip Morris Tobacco Company during the 1980s at its Institut für Biologische Forschung (INBIFO).
Methods: Analysis of internal tobacco industry documents now available at the University of California San Francisco Legacy Tobacco Documents Library and other websites.
Results: Inhaled fresh sidestream cigarette smoke is approximately four times more toxic per gram total particulate matter (TPM) than mainstream cigarette smoke. Sidestream condensate is approximately three times more toxic per gram and two to six times more tumourigenic per gram than mainstream condensate by dermal application. The gas/vapour phase of sidestream smoke is responsible for most of the sensory irritation and respiratory tract epithelium damage. Fresh sidestream smoke inhibits normal weight gain in developing animals. In a 21day exposure, fresh sidestream smoke can cause damage to the respiratory epithelium at concentrations of 2 µg/l TPM. Damage to the respiratory epithelium increases with longer exposures. The toxicity of whole sidestream smoke is higher than the sum of the toxicities of its major constituents.
Conclusion: Fresh sidestream smoke at concentrations commonly encountered indoors is well above a 2 µg/m3 reference concentration (the level at which acute effects are unlikely to occur), calculated from the results of the INBIFO studies, that defines acute toxicity to humans. Smoke-free public places and workplaces are the only practical way to protect the public health from the toxins in sidestream smoke.
Aerosolized hyaluronan (HA) has been previously shown to prevent cigarette smoke-induced airspace enlargement and elastic fiber injury in mice when given concurrently with smoke. In the present study, a more stringent test of the therapeutic potential of HA was performed by delaying treatment with this agent for 1 month. After treatment with cigarette smoke for 3 h per day for 5 days per week for 1 month, mice (DBA/2J) began receiving aerosolized HA (0.1%) for 1 h prior to smoke exposure (controls were given aerosolized water). The results indicate that much of the damage to the lung elastic fibers occurred within the first several months of smoke exposure, as measured by levels of desmosine and isodesmosine (DID) in bronchoalveolar lavage fluid (BALF). In contrast to previously published studies, where concurrent administration of aerosolized HA significantly reduced BALF DID levels within 3 months of smoke exposure, the same effect was not seen until 6 months when HA treatment was delayed. However, despite the prolonged breakdown of elastic fibers in the current study, a significant reduction in airspace enlargement was observed after only 2 months of HA treatment. These findings provide further support for testing this agent in patients with preexisting chronic obstructive pulmonary disease.
Hyaluronan; Lung; Desmosine; Cigarette Smoke; COPD
Rationale: Exposure to environmental tobacco smoke in early life has adverse effects on lung development. Apoptosis plays an essential role in development; however, the molecular mechanisms of pulmonary apoptosis induced by environmental tobacco smoke is unknown.
Objectives: To investigate the mechanistic role of nuclear factor (NF)-κB, a critical cell survival pathway, in the developing lungs exposed to environmental tobacco smoke.
Methods: Timed-pregnant rhesus monkeys and their offspring were exposed to filtered air or to aged and diluted sidestream cigarette smoke as a surrogate to environmental tobacco smoke (a total suspended particulate concentration of 0.99 mg/m3 for 6 h/d, 5 d/wk) from 45–50 d gestational age to 72–77 d postnatal age (n = 4/group).
Measurements and Main Results: NF-κB–DNA binding activity, regulated anti-apoptotic genes, and apoptosis were measured in lung tissues. Exposure to environmental tobacco smoke significantly suppressed NF-κB activation pathway and activity. Environmental tobacco smoke further down-regulated NF-κB–dependent anti-apoptotic genes and induced activation of caspases, cleavage of cellular death substrates (poly(ADP)-ribose polymerase and caspase-activated DNase) and an increase in the rate of apoptosis in the lung parenchyma. No significant alterations were observed for activator protein 1, p53 or Akt activity.
Conclusions: Our results indicate that exposure to low levels of environmental tobacco smoke during a critical window of maturation in the neonatal nonhuman primate may compromise lung development with potential implications for future lung growth and function. These findings support our hypothesis that NF-κB plays a key role in the regulation of the apoptotic process.
apoptosis; environmental tobacco smoke; infant monkeys; lung development; NF-κB
Objectives To investigate the association between environmental tobacco smoke, plasma cotinine concentration, and respiratory cancer or death.
Design Nested case-control study within the European prospective investigation into cancer and nutrition (EPIC).
Participants 303 020 people from the EPIC cohort (total 500 000) who had never smoked or who had stopped smoking for at least 10 years, 123 479 of whom provided information on exposure to environmental tobacco smoke. Cases were people who developed respiratory cancers or died from respiratory conditions. Controls were matched for sex, age (plus or minus 5 years), smoking status, country of recruitment, and time elapsed since recruitment.
Main outcome measures Newly diagnosed cancer of lung, pharynx, and larynx; deaths from chronic obstructive pulmonary disease or emphysema. Plasma cotinine concentration was measured in 1574 people.
Results Over seven years of follow up, 97 people had newly diagnosed lung cancer, 20 had upper respiratory cancers (pharynx, larynx), and 14 died from chronic obstructive pulmonary disease or emphysema. In the whole cohort exposure to environmental tobacco smoke was associated with increased risks (hazard ratio 1.30, 95% confidence interval 0.87 to 1.95, for all respiratory diseases; 1.34, 0.85 to 2.13, for lung cancer alone). Higher results were found in the nested case-control study (odds ratio 1.70, 1.02 to 2.82, for respiratory diseases; 1.76, 0.96 to 3.23, for lung cancer alone). Odds ratios were consistently higher in former smokers than in those who had never smoked; the association was limited to exposure related to work. Cotinine concentration was clearly associated with self reported exposure (3.30, 2.07 to 5.23, for detectable/non-detectable cotinine), but it was not associated with the risk of respiratory diseases or lung cancer. Frequent exposure to environmental tobacco smoke during childhood was associated with lung cancer in adulthood (hazard ratio 3.63, 1.19 to 11.11, for daily exposure for many hours).
Conclusions This large prospective study, in which the smoking status was supported by cotinine measurements, confirms that environmental tobacco smoke is a risk factor for lung cancer and other respiratory diseases, particularly in ex-smokers.
Cigarette smoking cannot fully explain the epidemiologic characteristics of chronic obstructive pulmonary disease (COPD) in women, particularly for those who rarely smoke, but COPD risk is not less than men. The aim of our study is to investigate the relationship between second-hand smoke (SHS) exposure and chronic bronchitis in Taiwanese women.
We used Taiwan's National Health Insurance Bureau claims data in 1999, and cross-checked using criteria set by the American Thoracic Society; there were 33 women with chronic bronchitis, 182 with probable chronic bronchitis, and 205 with no chronic bronchitis during our interview time between 2000 and 2005. We measured second-hand smoke (SHS) exposure by self-reported measures (household users and duration of exposure), and validated this by measuring urinary cotinine levels of a subset subjects. Classification of chronic bronchitis was also based on spirometry defined according to the GOLD guidelines to get the severity of COPD.
Women who smoked and women who had been exposed to a lifetime of SHS were 24.81-fold (95% CI: 5.78-106.38) and 3.65-fold (95% CI: 1.19-11.26) more likely to have chronic bronchitis, respectively, than those who had not been exposed to SHS. In addition, there was a significant increasing trend between the severity of COPD and exposure years of SHS (p < 0.01). The population attributable risk percentages of chronic bronchitis for smokers and those exposed to SHS were 23.2 and 47.3% respectively.
These findings indicate that, besides cigarette smoking, exposure to SHS is a major risk factor for chronic bronchitis in Taiwanese women.
Chronic Obstructive Pulmonary Disease (COPD) is a disease defined by an aberrant inflammatory response to inhaled cigarette smoke and other noxious particles. The factors triggered in the lungs that drive inflammation and lung tissue destruction are not fully understood, but mononuclear phagocytes play a central role by releasing mediators that promote both inflammation and tissue destructive emphysema. Although conflicting studies in alveolar macrophages exist regarding chronic cigarette smoke exposure and its effects on macrophage polarization patterns, we have recently identified a cell-type in mice defined by CX3CR1 expression whose population expands in the lungs and elaborates M1 signature cytokines in response to cigarette smoke exposure in vivo. In addition, the absence of functional CX3CR1 provides protection from tissue-destructive emphysema in a murine model of chronic cigarette smoke exposure. The heterogeneity and plasticity of discrete macrophage subsets, in terms of immuno-phenotype and function, may explain the seemingly disparate findings showing a suppressed inflammatory profile on the one hand and heightened inflammatory response on the other. This review proposes to examine the evidence that discrete mononuclear phagocyte subsets develop in response to cigarette smoke exposure, and the spatial cues provided by the lung tissue microenvironment in which the mononuclear phagocyte resides may influence the distribution and function of these subsets.
chemokines; macrophages; pattern recognition receptors