Atmospheric Pollutant Enhanced Ovalbumin-Induced Airway Inflammation In Asthmatic Murine

Air pollution is the primary environmental risk factor of public health. Air pollutants are formed by a complex mixture of many pollutants, and the currently accepted range of air pollutant is particulate matter, which closely connected with the health effects of epidemiology and is a sign of health hazards pollutants. Particulate matter (PM), especially the aerodynamic diameter≤10μm (PM10) is the primary pollutant in many major cities at home and abroad. Previous epidemiologic studies have indicated that exposure to ambient particulate matter in a metropolitan area closely relates to increases in mortality and morbidity of cardiovascular and respiratory diseases. A survey of 29 countries in European showed that, when PM10 increased 10μg per m3, the mortality of respiratory diseases increased 0.58%. German scientists surveyed 4757 women in the year of 1985-1994, and the results showed that the women living near the trunk roads and chronic exposed to PM10 suffered harmful effects of chronic obstructive pneumonia. In China, Meta-analysis of data of current dose response relationship between particulate matter exposure and mortality showed that, when PM10 increased 10μg per m3, acute mortality increased 0.38%. The study on the relationship between Taiwan air particles and daily maintenance rate of respiratory diseases found that, when PM10 increased 10μg per m3, the daily hospitalization rate of respiratory diseases increased by 0.72%-2.15%. It is reported that there are billions of tons of PM10 on the earth about every year. An adult one day and night is exposed to some tens of thousands or even hundreds of thousands of atmospheric particles. Therefore, the workers of environment, meteorology and medical are focus on the health effects of particulate matter in the air pollution.Asthma is a complex syndrome broadly defined by inflammation of the airways associated with airway hyperresponsiveness and mucus hypersecretion. In recent years, its prevalence and death rates showed a rising trend year by year. In some countries, such as the United States, asthma prevalence increased from 3.6% in 1980 to 5.8% in 2003; the prevalence of asthma in children in China increased from 2.03% in 1990 to 4.63% in 2000. Experts warn:by 2025, if the urbanization of global population increased from 45% to 59%, asthma patients will reach 400 million people. One of 250 cases of deaths caused by asthma. Asthma has become the world's second largest debilitating diseases following the death of cancer, while China has become the one of the highest mortality of asthma. Epidemiological studies showed that atmospheric concentration of particles increase the number of patients with asthma. Recent epidemiologic studies have shown that Asian dust storm events coincided with an increase in daily admissions and clinic visits for asthma and allergic rhinitis in Taipei, Taiwan. Experimental studies have also reported that exposure to ambient PM near a heavily trafficked Los Angeles freeway enhanced inflammatory and allergic responses in ovalbumin (OVA)-sensitized BALB/c mice. Therefore, the study of injury mechanisms of particulate matter on asthma in mice has important practical significance for pollution prevention and protecting the health of sensitive populations.Respiratory tract is the target organ of particulate matter, and the lung macrophage is an important defensive barrier within the respiratory tract. Lung macrophages digest phagocytic particles, induce the synthesis and release of all kinds of inflammatory factors, disrupte the balance of cytokine network, which launched the inflammatory process, promote the development of inflammation and cause damage to the lungs. The innate immunity is the first barrier in the protection against ocular infections. The Toll-like receptors (TLRs), that belong to pattern recognition receptors (PRRs) family, have been the molecules involved in the recognition of pathogen-associated molecular patterns (PAMPs) that lead to ocular inflammatory process. Nevertheless, other PRRs such as Nod-like receptor (NLR) could also be participating in this phenomenon. NALP3(nacht domain-, leucine-rich repeat-, and pyrin domain-containing protein 3) of NLR family members is cytyoplasmic receptor which is activated by microbial ligands like peptidoglycan as well as endogenous markers of cellular damage (for example ATP or uric acid crystals). The activate NALP3 interacts with adaptor proteins like ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain) to form NALP3 inflammasome. The inflammasome activates caspase-1. NALP3 inflammasome complex with the active caspase-1 can produce active IL-1βwith pro-IL-1β. Recent research has shown that silica and asbestos are sensed by NALP3 inflammasome, whose subsequent activation leads to IL-1βproduction.In the present study, we used UPM collected from the atmosphere in Beijing and AASD collected from the atmosphere from Iki-island in Japan. Comparison of allergic inflammation between aggravating effects of UPM/AASD that had been excluded of toxic materials (microbial materials, sulfate, etc.) by heating, and crude UPM/AASD were performed in the murine lungs. In this in vitro study, the gene expression of TLRs and NALP3 inflammasome in RAW264.7 cells was measured in the presence of heat UPM/AASD and crude UPM/AASD to investigate the role of TLRs and NALP3 inflammasome in the enhancement by UPM/AASD. This is the first experimental study on biochemical and pathologic evidences of Beijing dust and airborne dust in Japan exacerbating asthma caused by OVA.Materials and methods 1.Preparation of particlesThe samples were heated at 360℃for 30 min in an electric heater to exclude toxic materials (microbiological materials, sulfate, nitrate, etc.) adhering to them.2.Detection of fungi in particlesThe fungi of the samples were detected by an all-in-one fluorescence microscope and fluorescent dye Fungiflora Y. According to the manufacture's protocol, in brief, approximately 2.5μg of each particle sample was suspended in 40μl Fungiflora Y, and after 5 min slides of the particles were completed. Then the slides were observed with the all-in-one fluorescence microscope when the excitation wavelength was 472.5 run, and the emission wavelength was 520.0 nm.3.Analysis of water soluble components, lipopolysaccharide (LPS), andβ-glucan in particlesThe concentration of water soluble components, such as sulfate (SO42-) and nitrate (NO3-), in the samples was determined using an ion chromatograph and ICP-AES. Each particle sample was measured by kinetic assay using Endospec ES test MK for LPS activity and by Fungitec G test MK forβ-glucan activity.4.Animal and Study protocolMale ICR mice (5 wk of age) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). After 1 week of screening out sick mice, mice with abnormal body weight and mice stressed from different environmental breeding, mice were used at 6 weeks of age. OVA and particles were dissolved in the same saline. The instillation dose of particles was 0.1 mg per mouse. Mice were intratracheally instilled with these particles through a polyethylene tube under anesthesia with 4% halothane 4 times at 2-week intervals. The control mice were instilled intratracheally with 0.1 ml of normal saline per mouse. One day after the last intratracheal administration, the mice from all groups were killed by exsanguination under deep anesthesia by i.p. injection of pentobarbital at 12 weeks of age.5.Pathological evaluationThe lungs were fixed by 10% neutral phosphate-buffered formalin. After separation of the lobes,2-mm-thick blocks were taken for paraffin embedding. Embedded blocks were sectioned at a thickness of 3μm, and then were stained with hematoxylin and eosin (H&E) to evaluate the degree of infiltration of eosinophils or lymphocytes in the airway from proximal to distal. The sections were also stained with periodic acid-Schiff (PAS) to evaluate the degree of proliferation of goblet cells in the bronchial epithelium. A pathological analysis of the inflammatory cells and epithelial cells in the airway of each lung lobe on the slides was performed using a Nikon ECLIPSE light microscope.6.Bronchoalveolar lavage fluid (BALF) and bloodIn brief, the tracheas were cannulated after the collection of blood. The lungs were lavaged with sterile saline by syringe. The lavaged fluid was harvested by gentle aspiration. The total amount of the lavages collected from individual mice was measured for calculating the protein levels of cytokines and chemokines in BALF. The fluids from the two lavages were combined, cooled to 4℃, and centrifuged at 1,500 rpm for 10 min. The BALF supernatants were stored at-80℃until it was analyzed for cytokines and chemokines.7.Cell profile in BALFThe total cell counts of a fresh fluid specimen were determined using a hemocytometer. Differential cell counts were assessed on cytologic preparations. Slides were prepared using a Cytospin and stained with Diff-Quik to identify eosinophils with red granules. A total of 300 cells were counted under oil immersion microscopy.8.Quantitation of cytokines and chemokines in BALFThe protein levels of cytokine and chemokine in the BALF were determined using enzyme-linked immunosorbent assays (ELISA). Including:Interleukin (IL)-4, IL-5, IL-12, IL-13, Interferon (IFN)-y, IL-1β, Keratinocyte chemoattractant (KC), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-la, regulated on activation normal T cell expressed and presumably secreted (RANTES), eotaxin and MCP-3.9. OVA-specific IgE and IgGl antibodiesOVA-specific IgE and IgG1 antibodies were measured using a Mouse OVA-IgE ELISA kit and a Mouse OVA-IgG1 ELISA kit.10.Cell cultureRAW264.7 cells, which are macrophage-like cells derived from BALB/c male mice, were cultured at 37℃in a humidified atmosphere of 5% CO2-95% air and maintained in Dulbecco's modified Eagle's medium with 10% heat inactivated fetal bovine serum (FBS). They were plated at a concentration of 4×105 cells per 60-mm dish, and then sample with or without heating treatment were added to cells to give a final concentration of 3μg/ml for 3 hr.11.Gene expression analysisTotal RNA was extracted by standard procedures using 0.5 ml of Isogen (Nippon Gene) per dish. After DNase treatment of the total RNA, cDNA was synthesized by reverse transcription using M-MLV. Quantitative PCR was performed using an ABI Prism 7000 Sequence Detection System (ABI) under the same conditions. Two wells were used for each sample. The relative expression of each sample was calculated as the mean value divided by the mean value for GAPDH.12.Statistical analysisStatistical analyses on the pathologic evaluation in the airway, cytokine and chemokine proteins in BALF, and gene expression in RAW264.7 cells were conducted using SPSS 13.0 software as statistically significant at a level of p< 0.05. Results were expressed as mean±SD. Differences among groups were determined using one way analysis of variance followed by Student-Newman-Keuls test when F was significant.Results1.The detection of fungi in the particlesBright fluorescence of particles after being stained with Fungiflora Y. was clearly observed in crude samples.2.Contents of chemical elements, water soluble components, LPS andβ-glucan in the particlesThe oxide composition in this UPM sample was 32% SiO2 and 12% total carbonin, however, in this AASD sample was 60% SiO2. There were SO42-, NO3-, Cl-, NH4+, LPS and P-glucan in crude samples but non-detected in the heated samples.3.Enhancement of pathologic changes in the lung by theparticlesOVA+UPM and OVA+AASD caused marked accumulation of lymphocytes and eosinophils infiltration in the airway compared with OVA group. The degree of the accumulation was higher in OVA+UPM and OVA+AASD than in OVA+H-UPM and OVA+H-AASD4.Enhancement of cell numbers in BALF by the particlesOVA+UPM and OVA+AASD resulted in a remarkable elevation of neutrophils, macrophages, eosinophils and lymphocytes compared with the control, OVA and the single treatment.5.Enhancement of cytokines and chemokines in BALF by the particlesOVA+UPM and OVA+AASD enhanced the expression of IL-4, IL-5 and IL-12 compared with the control, OVA and the single treatment. OVA+UPM and OVA+ AASD synergistically increased protein levels of eosinophil relevant cytokines, such as Eotaxin, MCP-1 and MCP-3 in BALF compared with the each single treatment.6.Enhancement of OVA-specific IgE and IgG1 in serum by the particlesOVA+UPM and OVA+AASD markedly elevated the production of OVA-specific IgE and IgGl as compared with the control and OVA.7.TLRs mRNA expression in RAW264.7 cellsUPM and AASD significantly increased the expression of TLR2 mRNA compared with saline addition. And AASD decreased the expression of TLR4 mRNA.8.NALP3 inflammasome mRNA expression in RAW264.7 cellsAASD significantly increased the expression of NALP3 inflammasome compared with control. However, there was no significantly change of caspase-1.Conclusion1.Our findings suggest that UPM and AASD aggravate OVA-induced lung inflammation, possibly by activation of the Th2-associated immune response via cytokine and chemokine. The chemical products from fossil fuel combustion, fungi, bacteria, and silica-carrying contained in UPM and AD may be partly responsible for allergen-induced lung inflammation.2.TLR2 and NALP3 inflammasome may play an important role in this phenomenon.