Variation in response to cytotoxicity of cigarette smoke.

The cytotoxic effect of cigarette smoke condensate on human polymorphs was investigated in vitro by the method of vital dye exclusion. Exposure to 1/800 of the smoke from one high-tar cigarette killed a detectable proportion of a population of 10(6) cells. The response among the cells from 40 healthy people varied widely, the percentage of dead cells ranging from 3% to 66% and from 17% to 87% at exposure levels of 125 micrograms and 250 micrograms cigarette smoke condensate respectively. Differences in individuals'' responses were reproducible and unrelated to age or sex or smoking habit. The cells from 10 patients with irreversible obstructive airways disease and probable emphysema were significantly more sensitive than those from 10 patients with no respiratory disability matched for age and smoking habits. Genetically influenced variation in cellular response to cytotoxicity may be an important determinant of the risk of developing emphysema among smokers.     

STAT3 activation inhibits human bronchial epithelial cell apoptosis in response to cigarette smoke exposure

We have previously reported that cigarette smoke can induce DNA damage in human lung cells without leading to apoptosis or necrosis. In this study, we report that STAT3 is required for the survival of human bronchial epithelial cells (HBECs) following cigarette smoke-induced DNA damage. Cigarette smoke extract (CSE) exposure increases STAT3 phosphorylation (Tyr 705) and DNA binding activity in HBECs. CSE also stimulates IL-6 release and mRNA expression. Anti-IL-6 neutralizing antibody partially blocks STAT3 activation and renders the cells sensitive to CSE-induced DNA damage. Suppression of STAT3 by siRNA results in severe DNA damage and cell death in response to CSE exposure. These findings suggest that STAT3 mediates HBEC survival in response to CSE-induced DNA damage, at least in part, through the IL-6/STAT3 signaling pathway.     

The hazard of cigarette smoke to nonsmokers

The hazard of ambient cigarette smoke to nonsmokers is studied by comparing the average dose of exposure to ambient smoke of nonsmokers with the dose of exposure to primary smoke of smokers with due consideration of the possible nonlinear effect on low-density exposures. The maximum risk is estimated by assuming a linear response. The result is that the effect of ambient smoke is 1/13 of that due to primary smoke. Based on the meager information on the nonlinear effect, a minimum estimate is obtained which reduces the risk of ambient smoke to 1/60 of that of primary smoke. Thus the ambient smoke is estimated to cause an excess of deaths between 50,000 and 10,000 a year in a population of 220,000,000, or to reduce the life expectancy by an amount between 225 days and 48 days.     

The cytotoxic effect of volatile organic compounds of the gas phase of cigarette smoke on lung epithelial cells

Health effects of cigarette smoke (CS) in humans are well known from both clinical and epidemiological studies. However, the mechanism behind CS toxicity and carcinogenicity remains mainly unknown. Recent studies have pointed to the major importance of the gas phase of CS in generating its cytotoxic effects. In the current study, an exposure system capable of introducing the gas phase of mainstream cigarette smoke deprived of its volatile organic constituents (VOCs) was used to study the role of the nonorganic components of the gas phase on the cytotoxicity of smoke to monolayer cultures of mouse lung epithelial cells. Cell viability was measured by Wst-1 and the lactate dehydrogenase (LDH) assays. In cells treated with increasing doses of mainstream cigarette smoke gas phase (one to nine puffs), a dose-dependent increase in cytotoxicity was observed (one puff, 95% viability; nine puffs, 40% viability). Cell viability of cultures exposed to gas phase with only the nonorganic components was found to be equivalent to control, unexposed cultures, indicating that removal of VOCs resulted in almost eliminating the cytotoxic ability of the gas phase of CS. Furthermore, the removal of VOCs seems to reduce the effects of protein tyrosine nitration mediated through the gas phase constituents. The results obtained suggest the important and decisive role of VOCs in inducing cytotoxic effects.     

Role of bronchopulmonary C-fiber afferents in the apneic response to cigarette smoke

The role of vagal bronchopulmonary C-fiber afferents in eliciting the immediate changes in breathing pattern after acute inhalation of cigarette smoke was assessed with a selective blockade of myelinated vagal afferents (innervating both stretch and irritant receptors) utilizing the method of differential cooling. In 15 of 17 chloralose-anesthetized dogs tested, spontaneous inhalation of cigarette smoke (19.7% avg conc, 500-700 ml vol) reproducibly caused the following immediate responses: apnea, bradycardia, and hypotension. These responses occurred within 1 to 2 breaths of smoke inhalation and were followed by a delayed hyperpnea. The apneic duration reached 326 +/- 33% (SE) (n = 15) of the mean base-line expiratory duration. Differential cold block of both vagi (coolant temperature 8.4 +/- 0.3 degrees C) abolished the reflex apnea induced by a positive-pressure (7-10 cmH2O) lung inflation but did not affect the apneic response to smoke inhalation (345 +/- 35%). The smoke-induced apnea was completely abolished by lowering the coolant temperature to -1.3 +/- 0.2 degrees C (n = 10) or by bilateral vagotomy (n = 5) and returned to the control level after both vagi were rewarmed. Based on these results, we suggest that the immediate apneic response to inhaled cigarette smoke is elicited by a stimulation of vagal C-fiber afferents in the lungs and airways.     

Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure

An early event in the pathogenesis of emphysema is the development of inflammation associated with accumulation of polymorphonuclear leukocytes (PMN) in small airways, and inflammatory cell recruitment from the circulation involves migration across endothelial and epithelial cell barriers. Platelet-activating factor (PAF) promotes transendothelial migration in several vascular beds, and we postulated that increased PAF production in the airways of smokers might enhance inflammatory cell recruitment and exacerbate inflammation. To examine this possibility, we incubated human lung microvascular endothelial cells (HMVEC-L) with cigarette smoke extract (CSE) and found that CSE inhibits PAF-acetylhydrolase (PAF-AH) activity. This enhances HMVEC-L PAF production and PMN adherence, and adherence is blocked by PAF receptor antagonists (CV3988 or ginkgolide B). CSE also inhibited PAF-AH activity of lung endothelial cells isolated from wild-type (WT) and iPLA(2)β knockout mice, and with WT cells, CSE enhanced PAF production and RAW 264.7 cell adherence. In contrast, CSE did not affect PAF production or RAW 264.7 cell adherence to iPLA(2)β-null cells, suggesting that iPLA(2)β plays an important role in PAF production by lung endothelial cells. These findings suggest that inhibition of PAF-AH by components of cigarette smoke may initiate or exacerbate inflammatory lung disease by enhancing PAF production and promoting accumulation of inflammatory cells in small airways. In addition, iPLA(2)β is identified as a potential target for therapeutic interventions to reduce airway inflammation and the progression of chronic lung disease.     

Effect of cigarette smoke on fibroblast-mediated gel contraction is dependent on cell density

Cigarette smoke exposure has been associated with a variety of diseases, including emphysema. The current study evaluated the interaction of cell density and cigarette smoke extract (CSE) on fibroblast contraction of collagen gels. Protein levels of transforming growth factor (TGF)-beta1, fibronectin, PGE(2), and TGF-beta1 mRNA were quantified. Although both 5 and 10% CSE inhibited contraction by low-density fibroblasts (1 x 10(5) cell/ml), only 5% CSE augmented contraction in higher-density cultures (3-5 x 10(5) cells/ml). CSE also inhibited fibronectin and TGF-beta1 production in low-density cultures but stimulated fibronectin production in high-density cultures. Active TGF-beta1 was readily detectable only in higher-density cultures and was markedly augmented by 5% CSE. In contrast, although TGF-beta1 mRNA expression was inhibited in high-density cultures by 10% CSE, expression was increased in the presence of 5% CSE. These results suggest that CSE-induced inhibition of low-density fibroblast contraction is due to inhibition of fibronectin production, whereas CSE's stimulatory effect on high-density cells is the result of increased release of TGF-beta1. These effects may help explain the varied pathologies associated with exposure to cigarette smoke.     

Apoptotic cell death is a common response to pathogen attack in oats

We have examined the characteristics of cell death induced by pathogen infection in oats with respect to following hallmark apoptotic features: DNA laddering, chromatin condensation, and electron microscopic-terminal deoxynucleotidyl transferase-mediated UTP end labeling positive response. A wide range of plant pathogens representing different levels of parasitism in susceptible and resistant interactions were used for the inocula, which include (i) an obligate parasite, Puccinia coronata f. sp. avenae (the crown rust fungus); (ii) a facultative biotroph parasite, Magnaporthe grisea (the blast fungus); (iii) pathogenic bacteria, Pseudomonas syringae pv. atropurpurea and P. syringae pv. coronafaciens (the halo or stripe blights of oats); and (iv) Ryegrass mottle virus. Surprisingly, any of the pathogens used induced most of the apoptotic features in oat cells at and around the infection sites, indicating that apoptotic cell death is a common phenomenon in oats during pathogen attack. The localization and the timing of apoptotic cell death during a course of infection were, however, quite different depending on the interactions (compatible or incompatible) and the pathogens (fungi, bacteria, or viruses). Possible roles of apoptotic cell death in the susceptible and resistant interactions are discussed.     

Exposure to cigarette smoke, a major risk factor for sudden infant death syndrome: effects of cigarette smoke on inflammatory responses to viral infection and bacterial toxins

Exposure to cigarette smoke is a major risk factor for sudden infant death syndrome and also for respiratory infections in children. It has been suggested that toxigenic bacteria colonizing the respiratory tract might play a role in some cases of sudden infant death syndrome and nicotine has been demonstrated to enhance the lethality of bacterial toxins in a model system. Pyrogenic toxins of Staphylococcus aureus have been identified in tissues of infants who died of sudden infant death syndrome. It has been suggested that some of these deaths were due to induction of inflammatory mediators by infectious agents during a period when infants are less able to control these responses. The aim of this study was to assess the effects of a water-soluble cigarette smoke extract on the production of tumor necrosis factor alpha and nitric oxide from human monocytes in response to staphylococcal toxic shock syndrome toxin 1 or infection of the monocytes with respiratory syncytial virus. Cell culture supernatants were examined by a bioassay using mouse fibroblasts (L-929 cell line) for tumor necrosis factor alpha activity and by a spectrophotometric method for nitrite. Compared with monocytes incubated with medium only, monocytes incubated with any of the factors or their combinations tested in the study released higher levels of tumor necrosis factor alpha and lower levels of nitric oxide. Incubation with cigarette smoke extract increased tumor necrosis factor alpha from respiratory syncytial virus-infected cells while it decreased tumor necrosis factor alpha from cells incubated with toxic shock syndrome toxin. Incubation with cigarette smoke extract decreased the nitric oxide production from respiratory syncytial virus-infected cells while it increased the nitric oxide production from cells incubated with toxic shock syndrome toxin. Monocytes from a minority of individuals demonstrated extreme tumor necrosis factor alpha responses and/or very high or very low nitric oxide. The proportion of samples in which extreme responses with a very high tumor necrosis factor alpha and very low nitric oxide were detected was increased in the presence of the three agents to 20% compared with 0% observed with toxic shock syndrome toxin 1 or 4% observed with cigarette smoke extract or respiratory syncytial virus.     

IL-17RA is required for CCL2 expression, macrophage recruitment, and emphysema in response to cigarette smoke

Chronic Obstructive Pulmonary Disease (COPD) is characterized by airspace enlargement and peribronchial lymphoid follicles; however, the immunological mechanisms leading to these pathologic changes remain undefined. Here we show that cigarette smoke is a selective adjuvant that augments in vitro and in vivo Th17, but not Th1, cell differentiation via the aryl hydrocarbon receptor. Smoke exposed IL-17RA(-/-) mice failed to induce CCL2 and MMP12 compared to WT mice. Remarkably, in contrast to WT mice, IL-17RA(-/-) mice failed to develop emphysema after 6 months of cigarette smoke exposure. Taken together, these data demonstrate that cigarette smoke is a potent Th17 adjuvant and that IL-17RA signaling is required for chemokine expression necessary for MMP12 induction and tissue emphysema.     

Apoptosis is a mode of cell death in the polykaryon-forming unit assay

In the polykaryon-forming unit (PFU) assay, which defines cell survival as the ability to form a cytochalasin-induced polykaryon of predetermined ploidy, the mode of PFU deletion is not known. Incubation of L5178Y-S PFU in cytochalasin resulted in polyploidy (> or =32C) and most polykaryons (>75%) ultimately underwent apoptosis, detected using chromatin condensation and externalised phosphatidylserine. However, large polykaryons carrying terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)-labelled DNA strand breaks were not observed, presumably due to rapid loss of DNA. Gamma irradiation of PFU prior to cytochalasin exposure caused a reduction in the frequency of highly polyploid cells (>16C), consistent with either a supra-induction of apoptosis or a reduction in the ability of PFU to reach high ploidies. We conclude that L5178Y-S PFU are deleted by apoptosis.     

The cell death response to enteropathogenic Escherichia coli infection

Given the critical roles of inflammation and programmed cell death in fighting infection, it is not surprising that many bacterial pathogens have evolved strategies to inactivate these defences. The causative agent of infant diarrhoea, enteropathogenic Escherichia coli (EPEC), is an extracellular, intestinal pathogen that blocks both inflammation and programmed cell death. EPEC attaches to enterocytes, remains in the gut lumen and utilizes a type III secretion system (T3SS) to inject multiple virulence effector proteins directly into the infected cell, many of which subvert host antimicrobial processes through the disruption of signalling pathways. Recently, T3SS effector proteins from EPEC have been identified that inhibit death receptor‐induced apoptosis. Here we review the mechanisms used by EPEC T3SS effectors to manipulate apoptosis and promote host cell survival and discuss the role of these activities during infection.     

Arabidopsis ATG6 is required to limit the pathogen-associated cell death response

To begin to understand the interplay between autophagy and the hypersensitive response (HR), a type of programmed cell death (PCD) induced during plant innate immunity, we generated ATG6 antisense plants in the genetically tractable Arabidopsis thaliana system. AtATG6 antisense (AtATG6-AS) plants senesce early and are sensitive to nutrient starvation, suggestive of impairment of autophagic function in these plants. Additionally, these plants exhibited multiple developmental abnormalities, a phenomenon not observed in other AtATG mutants. AtATG6-AS plants produced fewer Monodansylcadaverine (MDC) and LysoTracker (LT) stained-autolysosomes in response to carbon and nitrogen starvation indicating that AtATG6 plays a role in the autophagic pathway in Arabidopsis. Interestingly, the level of AtATG6 mRNA in wild type Col-0 Arabidopsis plants is increased during the early phase of virulent and avirulent Pseudomonas syringae pv tomato (Pst) DC3000 infection suggesting that AtATG6 plays an important role during pathogen infection. In AtATG6-AS plants, HR-PCD induced upon infection with avirulent Pst DC3000 carrying the AvrRpm1 effector protein is not able to be contained at the infection site and spreads into uninfected tissue. Additionally, the disease-associated cell death induced by the infection of virulent Pst DC3000 bacteria is also partially misregulated in AtATG6-AS plants. Therefore, the AtATG6 antisense plants characterized here provide an excellent genetic model system to elucidate the molecular mechanisms by which autophagy regulates pathogen-induced cell death.     

The response of ataxia-telangiectasia lymphoblastoid cells to neutron irradiation

The response of control and ataxia-telangiectasia (A-T) cells to increasing doses of high-linear-energy-transfer (LET) ionizing radiation (neutrons) was compared. Ataxia-telangiectasia cells were markedly more sensitive to neutron irradiation than were control cells. The D0 value for the two A-T cell lines was 0.4 Gy while the value for controls was approximately 1.4 Gy. Fast neutrons were considerably more effective than gamma rays in inducing cell death in both cell types, but the sensitivity factor remained approximately the same as with gamma rays. A minimal depression of DNA synthesis was observed in ataxia-telangiectasia cells after neutron irradiation, similar to that reported previously after gamma irradiation. The extent of inhibition was not significantly greater in control cells, contrary to that seen with gamma rays. In time-course experiments a significant difference in degree of inhibition of DNA synthesis was observed between the cell types. Low doses of fast neutrons induced a G2-phase delay in both cell types, but the degree and extent of this delay was greater in ataxia-telangiectasia cells as observed previously with low-LET radiation.     

Impact of cigarette smoke on T and B cell responsiveness

Although its direct effects cannot be discounted, tobacco's effects on the immune system have been proposed to play a key role in mediating its deleterious health impact. Studies in rats using high levels of smoke exposure have suggested that tobacco smoke exhausts cellular signal transduction cascades, making lymphocytes unresponsive to stimulation. In the present study, we show that purified B or T cells, and total lymphocytes from the lungs, lymph nodes and spleens of smoke-exposed mice fluxed calcium, proliferated, and secreted immunoglobulin or IFN-gamma similarly to control mice when stimulated with ligands including anti-IgM, and anti-CD3. Importantly, we recapitulated these findings in PBMCs from human smokers; cells from long-term smokers and never-smokers proliferated equivalently when stimulated ex vivo. Previous reports of lymphocyte unresponsiveness in rats are inconsistent with these findings, and may reflect a phenomenon observed only at levels of smoke exposure well above those seen in actual human smokers.     

The effect of exposure to nicotine, carbon monoxide, cigarette smoke or cigarette smoke condensate on the mutagenicity of rat urine

Cigarette smokers have been reported to void urine which is more mutagenic than that voided by non-smokers, but the specific urinary mutagen(s) have not been identified. Since mechanistic studies are best performed in animal models, the objective of this study was to determine if a model to study the role of cigarette smoke and its components in urinary mutagenicity could be developed in rats. XAD-2 resin was used to concentrate the urine and the microsuspension modification of the Ames test used to quantify mutagenicity. Nicotine administered by intraperitoneal injection at 0.8 mg/kg (the maximum tolerated dose) or inhalation of carbon monoxide for 14 days at the maximum tolerated dose (1800 ppm, resulting in 68% carboxyhemoglobin) did not increase urinary mutagenicity. Cigarette smoke condensate (CSC) prepared by electrostatic precipitation of mainstream smoke increased urinary mutagenicity at doses of 100 and 200 mg/kg when administered acutely by either i.p. injection or gavage, verifying that the assay system was capable of detecting cigarette smoke-related mutagens in the urine. However, cigarette smoke administered by the appropriate route of exposure, nose-only inhalation, for 1, 7, 14 or 90 days (1 h per day) did not increase urinary mutagenicity. The smoke concentration administered was at or near the maximum tolerated dose as evidenced by carboxyhemoglobin concentrations of approximately 50%, and of 10% or more weight loss in exposed animals. Thus, although cigarette smoke condensate is mutagenic in vitro and mutagenic urine was observed when rats were given high doses of CSC by inappropriate routes of administration, acute or subchronic inhalation exposure to the maximum tolerated dose of whole cigarette smoke did not increase urinary mutagenicity in rats. These results indicate that the rat may be an inappropriate model to study urinary mutagenicity following the inhalation of tobacco smoke.     

Motoneuron programmed cell death in response to proBDNF

Motoneurons (MN) as well as most neuronal populations undergo a temporally and spatially specific period of programmed cell death (PCD). Several factors have been considered to regulate the survival of MNs during this period, including availability of muscle-derived trophic support and activity. The possibility that target-derived factors may also negatively regulate MN survival has been considered, but not pursued. Neurotrophin precursors, through their interaction with p75(NTR) and sortilin receptors have been shown to induce cell death during development and following injury in the CNS. In this study, we find that muscle cells produce and secrete proBDNF. ProBDNF through its interaction with p75(NTR) and sortilin, promotes a caspase-dependent death of MNs in culture. We also provide data to suggest that proBDNF regulates MN PCD during development in vivo.