The Impact of Nitric Oxide on Asthma - Essay Example

Table of Contents Introduction 3 Nitric Oxide and the Human Body 3 Mechanism and Metabolism 4 Functions 5 Nitric Oxide and Asthma 7 Mechanism of Nitric Oxide and Asthmatic Attacks 8 Nitric Oxide and Treatment of Asthma 9 Conclusion 9 References 11 Copy of Articles 12 Introduction Nitric oxide (NO), once generally viewed only as hazardous to humans, has now become new promising means of identifying and treating respiratory diseases such as asthma. Several studies have indicated the usefulness of nitric oxide monitoring in managing asthmatic patients particularly children. With the tool, it allows for objective monitoring of asthma attacks without the need of comprehensive articulation. The use of nitric oxide in this field is based on the pathophysiological, physical, molecular and genetic researches that have the role of nitric oxide in respiration and related diseases (Gill et al, 2005). The European Respiratory Society has indicated its conviction in the method that it has already published guidelines to standardize analysis, diagnosis and reference levels in using nitric oxide as part of respiratory treatment (Buchwald, 2005). In the United States alone, 6% those aged below 12 have been diagnosed with asthma and as high as 40% in urban areas. This reflects a 75% from data gather in the 1980's a trend that is reflected globally (World Health Organization [WHO], 2005). Nitric Oxide and the Human Body Nitric oxide is a highly reactive, prevalent gas in human chemical activity. It can be found in neurons as n NOS or NOS1, in macrophages as iNOS or NOS-2, and in endothelial cells as eNOS or NOS-3 (Bor-Kucukatay, 2005). Nitric oxide is cellularly synthesized by the enzyme nitric oxide sythases denoted by NOS from arginine, molecular oxygen and NADPH. Nitric oxide interacts rapidly molecularly and disperses through cell membranes acting in a paracinic or autocrinic behavior1. Endogenous nitric oxide is a product of L-argining amino acid and NOS. All three isofrms, NOS or NOS1, iNOS or NOS-2, eNOS or NOS-3 are found in the respiratory tract functioning a part in vascular and airway smooth muscle tone, inflammatory respiratory response, ciliary action and in eliminating bacteria, viruses and mycobacteria in the respiratory tracts (Smith et al, 2004). Mechanism and Metabolism Through connecting through a metal ion in the cell's protein or through cystine or other S atoms, nitric oxide causes allosteric change in the cell's protein. An example of this reaction is nitric oxide directed at the protein guanylyl cyclase which then creates the second messenger cyclic GMP (cGMP). The body produces oar manufactures nitric oxide to fight bacteria. TH 1cells as part of inflammatory response mechanisms secrete nitric oxide to attach bacterial macrophages or by the production of nitric oxide by through the conversion of nitrates found in food into nitrites (Maddox & Schwartz, 2002). Reaction with super oxide anions the result to the formation peroxynitrite2 (ONOO-) which can lead to break up of DNA and oxidation action in lipids. This can lead to nitric oxide toxicity. Peroxynitrite causes mitochondrial respiratory chain (I-IV) and manganese super oxide dismutase (MnSOD) to generate suroxide anions and hydrogen peroxide, both of which can cause fatal cell damage ("Nitric Oxide Metabolism", 2006). Figure 1 illustrates nitric oxide actions in the body. Functions Nitric oxide serves various functions in the human body. It can act as a catalyst for reactions to microbial attacks, as a messenger or inhibitor among others. It functions significantly in the circulatory and nervous system influencing blood flow, oxygenation of red blood cell and neural messaging (Maddox & Schwartz, 2002). The following are the functions of nitric oxide physiologically ("Nitric Oxide", 2006): Blood Flow Diffusion of nitric oxide into smooth muscle cells allowing for the efficient flow of blood as endothelial cells release nitric oxide at every systole (Beckett & Howarth, 2003). Restrains platelet aggregation to prevent blood clots NOS-3 prevents exocytosis of factors influencing inflammation of endothelial cells as well as of cytotoxic T lymphocytes (CTL) and macrophages Autonomic Nervous System nitric oxide can influence the parasympathetic motor neurons Triggers the medulla oblongata to increase respiration rate as needed through sending messages to nitric oxide-sensitive cells Production of nitric oxide by neuron in the hippocampus' CA1 region promotes NMDA receptors that are in charge of long-term potentiation (LTP) Magnifies synaptic action Kidneys Presence of nitric oxide in the region of the kidney's glomeruli enhances filtration and urine formation Endocrines Gonadotropin-releasing hormone (GnRH) by the hypothalamus Pancreatic exocrine production of pancreatic amylase Adrenaline production by the adrenal medulla. Reproduction The discharge of nitric oxide by nerve cell endings enhances blood flow, a principle used in sildenafil, vardenafil and tadalafil drugs nitric oxide is freed by acrosomes when sperm and egg cells meet concluding meiosis II by not allowing the entry of additional sperm regulating fusion of the pronuclei Immune systems Supports immunological actions against bacteria and other engulfed pathogens through lysosomes of macrophages. Aids in inflammatory response by TH 1 cells Nitric oxide produced by gastric juices during digestions kills bacteria in food Smooth muscle action Aids in peristalsis by relaxing muscles Influences the contractibility of uterine muscles Nitric Oxide and Asthma Pathogenically, asthma is viewed as closely linked with bacterial exposure and antigens as well as the degree of effectivity of the activation of TH 1 lymphocyte cells. The theory, known as the hygiene hypothesis, states that the prevalence of TH 2 instead of TH 1 cells can cause the development of asthma and allergies (Maddox & Schwartz, 2002). Asthmatic attacks are caused by the inflammation of bronchial and other respiratory and circulatory channels limits the oxygen levels and blood circulation which then is evidenced by respiratory distress. Researches have hypothesized that this imbalance of TH 1 and TH 2 cells caused by raised levels of nitric oxide leads to the TH 2 reactions characterized by allergic asthmatic symptoms. The utilization of measuring nitric oxide levels to monitor asthma attacks especially in children allows doctors to measure severity without the need for patient to directly verbalize their level of distress which may be difficult in the course of an attack or with children with limited verbalization (Maddox & Schwartz, 2002). Mechanism of Nitric Oxide and Asthmatic Attacks Nitric oxide becomes involved as the body react to a bacteria, allergen or macrophage. Nitric oxide is produced to attach and attack the bacteria. As the nitric oxide is released, there are subsequent neurological and physiological effects. Nitric acid interacts with red blood cells through binding to the heme portion of hemoglobin to form S-nitrosohemoglobin producing of methemoglobin (Beckett & Howarth, 2003). As the TH 1 cells attack the bacteria and endothelial cells increase blood flow, neurological receptors in the brain sensitive to fluctuation of nitric oxide begin to prompt all other body functions rapidly. This rapid reaction is enhanced by the magnifying effect of nitric oxide to synaptic reaction. As the brain is flooded by the effects of nitric oxide, the medulla oblongata is prompted to increase respiration rate and total body circulation ("Nitric Oxide", 2006). This has the effect of not only supporting the initial action of endothelial cells but also increases the responsiveness of the rest of the body's systems (McFadden, 2004). Adrenaline is increased together with pancreatic amylase is released to enhance the body's reactions and processing of needed nutrient to control the bacteria. The increase of nitric oxide in the bloodstream reaches the kidneys and nitric oxide triggers glomeruli to increase filtration rate ("Nitric Oxide", 2006). In these events, nitric oxide bonds with the protein elements coordinating the function of the organs to create the appropriate reactions to the invasion. The increase of nitric oxide also affects cellular mitochondrial structures to increase their defense against foreign bodies. NO production and the production of precursors such as L-arginine and the potassium blocker TEA are able to overturn the effect of NOS inhibitors such as Guanylate cyclase. This indicates the role of NO in determining the deformability of red blood cells mechanically and as a regulator (Bor-Kucukatay, 2003). The NO increase is the body's way of responding defensively but at the same time excites physiologically effects that cause asthmatic reactions. Nitric Oxide and Treatment of Asthma The increase of nitric oxide in the blood is not as much indicative of an asthmatic attack as when there is substantial increase in exhaled NO observed. There is significant increase in NO during asthma attacks due to the gravity of the bacteria attack and the existing predisposition of the patient to the bacterial allergens. The use of exhaled nitric oxide in evaluation of asthma attacks is a non-invasive method that can yield valuable information regarding patient conditions beyond clinical indicators. Together with the development of more advance nitric oxide analyzers, diagnosis and treatment of unstable and severely asthmatic patients will be enhanced (Kharitonov & Barnes, 2001). Recent researches have shown that nitric oxide plays a vital role in respiration and thus the interest in its application in treating asthma particularly children. It is affording doctors to learn not only about the physiology of asthma, which can be eliminated in childhood, but also the cellular factors that lead to the condition (Gill, 2005). Analysis of the level of NO gas can indicate the level of body reaction and at the same time monitor the effect of treatment. Bauchner (2005) has pointed out that asthma is not just as respiratory condition, the respiratory distress brought on by the disease is actually an indication of the immunological distress that can be traced to the cellular levels. Conclusion The increase incidence of childhood asthma since the 1980's makes these researchers important. Though the prognosis of curing childhood remains high, the increase in the volume of incidence alone poses a challenge to current medical services (Beckett & Howarth, 2003). According to Jongste (2005), existing methods of using nitric oxide depend entirely on pre-defined discriminatory levels, indicators and determined or traditional parameters of asthma control. He points out that "algorithms more or less arbitrarily and it is unclear what the effect of other cut-off levels or alternative dosing schedules" (p.380). In effect, though there is existing standardization of results, interpretation and subsequent may be subject to a high degree of variation. Researchers are now emphasizing the need to understand more about nitric oxide to be able to set clearer methodologies for its use (Smith et al, 2004). A more in-depth understanding of the incidence and implication of nitric oxide production by the body is needed to create structured and appropriate procedures in utilizing it in respiratory treatments as well as other application with other diseases. References Bauchner, Howard (2005). Measuring Exhaled Nitric Oxide Can Guide the Treatment of Asthma Massachusetts Medical Society. Retrieved on October 25, 2006 from http://pediatrics.jwatch.org/cgi/content/full/2005/708/1q=etoc Beckett P.A. and Howarth P.H. (2003). Pharmacotherapy and airway remodeling in asthma Thorax Volume 58 Number 2. pp. 163-74 Bor-Kucukatay, Melek, Wenby, Rosalinda B., Meiselman, Herbert J. and Baskurt, Oguz K. (2003). Effects of nitric oxide on red blood cell deformability. American Journal of Physiology - Heart and Circulatory Physiology Volume 284 Issue 5. pp. H1577-H1584 Gill, Michelle et al (2005). Exhaled Nitric Oxide Levels during Acute Asthma Exacerbation. Academic Emergency Medicine Volume 12 Number 7. Society for Academic Emergency Medicine. pp. 579-586 Jongste, J. C. de (2005). Yes to NO: the first studies on exhaled nitric oxide-driven asthma treatment. European Respiratory Journal Volume 26. London: ERS Journals Ltd. pp. 379-381 Kharitonov, Sergei A. and Barnes, Peter J. (2001). Does Exhaled Nitric Oxide Reflect Asthma Control American Journal of Respiratory Critical Care Medicine September Volume 164 Number 5: pp. 727-728 Maddox, L. and Schwartz, D.A. (2002). The Pathophysiology of Asthma. Annual Review Medicine Volume 53. pp. 477-498 McFadden, E.R. et al (2004). Harrison's Principles of Internal Medicine 16th Edition. New York: McGraw-Hill. pp. 1508-1516. Nitric Oxide (2006). Wikipedia Free Encyclopedia. Retrieved on October 25, 2006 from http://en.wikipedia.org/wiki/Nitric_Oxide Nitric Oxide Metabolism (2006). Sigma-Aldrich Resources. Retrieved on October 25, 2006 from http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Cell_Signaling/Pathway_Slides_and_Charts/Nitric_Oxide_Metabolism.html Smith, Andrew D. et al (2004). Diagnosing Asthma: Comparisons between Exhaled Nitric Oxide Measurements and Conventional Tests. American Journal of Respiratory and Critical Care Medicine Volume 169. pp. 473-478 World Health Organization (2002). Asthma Fact Sheet. Geneva: World Health Organization. Copy of Articles Bauchner, Howard (2005). Measuring Exhaled Nitric Oxide Can Guide the Treatment of Asthma Massachusetts Medical Society. Retrieved on October 25, 2006 from http://pediatrics.jwatch.org/cgi/content/full/2005/708/1q=etoc The decision to titrate the dose of inhaled corticosteroids (ICS) in patients with asthma is usually based on asthma symptoms and pulmonary function tests (PFTs), but these tests are difficult to do and of uncertain accuracy. Investigators in New Zealand randomly assigned 97 patients (age range, 12 to 73 years) with persistent asthma to dose adjustment of fluticasone based on either conventional guidelines (disease symptoms and PFTs) or measurement of exhaled nitric oxide. Exhaled nitric oxide reflects bronchial-wall inflammation, airway hyper-responsiveness, and induced-sputum eosinophilia. After a 3- to 12-month run-in phase to establish optimal ICS dose, patients were followed for 12 months, with visits every 2 months for adjustment of the fluticasone dose. Compared with controls, those in the nitric oxide group were receiving a significantly lower dose of ICS at the end of the study (370 g/day vs. 641 g/day) without compromising asthma control. There were no significant differences between the groups in nighttime waking, pulmonary function, levels of airway inflammation, or use of bronchodilators or prednisone. Although there was a 46% difference in asthma exacerbations favoring the nitric oxide group, this difference did not reach statistical significance (0.49 vs. 0.90 episodes per patient per year). Comment: Although an inexact science, titration of inhaled corticosteroids is a key component of asthma management. Exhaled nitric oxide measurements offer promise because they are accurate and easy to perform. This approach may reduce exposure to ICS and is likely the way children with asthma will be monitored in the future. Its adoption will likely be dependent upon reimbursement eligibility and more research on its use in young children. Beckett P.A. and Howarth P.H. (2003). Pharmacotherapy and airway remodeling in asthma Thorax Volume 58 Number 2. pp. 163-74 Background Short-term exposure to high concentrations of ozone has been shown to increase airway hyper-responsiveness (AHR). Because the changes in AHR and airway inflammation and structure after chronic ozone exposure need to be determined, the goal of this study was to investigate these effects in a murine model of allergic airway disease. Methods We exposed BALB/c mice to 2 ppm ozone for 4, 8, and 12 weeks. We measured the enhanced pause (Penh) to methacholine and performed cell differentials in bronchoalveolar lavage fluid. We quantified the levels of IL-4 and IFN- in the supernatants of the bronchoalveolar lavage fluids using enzyme immunoassays, and examined the airway architecture under light and electron microscopy. Results The groups exposed to ozone for 4, 8, and 12 weeks demonstrated decreased Penh at methacholine concentrations of 12.5, 25, and 50 mg/ml, with a dose-response curve to the right of that for the filtered-air group. Neutrophils and eosinophils increased in the group exposed to ozone for 4 weeks compared to those in the filtered-air group. The ratio of IL-4 to INF- increased significantly after exposure to ozone for 8 and 12 weeks compared to the ratio for the filtered-air group. The numbers of goblet cells, myofibroblasts, and smooth muscle cells showed time-dependent increases in lung tissue sections from the groups exposed to ozone for 4, 8, and 12 weeks. Conclusion These findings demonstrate that the increase in AHR associated with the allergic airway does not persist during chronic ozone exposure, indicating that airway remodeling and adaptation following repeated exposure to air pollutants can provide protection against AHR. Asthma is characterized by the presence of a variable airflow limitation, airway hyper-responsiveness (AHR), and airway inflammation [1]. Acute exposure to ozone, which is an important component of the photochemical oxidation products of substrates emitted as air pollution from automobile engines [2], decreases pulmonary function, increases AHR, and induces airway inflammation in dogs [3], guinea pigs [4], and humans [5-7]. Chronic airway inflammation is associated with airway remodeling that includes airway wall thickening as a result of inflammatory and structural changes, such as edema; inflammatory cell infiltration; mucous gland hyperplasia; reticular basement membrane thickening; subepithelial fibrosis; vascular smooth muscle cell proliferation, hyperplasia, and hypertrophy; and myofibroblast and goblet cell hypertrophy [8-11]. Airway wall thickening and airway reactivity were inversely associated in patients with asthma, suggesting that airway wall thickening prevents excessive airway narrowing in human subjects in vivo [12]. Interleukin (IL)-4 is key factor contributing to the chronic inflammatory state that characterizes asthma and may be involved in the connective tissue alterations that characterize airway remodeling in asthma. IL-4 can stimulate fibroblasts [13]. Interferon (IFN)-, thought to be deficient in asthma, can antagonize some of the effects of IL-4 [14]. The effects of long-term, repeated exposure to ozone on AHR and airway structural changes remain poorly defined. Our underlying hypothesis is that repeated episodes of ozone exposure give rise to some of the remodeling changes associated with asthma, which may in turn be associated with sustained airway dysfunction. The aims of this study were to examine the relationship between ozone exposure and AHR by using barometric whole-body plethysmography (WBP) and to characterize the airway structural changes following a daily 8-h exposure to 2 ppm ozone for 4, 8, and 12 weeks in a murine model of asthma. Airway inflammation was also assessed by analysis of bronchoalveolar lavage (BAL) fluid. Figures Figure 1 Schematic of the sensitization protocol Mice Female BALB/c mice (aged 5 to 6 weeks; DaeMul Laboratories, Daejeon, Korea) known to be high IgE responders were used. The mice were maintained on an ovalbumin (OVA)-free diet and were individually housed in rack-mounted stainless steel cages with free access to food and water. Ovalbumin-induced allergic airway disease model An OVA-induced allergic airway disease model of asthma was used with some modification [15]. Briefly, mice were sensitized on days 1 and 14 by intraperitoneal injection with 10 g of grade V OVA (Sigma Chemicals, St. Louis, MO) and 1 mg of aluminum potassium sulfate (Sigma Chemicals) in 500 L of saline solution. On days 21 to 23, the mice were challenged by daily exposure (30 min) to an aerosol of 1% (wt/vol) OVA in saline solution. Vehicle control mice were treated with a suspension of aluminum potassium sulfate (1 mg) in saline solution (500 L) and challenged with aerosolized saline solution daily from days 21 to 23. Aerosol challenge was conducted on groups of up to 12 mice in a closed chamber attached to an ultrasonic nebulizer (NE-UO7; Omron Corporation, Tokyo, Japan) with an output of 1 mL/min and 1- to 5-m particle size. Ozone exposure The mice housed in whole-body exposure chambers were exposed to ozone concentrations of 2 ppm for 4, 8, and 12 wks (n = 6; Fig. 1); the ozone doses and exposure times were selected based on our previous study [16]. Ozone was generated with Sander model 50 ozonizers (Sander, Eltze, Germany). The concentration of ozone within the chambers was monitored throughout the exposure with ambient-air ozone motors (model 49 C; Thermo Environmental Instruments Inc., Franklin, MA). The air-sampling probes were placed in the breathing zone of the mice. The mean chamber ozone concentration ( SE) during the 8-h exposure period was 1.92 0.15 ppm. The breathing parameter values of spontaneously breathing BALB/c mice were determined under standard conditions at room air and temperature. Determination of airway responsiveness Airway responsiveness was measured by barometric plethysmography using whole-body plethysmography (WBP; Buxco, Troy, NY) after ozone exposure, while the animals were awake and breathing spontaneously as a modification of the method described by Hamelmann et al. [17]. Enhanced pause (Penh) to methacholine as measured using barometric plethysmography is a valid indicator of bronchoconstriction in mice and can be used to measure AHR [17-19]. Aerosolized methacholine in increasing concentrations (2.5-50 mg/ml) was nebulized through an inlet of the main chamber for 3 min. Bronchoconstriction alters breathing patterns, and changes in the timing of early and late expirations (Pause) and in Penh are the results of alterations in the timing of breathing, as well as the prolongation of the expiratory time. Furthermore, airway constriction increases the thoracic flow asynchronously with the nasal flow, resulting in an increase in the box pressure signal. Penh is an empiric parameter that reflects changes in the waveform of the measured box pressure signal that are a consequence of bronchoconstriction. Before taking readings, the box was calibrated with a rapid injection of 150 l of air into the main chamber. The difference between the pressure in the main chamber of the WBP containing the animal and that in a reference chamber was measured as the box pressure signal, which is caused by the pressure change in the main chamber during the respiratory cycle of the animal. A pneumotachograph with defined resistance in the wall of the main chamber acted as a low-pass filter and allowed thermal compensation. The time constant of the box was determined to be approximately 0.02 s. Mice were placed in the main chamber, and baseline readings were taken and averaged for 3 min. BAL fluid preparation and analysis BAL was performed immediately after the last measurement of airway responsiveness. The mice were deeply anesthetized with 50 mg/kg of pentobarbital sodium injected intraperitoneally and were killed by exanguination from the abdominal aorta. The trachea was cannulated with a polyethylene tube through which the lungs were lavaged three times with 1.0 ml of physiological saline (4.0 ml total fluid removed). The BAL fluid was filtered through wet gauze (4 4 inches). Trypan blue exclusion for viability and total cell count was performed. The BAL fluid was centrifuged at 150 g for 10 min. The obtained pellet was immediately suspended in 4 ml of physiological saline, and total cell numbers in the BAL fluid were counted in duplicate with a hemocytometer (improved Neubauer counting chamber). A 100-l aliquot was centrifuged in a cytocentrifuge (model 2 Cytospin; Shandon Scientific Co., Pittsburg, PA), and differential cell counts were performed using the centrifuged preparations stained with Diff-quick, counting 500 or more cells for each animal at a magnification of 1000 (oil immersion). Cytokine measurement The levels of IL-4 and IFN- were quantified in the supernatants of BAL fluids by enzyme immunoassays according to the manufacturer's protocol (Endogen Inc., Woburn, MA). The sensitivity of the assays was 5 pg/ml. Preparation of lung tissues and morphological analysis The mice were euthanized after the final exposure, and the lungs and trachea were filled intratracheally with a fixative (0.8% formalin, 4% acetic acid) using a ligature around the trachea. The lungs were removed, and lung tissues were fixed with 10% (vol/vol) neutral buffered formalin. The specimens were dehydrated and embedded in paraffin. For histological examination, 4-m sections of fixed, embedded tissues were cut on a Leica model 2165 rotary microtome (Leica Microsystems, Nussloch, Germany), placed on glass slides, deparaffinized, and stained sequentially with toluidine blue (Richard-Allan Scientific, Kalamazoo, MI). Selected toluidine blue-stained sections were used for measuring epithelial, goblet, and smooth muscle cells providing that the epithelium and submucosa could be easily identified and that the number of epithelial, goblet, and smooth muscle was adequate to allow multiple measurements (i.e., approximately 1 mm). Areas of the lung tissue with intact surface epithelium were selected for examination and quantification under a transmission electron microscope (H-7000; Hitachi, Tokyo, Japan). Ultrathin sections were cut, placed on high-transmission, 200-mesh, thin-bar copper grids, and stained with uranyl acetate and lead citrate. Light microscopic quantification was performed at 200, and electron microscopy was performed at 5000. The cells that were counted (e.g., myofibroblasts) were used as evidence of airway remodeling rather than inflammation in a subepithelial zone of the entire transmission electron microscopy section, and the counts were expressed per 0.1 mm2 of tissue. Myofibroblasts were identified by spindle-like projections, dilated rough endoplasmic reticulum, a greatly infolded and crenated nuclear membrane, and bundles of parallel cytoplasmic filaments associated with dense body condensations. The sections were coded and examined under light microscopy in random order by the same observer, who was unaware of the origin of the sections. Intra-observer repeatability was assessed by measuring the same section four times and was expressed as a percentage of the coefficient of variation for the four measurements. Statistical analysis All data were analyzed using SPSS version 7.5 for Windows (SPSS Inc., Chicago, IL). The data are expressed as means SE. For measured variables with a normal distribution, Student's paired t-test was used to compare paired data. For variables that did not have a normal distribution, the Mann-Whitney U-test was used for comparisons. Differences with p-values less than 5% were regarded as statistically significant. Figures Figure 2 Methacholine-induced airway responses measured by whole-body plethysmography in BALB/c mice challenged with saline and ovalbumin Figure 3 Methacholine-induced airway responses measured by whole-body plethysmography in BALB/c mice exposed to filtered air and to 2 ppm ozone for 8 h per day for 4, 8, and 12 weeks Figure 4 (A) Bronchioles exposed to filtered air have normal-appearing bronchioles and bronchiolo-alveolar portal and a normal transition from the low columnar epithelium lining the terminal bronchioles to the attenuated epithelium lining the alveoli Figure 5 Goblet cell counts in the epithelium of bronchioles of mice exposed to filtered air and to 2 ppm ozone for 4, 8, and 12 weeks Figure 6 (A) Transmission electron micrograph of a bronchiole specimen from mice exposed to filtered air showing normal epithelium, smooth muscle, and capillaries Figure 7 Mean number of myofibroblasts counted under electron microscopy in the subepithelial zone of specimens from mice exposed to filtered air and to 2 ppm ozone for 4, 8, and 12 weeks The OVA-exposed group demonstrated significantly increased Penh at methacholine concentrations of 6.25, 12.5, 25, and 50 mg/ml compared to that of the saline-exposed group (Fig. 2). The ozone-exposed group demonstrated significantly decreased Penh at methacholine concentrations of 12.5, 25, 50 mg/ml compared to that of the filtered-air group (Fig. 3). We did not observe any differences in inflammatory cells or the levels of cytokines in the BAL fluids, or any changes in airway remodeling among the groups exposed to filtered air for 4, 8, and 12 weeks (data not shown). Therefore, we used the data for the group exposed to filtered air for 4 weeks in the comparisons to the ozone-exposed groups. The proportions of eosinophils and neutrophils in BAL fluids were significantly higher in the group exposed to ozone for 4 weeks than in the filtered-air group (filtered-air group vs. ozone-exposed for 4 vs. 8 vs. 12 weeks: eosinophils, 1.5 0.28 vs. 2.5 0.13 vs. 1.11 0.05 vs. 1.8 0.08%; neutrophils, 2.2 1.32 vs. 4.5 1.02 vs. 1.9 1.22 vs. 2.5 2.01%, respectively; p < 0.05). The INF- level decreased significantly after 4, 8, and 12 weeks of ozone exposure compared to that of the filtered-air group. The IL-4 level in BAL fluids was not different between any of the ozone-exposed groups and the filtered-air group (filtered-air group vs. ozone-exposed for 4 vs. 8 vs. 12 weeks: IFN-, 75.4 2.57 vs. 30.3 9.52 vs. 64.9 2.9 vs. 55.6 6.64 pg/ml; IL-4, 33.3 3.27 vs. 65.1 2.96 vs. 55.6 6.64 vs. 45.9 5.26 pg/ml, respectively). The ratio of IL-4 to INF- increased significantly after 4, 8, and 12 weeks of ozone exposure compared to the ratio of the filtered-air group (filtered-air group vs. ozone-exposed for 4 vs. 8 vs. 12 weeks: 0.43 0.1 vs. 3.24 3.4 vs. 1.30 0.89 vs. 0.96 0.38, respectively; p < 0.05). The ozone-exposed groups also demonstrated significantly increased protein levels compared to that of the filtered-air group (filtered-air group vs. ozone-exposed for 4 vs. 8 vs. 12 weeks: 10.07 0.06 vs. 14.55 0.76 vs. 11.12 0.03 vs. 12.05 0.11 g/l; p < 0.01). The development of airway remodeling in the lungs of ozone-exposed mice was assessed by histological examination of toluidine blue-stained sections of lung tissue. The lungs of mice exposed to ozone for 4, 8, and 12 weeks were isolated, and representative 5-m paraffin sections of lung tissue (3 sections every 100 m) were examined. The number of goblet cells was significantly greater in the airway epithelium of mice after 4, 8, and 12 weeks of chronic exposure to ozone than after exposure to filtered air (Fig. 4). In addition to the marked increase in goblet cell number, an increased peribronchiolar collagen layer and a thickened smooth muscle coat were observed in the lung tissue sections from the ozone-exposed groups (Figs. 4 and 5; p < 0.05). Electron microscopic observations revealed increased collagen fiber deposition, increased smooth muscle cell hypertrophy and hyperplasia, and smooth muscle cell disorganization in the lung tissue sections from the ozone-exposed groups (Fig. 6). The number of myofibroblasts significantly increased in the subepithelial zone after 4, 8, and 12 weeks of chronic exposure to ozone compared to the number in mice exposed to filtered air (Fig. 7; p < 0.05). We examined the effects of long-term exposure to ozone on airway remodeling and dysfunction in a mouse model of allergic airway disease. By measuring airway responses to methacholine, we found a decrease in AHR after long-term ozone exposure. We also observed collagen deposition and smooth muscle cell hyperplasia and hypertrophy in mice subjected to long-term ozone exposure. These changes suggest that chronic airway remodeling may be associated with AHR and airway inflammation following long-term exposure to ozone. Chronic but not brief allergen exposure was associated with a markedly increased amount of extracellular matrix in the subepithelial region of the airway wall and with increased mucin content within the airway epithelium at 4 and 8 weeks after the last allergen challenge [20]. Repeated inflammatory events may contribute to airway remodeling in asthma [21]. Animal studies of allergen-induced AHR have shown that prolonged OVA exposure results in the increased deposition of fibronectin and collagen, which was accompanied by a progressive decrease in AHR, indicating that thickening or stiffening of the airway may be protective against AHR [22,23]. The increase in goblet cell number and in mucus lining the airway may serve a protective function against inhaled toxins and excessive mucosal dehydration [24]. We measured lung function using unrestrained plethysmography, which in conscious mice represents the extreme of noninvasiveness and is highly convenient; however, it provides respiratory measurements that are so tenuously linked to respiratory mechanics that they cannot be considered as meaningful indicators of lung function [25]. In our study using a murine model of asthma, the increase in AHR following OVA sensitization and challenge decreased after repeated exposure to ozone over a period of up to 12 weeks, indicating that structural airway changes can occur as protection against AHR after repeated exposure to air pollutants. Such changes included goblet cell hyperplasia, increased myofibroblast proliferation, increased collagen deposition, and smooth muscle hypertrophy and hyperplasia. Many asthma patients present evidence of residual airway obstruction, which can exist in asymptomatic patients, after antiasthma drugs; this probably represents remodeling. Remodeling may also be important in the pathogenesis of nonspecific AHR, especially the component that reverses slowly or incompletely with inhaled glucocorticosteroid treatment [26]. Airway injury or inflammation caused by air pollutants has been evaluated mainly by the analysis of fluids collected by bronchoalveolar lavage, which is an especially invasive technique totally unsuitable for children. Research in the field of biomarkers is providing new perspectives with the development of noninvasive tests for monitoring inflammation and damage in the deep lung. Our data in a murine model of asthma suggest that repeated exposure to air pollutants can induce airway remodeling and may account for irreversible airway obstruction. It is necessary to speculate on how various aspects of the remodeling process could contribute to airway dysfunction and nonspecific AHR. IL-4 produced by several cell types, predominantly by Th2 lymphocytes, is believed to contribute to the characteristic inflammatory response in asthmatic airways [27]. IL-4 can modulate the behavior of fibroblasts [13] and may stimulate fibroblast-mediated contraction of extracellular matrix, as in a model of the tissue remodeling characteristics of fibrotic lesions [28]. The Th2-derived cytokines, IL-4 and IL-13, can stimulate the production of TGF- in airway epithelial cells but not in lung fibroblasts. IFN-, in contrast, can inhibit TGF-2 release both under basal conditions and following IL-4 or IL-13 stimulation. The ability of these cytokines to modulate TGF- release may contribute to both normal airway repair and the development of subepithelial fibrosis in asthma [29]. In the present study, the decrease in INF-, the trend toward an increase in IL-4, and the increase in the ratio of IL-4 to INF- after chronic ozone exposure may contribute to structural airway changes following repeated ozone exposure in a murine model of asthma. Further studies are needed to clarify the potential mechanisms responsible for the AHR decrease in ozone-exposed mice despite the increase in airway smooth muscle mass and airway inflammation, as shown in the present study. In conclusion, we have demonstrated that the airway physiology and airway structure are altered in a murine model of asthma chronically exposed to ozone. Sustained airway dysfunction was observed after 4 weeks of ozone exposure, and airway remodeling was sustained following 12 weeks of ozone exposure. The observation that airway remodeling persists after the recovery of AHR supports the postulate that structural changes contribute to changes of AHR in mice chronically exposed to ozone. Bor-Kucukatay, Melek, Wenby, Rosalinda B., Meiselman, Herbert J. and Baskurt, Oguz K. (2003). Effects of nitric oxide on red blood cell deformability. American Journal of Physiology - Heart and Circulatory Physiology Volume 284 Issue 5. pp. H1577-H1584 NITRIC OXIDE (NO) plays a major role in cardiovascular regulation (5, 27), with its action mainly attributed to the effects on vascular smooth muscle cells (5, 25, 27). However, it has also been shown that NO synthesized in endothelial cells not only diffuses to the adjacent smooth muscle cells but also to the vascular lumen (7, 24). In addition to its effects on leukocytes and platelets, NO interacts with red blood cells (RBCs) via binding to the heme portion of hemoglobin to form S-nitrosohemoglobin (16, 45) and inducing the formation of methemoglobin (23, 43, 45). Human RBCs are positive for both inducible (NOS2 or iNOS) and constitutive (NOS3 or eNOS) forms of NO synthase (NOS), and thus are capable of synthesizing their own NO (17). Petrov et al. (37, 38) have demonstrated the existence of particulate and soluble guanylate cyclase as well as phosphodiesterases in human RBCs. It has therefore been suggested that NO synthesized within RBCs may modulate RBC physiological behavior and thus that both extracellular and intracellular sources of NO can affect the cell (9, 17). Korbut and co-workers (21, 22, 41) have suggested that NO may have a regulatory effect on RBC deformability and aggregation and have shown that this effect is concentration dependent. The same authors also demonstrated that NOS inhibitors have a protective effect on erythrocyte deformability in septic shock and in the acute phase of endotoxemia in rats (22, 42). Furthermore, Mesquita et al. (29) have shown that both acetylcholine (ACh) and the NO donor spermine-NONOate improve RBC deformability and suggest that the effect of ACh may be due to induced NO synthesis mediated by M1-type ACh receptors on the RBC membrane. Chronic inhibition of NOS by N -nitro-L-arginine methyl ester (L-NAME) has been found to significantly reduce RBC deformability in rats (6); the mechanical properties of RBC from these animals were normalized in vitro by a low dose (10M) of sodium nitroprusside (SNP), whereas higher doses were ineffective (6). The present study was designed to provide further insight into the effects of NO on the rheological behavior of human RBCs. In particular, the in vitro effects of NOS inhibitors, NO donors, and guanylate cyclase inhibitors on RBC deformation in defined shear fields were evaluated. Our results indicate the marked influence of NO on RBC deformability and thus lend support to the possibility that NO has an important regulatory effect on this cellular mechanical property. Blood Sampling Blood samples, anticoagulated with heparin (15IU/ml), were obtained from healthy adult male volunteers. RBCs were isolated from whole blood by centrifugation (1,400g, 6min) followed by two washing steps in PBS (pH=7.4,290mosmol/kg). The washed RBCs were then resuspended in plasma at a hematocrit of 40% for the experimental studies. All RBC preparations, incubations, and measurements were carried out within 4-6 h after blood collection. Incubations RBC suspensions were incubated at room temperature for 1h in the presence of various chemical agents, after which RBCs from these suspensions were used for the determination of RBC size, geometry, and cellular deformability. This 60-min incubation period was selected based on preliminary studies that indicated a gradual increase of effects up to 40min, with no additional change thereafter. The following agents were used for the incubations. NOS inhibitors and/or NO donors. Both a nonspecific NOS inhibitor (L-NAME) and a specific iNOS inhibitor [S-methylisothiourea (SMT)] were used. The effects of these NOS inhibitors were first tested at concentrations between 10 6 and 10 2 M, and the most effective doses were then selected to be used for the rest of the L-NAME and SMT studies. SNP and diethylenetriamine (DETA)-NONOate were used as NO donors at concentrations between 10 4 and 10 7 M and were employed alone or with NOS inhibitors. NO precursor. The effect of the NO precursor L-arginine was evaluated at a concentration of 10 5 M. Soluble guanylate cyclase inhibitors. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) was used with or without NO donors. ODQ was tested at various doses (10 6-10 4 M), after which 10 5 M was selected to be used throughout the study. Additionally, in a separate series of experiments, methylene blue, an inhibitor of both NOS and soluble guanylate cyclase, was tested at 10 5 M. Potassium channel inhibitor. The effect of the nonselective potassium channel inhibitor tetraethylammonium chloride (TEA) was first tested at concentrations between 10 4 and 10 10 M and then used at 10 8 M with or without L-NAME. All chemicals were obtained from Sigma (St. Louis, MO) except for DETA-NONOate, which was obtained from Cayman Chemicals (Ann Arbor, MI). The soluble guanylate cyclase inhibitor ODQ was dissolved in DMSO and later diluted in PBS to the desired final concentration; the other chemicals were directly dissolved in PBS. RBC Deformability Measurements RBC deformability (i.e., the ability of the entire cell to adopt a new configuration when subjected to applied mechanical forces) was determined at various fluid shear stresses by laser diffraction analysis using an ektacytometer (LORCA, RR Mechatronics; Hoorn, The Netherlands). The system has been described elsewhere in detail (18). Briefly, a low hematocrit suspension of RBC in an isotonic viscous medium (70-kDa dextran) was sheared in a Couette system composed of a glass cup and a precisely fitting bob, with a gap of 0.3mm between the cylinders. A laser beam was directed through the sheared sample, and the diffraction pattern produced by the deformed cells was analyzed by a microcomputer. On the basis of the geomerty of the ellipitical diffraction pattern, an elongation index (EI) was calculated: EI=(L W)/(L+W), where L and W are the length and width of the diffraction pattern, respectively. EI values were calculated for shear rates between 0.5and 15Pa; an increased EI at a given shear stress indicates greater cell deformation and hence greater RBC deformability. All measurements were carried out at 37C. Determination of Cytosolic Calcium Concentration Cytosolic calcium concentration was determined in a separate series of experiments for RBC incubated with L-NAME (10 3 M) or SNP (10 6 and 10 5 M) using a method modified from David-Dufilho et al. (11). RBCs were separated using a density gradient (Histopaque-1077) to eliminate white blood cell contaminants. The cells were washed twice in PBS and once in HEPES buffer [containing (in mM) 123 NaCl, 5KCl, 1MgCl2 6H2O, 1.3CaCl2, 10glucose, and 25HEPES; pH=7.4] and then resuspended in the HEPES buffer at a hematocrit of 40%. After the addition of the NO donor SNP or the nonspecific NOS inhibitor L-NAME, the suspension hematocrit was immediately reduced to 1%, and RBCs were incubated at room temperature (22 1C) for 30min. Fura 2-AM at a concentration of 10nM was added to this suspension and incubated at 37C for 30min (i.e., total incubation time of 60min). The suspension was then centrifuged at 350g for 5min, and the packed RBCs were resuspended in PBS to a hemoglobin concentration of 0.05g/dl. The fluorescence spectrum was measured using an excitation range of 335-385 nm with emission monitored at 510nm using a spectrofluorophotometer (Shimadzu RF-5000; Tokyo, Japan); the ratio of fluorescence intensities for the fura 2-Ca2+ complex and the unchelated fura 2reflects cytosolic calcium concentration. Determination of RBC Volume and Shape Mean RBC volume and volume distribution were determined by an orifice-impedance technique using an automated analyzer (ElZone 280PC, Micromeritics; Norcross, GA). RBCs were also fixed in isotonic 0.5% glutaraldehyde in PBS and kept at room temperature until their shape was examined via phase-contrast or differential interference contrast light microscopy. Statistics Results are expressed as meansSE. Statistical comparisons between groups were done by repeated-measures ANOVA, followed by Newman-Keuls posttest, with P values Read More