The Significance Of HMGB In Cigarette Smoking Induced Chronic Lung Inflammation And Emphysema In Mice

Background:Chronic obstructive pulmonary disease (COPD) is being defined as a preventable and treatable disease with high mortality and morbidity. COPD is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lung to noxious particles or gases manly duo to cigarette smoking. It is estimated that COPD will become one of the major health challenges in the coming few decades. Prevalence surveys suggest COPD is presently the fourth leading cause of death in most industrialized countries, and will be the third leading cause of death by2020according to WHO report. In China, the mortality of COPD is rising rapidly because the rapid urbanization, air pollution, aging of the whole population. The mortality is even higher in rural areas for economical conditions and biomass fuel using. The burden of COPD has become a serious social and economical problem. It is necessary that more attention is paid to the prevention and treatment of the disease. COPD is considered a disease not limited to the lung but affected the whole body including organs like the heart and muscle.The mechanism of COPD has not been completely understood. Tobacco smoking is believed the primary risk factor for the development of COPD. This has more important meanings especially in China for the fact that China is now the biggest cigarette consuming country. Other factors as burning biomass fuels for cooking and heating in rural area and some district in South China are also important causes of COPD in China. There are more than4,000kinds of chemicals in the cigarette smoke including carbon monoxide, nitro oxide, nicotine, etc. Those chemicals could impose heavy burden on the lung and cause various pathological processes, resulting lung tissue damage, emphysema, bronchiole-fibrosis, mucous hyper secretion, and muscle atrophy.It is obvious that most patients with COPD have a history of cigarette smoking for a long time. But not all cigarette smokers develop COPD. This phenomenon indicates that not the environmental factors but the personal heredity is dependent factors in development of COPD. The inflammation in the lung of COPD patients maintains for a long time even after smoking quitting also denote the complexity of the mechanism of COPD.Smoking can induce severe oxidative stress in the lung. In the smoke fog larger amount of bacterial toxin such as lipoproteins (LPS) could be found. After breathing in LPS containing smoke, one often cough, feel chest-tightness, dispnea, spit sputum. The spirometric index forced-expiratory volume in second one also lowered. The cytokines as tumor necrosis factor-alpha, interferin-1beta, interferin-8, and interferin-6are elevated in the bronchial alveolar lavage fluid. The cigarette smoke also contains high concentration of reactive oxidative stress ROS. The inflammation caused by smoking could activate cells like phagocytes and neutrophiles. These activated cells can release ROS and further increase the concentration of ROS in the airway and lung tissue, causing redox disturbances.Oxidative stress is an important mechanism of airway inflammation. Active oxygen can induce inflammation and airway hyper-reactivity, cell apoptosis, damage the function of protective anti-protein enzyme, and reduce the sensitivity to glycol-corticosteroids. Compared with the healthy, there are more macrophages, T cells, neutrophiles, and eosnophils, which indicate an enhanced inflammation in the lung of smokers. This was also proved in the mouse model of chronic smoking. All of those cells take part in the inflammation process in COPD. The cytokines are abnormal in patient with COPD implies that an immune disturbance is exist in the body of COPD patients.Dendritic cells are professional antigen present cells that play a pivot role in both the innate and adoptive immune. In tissue, immature DC accept various antigens and process the inflammatory signal, the cell became matured with increased expression of surface bio-marks such as CD83and co-stimulator CD80, CD86. Matured DCs then present signals to naive T cells, and TO cells proliferated into T helpl and T help2cells and activate CD4+T cells, CD8+cytotoxic cells. Matured DCs can produce various cytokines like IL-12, IL-10, IL-1, IL-6, IL-8, and TNF-α etc that exert immune regulating effect directly, In this way, DC modulates the immune response.It is now known that there are two kinds of immune responses mediated by DC. DCs are rich in pattern recognition receptors (PPRs), which can bind with pathogen-associated molecular pattern (PAMP) and damage associated molecular pattern (DAMP). LPS is a typical PAMP than can stimulate DC, drives DCs to maturate. DAMPs are another kind of stimulators that can drive DC maturation. In fact, DAMPs are normal cell contents under normal circumstance. When cells are damaged, for example, necrosis and apoptosis, the cell contents were released outside. The most common DAMPs include HMGB1, heat-shock protein and uric acid etc.HMGB1is a kind of highly preserved DNA binding protein and a typical DAMP. Normally, HMGB1is located in the nucleus, binding to DNA and play am important role in gene transcription, DNA repairing, cell proliferation. When released outside the cells, HMGB1acts as a pro-inflammatory cytokine. It is found that HMGB1rise in late stage during acute severer infection like septic shock and for this reason named late-inflammatory cytokine. HMGB1is also nominated the pro-inflammatory factor for it can promote some other cytokines released.HMGB1can bind to RAGE and toll-like receptors. Both RAGE and toll-like receptors are abundantly expressed on the surface of DC. After binding to the receptors on DC, HMGB1acts in many ways, resulting in DC maturation. HMGB1/RAGE pathway had been proven crucial for the DC maturation. HMGB1takes part in oxidative stress by way of act on NF-&B in the pathway of RAGE. Recent years, there are many researches on the effects of HMGB1/RAGE pathway in lung disease. HMGB1has been proved involved in many diseases such mechanical ventilation induced lung damage, non-small cell lung cancer, lung fibrosis. HMGB1/RAGE also is found to play a key role in some self-immune disease like systemic lupus erythematosus,The chemicals in cigarette smoke can affect the function of DC. It is reported that numbers of DC in BALF and lung tissue of chronic smoking mouse model increase obviously. In human being, smoking can raise the number of DC in the airway and the CD40and CD86positive DC aggregation. Smoking not only affects the amount of DC but influence the secretion of cytokines from DC. For example, cigarette smoking extract can increase the secretion of IL-12.DC are involved in the chronic airway inflammation and tight related with numerous inflammation cytokines, but the exact role in the development of COPD is not known. It is hard in fact to decide the roles of DC in clinic observation for patients often has infection when they are in hospital. Studies in the past demonstrated that HMGB1concentration in the blood and BALF of mouse model caused by cigarette smoking rise significantly. The expression of HMGB1on lung tissue also increases remarkably, indicating that HMGB1could be important antigen in the development of COPD.Considering that oxidative stress is involved in the mechanism of some respiratory disease like asthma and COPD, researcher tried anti-oxidants like N-acetylcysteine in treatment of COPD. Some researches reported that anti-oxidants could suppress the inflammation in the lung. But no anti-oxidants were recommended in clinic until today.On the literatures mentioned above, we hypothesized that there may exist such a pathway in the developing of COPD, smoking——oxidative stress——cell damage including necrosis and apoptosis——HMGB1elevated——activate DC——immune response——inflammation——cell damage. The HMGB1may play a key role in expanding the inflammation by activating DC in the loop. Oxidative stress is involved in the process and possibly be suppressed by anti-oxidant ambroxol. We designed two experiments in this study to confirm our hypothesis. In one experiment, using cigarette smoking mice model, we observe the changes of HMGBl, other cytokines as interferin-10and interferin-12, and maturation of DCs in the lung tissue. In the other, we treat the DC derived from isolated mouse bone marrow monocytes with HMGB1directly and see whether changes are similar to the result observed in mouse model. The clarification of mechanism of HMGB1in COPD could supply new ways of treatment for COPD.This study is divided into two parts;Part one:HMGB1, dendritic cell, and cytokines in the lung of cigarette smoking mousePart two:The interference of ambroxol in the HMGB1induced changes of dendritic cells HMGB1, Dendritic Cell, and Cytokines in Cigarette Smoking exposed MiceObjective:1. To investigate the expression of HMGB1in the lung of cigarette smoking exposed mice2. To observe the relationship between the expression of HMGB1and other cytokines changes3. To study the changes of dendritic cells in the lung of cigarette smoking-exposed mice4. To investigate the effect of ambroxol on inflammation changes in cigarette smoking-exposed miceMethods:1. Animal Male Balb/C mice were purchased from animal center, Anhui Medical University. The mice were6-8weeks old on arrival, and were acclimatized for one week in our animal house under standard conditions of temperature, humidity, and light/dark (12/12-h) cycle. The study was approved by the Animal Ethics Committee of Anhui Provincial Hospital and the animals were treated with humane care and attention during this study.2. Research protocol Mice were divided randomly into three groups including the control, the smoking, and the ambroxol groups. The mice in the smoking and ambroxol groups were exposed to mainstream tobacco smoke in a smoke exposure box designed by ourselves, five cigarettes twice a day,5d/wk. To the control group of mice, no addition treatments were given.The mice of the ambroxol group were instilled ambroxol,0.8mg/g, by way of gastric tube before cigarette smoking exposure each time. After21weeks exposure, all mice were executed and samples of blood, bronchial lung lavage fluid, and lung tissue were collected.3. Detection Hematoxylin&eosin (HE) stain was used to observe the pathological changes in the lung sections and confirm the establishment of chronic lung inflammation and emphysema caused by cigarette smoking. Enzyme-linked immuno sorbent assay (ELISA) was adopted to detect the concentration of HMGB1, IL-10, and IL-12in blood and BALF. Immuno-histological chemical method was applied to detect expression of HMGB1, the numbers of CD11c positive DCs, CD83positive DCs, CD80positive and CD86positive DCs in the lung tissue.Results:1. The establishment of chronic inflammation mouse model of cigarette smokingCompared with the controls, there are obvious pathological changes in the lung tissue section of mice in the smoke and treat group. Under microscope, we found remarkable inflammatory change in the lung, including the enlargement of alveolar space, gland hyperplasia, aggregation of inflammatory cells, destruction of tissue structure, and emphysema formation. The manifestation of pathology confirmed reliability of the animal model inducing method we used in this study.2. HMGBl increased in the lungThe expresrion of HMGB1in lung tissue (34.71±8.36vs5.57±3.51MOD, p<0.01),blood (25.75±3.30vs4,0±1.83ng/ml), and BALF(3.15±0.21vs1.15±0.60ng/ml) in cigarette smoking exposed rose significantly compared with the controls. The differences were statistically significant. HMGB1concentration in ambroxol treat group was reduced and the difference between the smoke and treat group was also significant statistically (p<0.05).3. The rise of HMGB1is correlated with IL-12The concentration of IL-12rose in the blood and BALF of CS-exposed mice compared with the controls. The differences were statistically significant (507.1±149.8vs225.0±17.00pg/ml, p<0.01). When treated with ambroxol, the changes were attenuated and the results remained statistically significant.4. Changes of dendritic cells in the lungUnder microscope, we found more CDllc positive cells in CS-exposed and ambroxol treated mice than in the controls, which indicated that smoking could increase the number of DCs in the lung. The number of CD83positive cells increased indicating that matured DCs increased in the lung as CD83was usually defined as the maturation mark of DC. We also found more CD80positive cells in the slides of lung in CS-exposed and ambroxol treated mice. For CD80is a co-stimulator factor which act as a bridge between DC and T cells, the rose of CD80implies the ability of co-stimulatory function was enhanced. The CD83and CD80positive DCs were mostly aggregated at the lymphoid follicle.5. The effect of ambroxol on the changes induced by cigarette smokingIn the HE staining slice of mouse lung, a relatively attenuated damage could be observed in ambroxol treated mice compared to CS-exposed mice. The inflammatory cell aggregation was also not as severe as in the CS-exposed mice. The expression of HMGB1in the lung tissue was lowered, the elevated IL-12level was lowered and the expression of CD11c, CD83, and CD80were reduced compared with the CS-exposed mice. The differences are all significant statistically.Conclusion:1. Cigarette smoking can induce obvious lung damage in Balb/c mouse; there are remarkable chronic inflammations in the lung including bronchiolitis and emphysema;2. HMGB1increased remarkably in the lung and elevated in the blood and BALF in smoking mouse. The rise of HMGB1is synchronously increased with other cytokines;3. Cigarette smoking could considerably increase the IL-12in the lung;4. The number of DCs in the lung increased considerably duo to cigarette smoking. DCs tend maturated and the co-stimulatory ability possibly enhanced. The maturation of DC was synchronously with the increase of HMGBl.5. Ambroxol could attenuate chronic inflammation in the lung caused by cigarette smoking. The mechanism may be related with the changes of HMGB1and DCs. The interference of ambroxol in the HMGB1induced changes of dendritic cellsObjectives:1. To investigate the effect of HMGB1on marker protein expression of dendritic cells;2. To investigate the affects of HMGB1on expression of co-stimulator on DC and the cytokine production3. To investigate the effect of ambroxol on changes of dendritic cells induced by HMGB1Methods:1. DC generationThe same Balb/c mice as in the experiment one were used in this study. First, bone marrow-derived cells were flushed out of the cavities from the femurs and tibias with PBS. Cells were then added0.83%ammonium chloride and the lysed red blood cells and other contents were abandoned. The selected monocvtes-like cells were washed twice with1640medium and seeded out at a density of2X106cells per60-mm dish. Cells were cultured for6days in RPMI1640medium containing10%fetal calf serum (FCS),1%penicillin/streptomycin,1%glutamine,1%non-essential amino acids and0.05%β-mercaptoethanol. Cultures were supplemented with granular monocytes colony stimulating factor (GM-CSF)20ng/ml, and Interleukin-410ng/ml and fed with fresh medium containing GM-CSF and IL-4on days3and then every other day. On day3, the non-adherent cells were discarded and the remaining cells were fed with the complete medium.2. Treatment of DCOn day6, cells were divided into three groups, the controls, the HMGB1group, and the ambroxol group. HMGB1were added into the medium at a final concentration of lug/ml in HMGBl group. In ambroxol group, ambroxol100μmol/ml was added1hr before the addition of HMGB1. On day7.>80%of the cells expressed CD11c, a marker for mouse DC, by flow cytometry. Experiments were performed at days8of DC culture. The cell suspensions were transferred to96-well culture plates,5X105cell each well and CD80-FITC, CD83-PE antibody were added for flow cytometry. The supernatants were collected for detection of IL-10, IL-12.3. Measurement of cytokinesELISA is used in measuring the concentration of mouse IL-10and IL-12. IL-10and IL-12levels in supernatants were measured using a commercially available ELISA kit according to the manufacturer’s instructions.4. Detection of DC surface proteinFlow cytometry was used. Cell suspensions were labeled according to standard procedures using the following monoclonal antibodies:CDllc-PE, CD80-FITC, CD83-PE, or an isotype control. After incubating with the antibody for60minutes at4℃, the cells were washed twice and resuspended in FACS buffer for flow cytometric analysis.5. Assessment of DCs’proliferation abilityLymphocyte proliferation test was performed by MTT. After sacrificing, the Balb/c mouse spleen was collected under sterile conditions. The spleen was grinded, filtered, and the cell suspension was collected. T cells for reaction were isolated using lymphocyte separation liquid and cotton column. Then cells were mixed with DCs from different groups at7days in a proportion of1:10and put into96well culture plate with200u1mixed cell suspension in each well. The cells were incubated under37℃,5%CO2conditions for4days. Four hours before the end of experiment, MTT at a concentration of0.5mg/ml per well was added and the OD value was measured with a spectrophotometer at570nm and stimulation index (SI) was calculated.Results:1. DCs generated from bone marrow monocytes-like cellsThe generated cells from mice bone marrow grow well in the culture medium. At the first day, most cells were small, round and attached to the dish wall. The capacity of cells increased with the time progressed. On day5to6, part of the cells detached from the cell wall and floated in the medium. Some of the cells not looked enlarged but also have irregular round shape and dendritic-like protruding. The flow cytometry indicated that more than80percent cells demonstrated CD11c positive. As CD11c is a marker of DC, the result indicated that most of the cells generated were DCs.2. Expression of CD83The expression of CD83was less than20percent in DC in the controls which indicated that the DCs were not matured. When stimulated with HMGB1, CD83on DC surface increased remarkably, up to70%. CD83is a kind of surface protein on DC and considered the marker of maturation. CD83positive rate changes before and after HMGB1stimulation demonstrated than HMGB1could promote maturation of DC.3. Expression of CD80CD80is one of the co-stimulators expressed on the surface of matured DC. We detected the expression of CD80on DCs and found that in the controls, expression of CD80were low and increased when treated with HMGB1(15.31+8.51vs60.50+18.01; p<0.05), the differences were statistically significant. When pre-treated with ambroxol, CD80expression was lowered to some extent compared with the HMGB1group (41.09±9.769vs60.50±18.01, p<0.05), but remained higher than the controls.4. Changes of IL-10and IL-12concentration in the supernatantsELISA results indicated that both IL-10and IL-12concentration in the supernatants were increased when cells were treated with HMGB1. IL-12was elevated in the supernatant of HMGB1-treated DC compared with that in the control groups (375.8+36.93vs12.90±1.108pg/ml, p<0.001). When pre-treated with ambroxol the increased IL-12concentration was reduced and the difference was statistically significant (274.0±40.53, vs375.8±36.93pg/ml, p<0.05). Likewise, the concentration of IL-10is increased in the HMGB1group, and reduced when pre treated with ambroxol (47.05±4.25;38.71±4.49pg/ml,p<0.05)5. Lymphocytes proliferation assay.MTT results demonstrated that SI of DCs stimulated by HMGB1was considerably higher than the controls (57.17±6.959vs14.56±3.487, p<0.001). when pre-treated with ambroxol, SI reduced statistically compared with HMGB1stimulated DCs (37.72±3.294vs57.17±6.959, p<0.05). The results implied that HMGB1can promote the proliferation capacity of DC and this can be interfered by ambroxol in part.Conclusion:1. Bone marrow monocytes-like cells are reliable sources of DC. By culturing in GM-CSF and interleukin-4containing1640medium, cells high expressed CD11c. The DC generation method is convenient and reliable.2. HMGB1can drive DC maturate directly; HMGB1can promote DC maturation and enhance DCs ability to promote T cell proliferation.3. Ambroxol can partially block the pro-mature action of HMGB1on DC.