P Hasgall, L Bellehsen, I Hammel, D Amichai, F Levi-Schaffer
P Hasgall, L Bellehsen, I Hammel, D Amichai, F Levi-Schaffer. In Vitro Exposure To Cigarette Smoke Activates Eosinophils: Implications For Lung Inflammation. The Internet Journal of Asthma, Allergy and Immunology. 2006 Volume 5 Number 2.
Effects of cigarette smoke (CS) on eosinophils (EOS), important cells involved in the pathogenesis of chronic lung diseases such as asthma, were studied in vitro. EOS were isolated from healthy and mildly atopic donors and exposed to soluble components of cigarette smoke (CSE). Viability and apoptosis were assessed by flow cytometry after staining with propidium iodide and Annexin-V. Activation was determined by release of newly-synthesized (IL-8, IL-6) mediators and by phosphorylation of MAPKs. CSE effects on ultrastructural morphology and production of neutrophil chemotactic factors in CSE-activated EOS were also evaluated. CSE concentrations from 0-2.5% were non-toxic for up to 18-24 hours of exposure. However, CSE at 2.5% activated EOS as evidenced by ultrastructural degranulation: release of IL-8 and IL-6, and increased expression of the MAPK, c-Jun. Supernatants from CSE-activated EOS were found to be significantly chemotactic for neutrophils. These results suggest that CS may aggravate lung inflammation by activating EOS which, in turn, release inflammatory mediators promoting inflammatory cell recruitment and lung remodeling.
Eosinophils (EOS) are terminally differentiated granular leukocytes that are produced in the bone marrow and migrate to inflamed tissues in response to chemotactic signals. Their recruitment, growth and survival are supported by cytokines and chemokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukins (IL)- 3 and -5 [1,2,3,4]. Eosinophilia occurs in response to parasitic infection and exposure to allergens. Eosinophils are particularly evident in chronic lung inflammation where their products appear to play roles both in bronchial hyper-responsiveness and in tissue remodeling, leading to compromised lung function [5,6]. Increased numbers of eosinophils in the bronchial mucosa have been correlated with a number of conditions such as: bronchial wall thickening, epithelial cell hypertrophy, and myofibroblast hyperplasia ; Goblet cell hyperplasia and increased mucus production ; and tissue remodeling ; and fibrosis . Neutralization of IL-5, the main eosinophil growth factor, has been shown to block airway hyper-reactivity .
Many of the eosinophil mediators involved in the pathogenesis of airway inflammation are preformed and stored in granules that are released upon eosinophil activation. These include: major basic protein (MBP) whose levels are correlated with bronchial epithelial damage ; eosinophil cationic protein (ECP) which has been shown to be elevated in chronic asthma and associated with airflow obstruction ; eosinophil-derived neurotoxin (ECN) which stimulates fibroblast proliferation ; and eosinophil peroxidase (EPO) which catalyzes the peroxidation of halides and forms toxic nitrogen reactive species that contribute to asthmatic inflammation . EPO, which is used as a biomarker of eosinophil degranulation (i.e., activation) , inactivates leukotrienes that cause bronchoconstriction . MBP, the main granule constituent also induces degranulation of tissue mast cells, which contribute to airway hyper-responsiveness and inflammation . Additionally, eosinophil cytoplasmic lipid bodies contain cyclooxygenases (COX), lipoxygenases, and phospholipase A2, all of which contribute to inflammation through their role in synthesis of proinflammatory eicosanoids . Upon activation, eosinophils also synthesize and release a variety of proinflammatory cytokines including: the interleukins IL-1α, IL-6, IL-8; tumor necrosis factor- alpha (TNF-α); transforming growth factors (TGF)-α and β and eotaxin [19,20]. TNF-α, which is also produced by several other inflammatory cell types, causes recruitment of eosinophils and neutrophils to inflamed tissues, induces eosinophil synthesis and release of matrix metalloproteinases (MMPs) involved in lung tissue remodeling, and synergizes with GM-CSF to promoting eosinophil survival . It induces eosinophil synthesis and release of IL-8, an important chemotactic and activating factor for neutrophils and lymphocytes [22,23] which induces adhesion molecule expression also triggers the oxidative burst response, contributing to tissue damage . IL-6, a highly pleiotrophic cytokine, stimulates a variety of immune and inflammatory responses and plays an important role in airway inflammation [25,26,27]. Eosinophil-derived TGF-β promotes lung fibrosis by stimulating fibroblast proliferation and synthesis of extracellular matrix (ECM) components such as collagen . Thus, intrinsic agents (such as TNF-α and C5a) and extrinsic agents which persistently activate airway eosinophils may induce an inflammatory cascade leading not only to chronic inflammation but also lung tissue remodeling and compromise of lung function.
The adverse effects of long term exposure to cigarette smoke (CS) on human health are increasingly being documented both in human and animal studies. CS exposure is associated with reduced immune function [29,30]. Exposure to CS also influences inflammatory responses by enhancing release of proinflammatory cytokines [31,32]. CS is composed of a multitude of potentially harmful chemicals including aldehydes (formaldehyde, acrolein), ketones, organic acids, phenols, cyanides, nitrogen oxides (NO) and reactive oxygen species (ROS); thus, exposure to CS likely induces a state of oxidative stress leading to release of proinflammatory cytokines such as TNF-α, IL-6 and IL-8 through activation of stress kinases such as JNK and p38 and redox-sensitive transcription factors such as NF-κB and AP-1 . Other CS components such as formaldehyde and acrolein enhance neutrophil migration into the lung  and inhibit apoptosis of neutrophils  and high concentrations of nicotine stimulate neutrophil degranulation . In both humans and animals, CS induces release of chemokines such as monocyte chemotactic protein (MCP)-1 and IL-8 that attract monocytes/macrophages and neutrophils to the lung .
Materials and methods.
In the absence of GM-CSF, eosinophil survival was reduced over time in culture regardless; however, exposure to CSE for 24-48h contributed significantly to the decline in eosinophil viability at all CSE concentrations tested (Fig. 1B). In the presence of GM-CSF, eosinophil viability was not markedly affected by exposure to CSE concentrations of ≤2.5% for 24- 48h. However, this was not the case at [CSE] > 2.5%. Thus, at lower CSE concentrations (i.e., 2.5% and lower) the addition of the survival factor, GM-CSF appeared to partially rescue eosinophils from the additional toxic effects of CSE. It also appeared that the interval between 12-24h was a critical determinant of eosinophil survival in the absence of GM-CSF and after exposure to CSE.
Concentrations of IL-6 (Fig. 5) in supernatants of eosinophils cultured in the presence of 2.5% CSE were also significantly higher in comparison to those in supernatants of cells cultured in medium alone (400 ± 67 pg/ml vs. 1329 ± 302 pg/ml p < 0.05). In eosinophils activated with GM-CSF + TNF-α and exposed to 2.5% CSE, IL-6 release was significantly increased (770 ± 19 pg/ml vs. 2174 ± 610 pg/ml p < 0.05) in comparison with eosinophils activated in medium alone. This effect on IL-8 and IL-6 release was not observed in cyclohexamide-treated eosinophils, suggesting that CSE influences
Smoking, especially in association with chronic lung conditions such as COPD, is associated with the release of many pro-inflammatory cytokines and chemokines including, TNF-α and IL-8 . Accumulation of neutrophils in the large airways has been associated with disease severity . While several studies have shown that cigarette smoke may activate lung macrophages , epithelial cells  and fibroblasts  to release pro-inflammatory mediators, little is known about the effects of cigarette smoke on eosinophils, one of the main effector cells in allergic inflammation in the lung, especially in the late phase of asthma. Here we show that
While at lower concentrations (<5%) CSE appeared to enhance eosinophil activation, higher concentrations and increased exposure times proved to be toxic to these cells. Addition of the eosinophil survival factor GM-CS partially reversed this effect and rescued CSE-exposed eosinophils from death. As exposure to CSE did not result in increased detection of Annexin V, it is likely that eosinophil death (at least at CSE concentrations of 2.5% or less) was not due to apoptosis. Following activation eosinophils release survival factors such as IL-5  and GM-CSF  that promote their own survival. Hence, it is possible that CSE also enhanced release of these survival factors or by actively inhibiting apoptosome formation as has been demonstrated for Jurkat cells , a cell line that undergoes apoptosis readily and which displays characteristic apoptosis markers.
We reported earlier  that tryptase-activated human peripheral blood eosinophils produce IL-8 and that this production is mediated by the mitogen-activated protein kinase (MAPK)/AP-1 pathway. In this study we observed that exposure to 2.5% CSE induced the expression of c-Jun protein in eosinophils within 30 min. These results are in accordance with the previous finding that nitric oxide (NO) and reactive oxygen species (ROS), major components of cigarette smoke, cause induction of AP-1 [66,67,68]. AP-1 may be activated through the MAPK pathway via the phosphorylation of either JNK or P38 [64,67] whereas phosphorylation of ERK1/2 does not appear to be required. While technical difficulties in our study precluded clear interpretation of JNK and P38 phosphorylation, transient phosphorylation of ERK1/2 was observed. It is possible that the sensitivity of stress induced kinases to temperature changes made it difficult to detect transient phosphorylations of JNK and P38 , while ERK1/2 being a cytokine-induced kinase was more resilient to temperature variations during manipulation of the cells.
The results of our preliminary studies, taken together with those of other studies, suggest that exposure to cigarette smoke may augment lung inflammation through its effects on eosinophils. How this might occur is summarized in the model shown in Fig. 9. In this model, cigarette smoke has a direct effect on eosinophils by causing the release IL-8 and IL-6 and by inducing degranulation and release of other proinflammatory mediators. As a result, neutrophils are recruited into the inflamed tissue enhancing the ongoing inflammatory process. IL-6, being a strong pro inflammatory and pro-angiogenic mediator enhances the inflammatory response. TNF-α, released mainly by macrophages and lung epithelial cells after exposure to CS potentates the release of IL-8 and IL-6 by eosinophils. GM-CSF, besides being a survival factor, acts synergistically together with TNF-α to enhance CS-induced cytokine release from eosinophils.
Although our study model is similar to those used in similar investigations to evaluate cigarette smoke effects on inflammatory cells , it is difficult to extrapolate its results to the
This study was supported by funds from the Aimwell Charitable Trust (UK).
Leah Bellehsen, PhD, Department of Pharmacology, Faculty of Medicine, The Hebrew University of Jerusalem, PO Box 12065, Jerusalem, Israel. Email: Leah_Bellehsen@yahoo.ca FAX: 972-2-675-8144