京都大学 / 0048 / 新制・論文博士 / 博士(人間健康科学) / 乙第13006号 / 論人健博第1号 / 新制||人健||3(附属図書館) / 32934 / (主査)教授 藤井 康友, 教授 澤本 伸克, 教授 一山 智 / 学位規則第4条第2項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Final publication is available at http://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=26018088 / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19635号 / 医科博第73号 / 新制||医科||6(附属図書館) / 32671 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 妹尾 浩, 教授 渡邊 直樹, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality, with the potential to afflict as many as half of the 1.1 billion smokers in the world. The inflammatory response to cigarette smoke is believed to mediate the progressive and irreversible loss of lung function that characterizes COPD. The greatest burden of the disease arises from episodes of worsened symptoms and inflammation, usually triggered by microbial infection. Currently, the mechanisms that drive cigarette smoke-induced inflammation are being elucidated but ambiguity remains about this response and the inflammatory response engaged in a smoke-exposed lung experiencing a microbial infection. This thesis sought to investigate inflammatory mediators induced by cigarette smoke and those induced by bacteria, the most common cause of infectious exacerbations of COPD, in the context of smoke exposure. In chapter two we investigated the role of Breast Regression Protein-39 (BRP-39), a gene commonly observed to be increased under inflammatory conditions, in the inflammatory response to cigarette smoke. In order to determine the mechanisms of BRP-39 induction, its expression and inflammation was assessed in IL-13, IL-18, and IL-1R1 deficient mice. BRP-39 was found to be redundant in cigarette smoke-induced inflammation, but these data confirmed that IL-1R1 was a crucial mediator of this response. After examining the inflammatory response elicited by smoke alone, we investigated the importance of IL-1 signaling in a model of bacterial exacerbation of cigarette smoke-induced inflammation. We found that the exacerbated neutrophilia that typifies the response of a smoke-exposed lung to bacteria was dependent on IL-1α-mediated production of the CXCR2 ligand CXCL5. This study identified the unique phenomenon that cigarette smoke primes alveolar macrophages to produce excessive amounts of IL-1α in response to bacterial stimuli. The purpose of the final study of the thesis was to more comprehensively characterize the extent to which cigarette smoke changes the phenotype of macrophages. Examining total gene expression by microarray found that smoke-exposed alveolar macrophages were in a proliferative state expressing a unique profile of inflammatory mediators. Further analysis revealed that this was likely the result of a pulmonary environment rich in growth factors. Taken together, these data provide detail to the understanding of the biological process of inflammation that drives the pathogenesis of COPD. These studies identify a phenomenon that predisposes smokers to experience more severe responses to bacteria and reinforces the targeting of IL-1 signaling in the treatment of COPD. / Thesis / Doctor of Philosophy (Medical Science)

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Aims: to test the hypothesis that peripheral blood neutrophils (PMN) exhibit delayed spontaneous apoptosis in individuals with type 2 Diabetes, and that the delay is further exacerbated in individuals who co-express chronic periodontitis. Materials and methods: 73 individuals were enrolled, including those with type 2 diabetes (T2DM) (n=16), chronic periodontitis (CP) (n=15), diabetics with chronic periodontitis (T2DM+CP) (n=21) and healthy volunteers (n=21). PMN apoptosis was determined by flow cytometry using TUNEL and Annexin V assays. Caspase 3, 8 and 9 activity was measured by colorimetric assay. PMN surface death receptor quantification was performed by flow cytometry staining with fluorescence conjugated anti-CD120a (TNFR1) and anti- CD95 (FasR) antibody. Inflammatory biomarker analyses of serum samples were performed using multiplexed sandwich immunoassays. Results: In healthy volunteers, individuals with T2DM, CP and T2DM+CP, spontaneous PMN apoptosis observed at 12 hours reached 85.3% ± 3.1, 67.3% ± 3.9, 62.9% ± 3.5 and 62.5% ± 5.4, respectively (p<0.05 ). Caspase-3 activity was significantly reduced in individuals with T2DM and T2DM+CP (p<0.05), when compared to healthy volunteers. Caspase-8 activity was also significantly decreased in CP and T2DM+CP (p<0.05), associated with reduced cell surface Fas receptor, TNF receptors and Fas ligand serum levels. Glucose alone was not observed to effect PMN apoptosis; concurrent incubation with the RAGE agonist S-100B induced a significant PMN apoptosis (p<0.05). Conclusion: These data support the premise that the inhibition of PMN apoptosis in individuals with T2DM occurs through an AGE/RAGE ligand/receptor mediated interaction.

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The bovine polymorphonuclear leukocyte (neutrophil) is a central component of the acute inflammatory response, and is capable of reacting to a myriad of pro-inflammatory chemical signals that have been characterized in the context of bovine respiratory disease (BRD). Human neutrophils and bovine macrophages are known to react to pro-inflammatory signals as well; however, they are also capable of responding to anti-inflammatory signals from the autonomic nervous system. In particular, activation of the beta2-adrenergic receptor on these cells decreases several aspects of inflammatory activity, including reactive oxygen species production, chemotaxis, degranulation, and inflammatory mediator production. Dysfunction of beta-adrenergic receptors is known to contribute to the pathophysiology of numerous diseases in both people and animals. For example, congestive heart failure, asthma, cystic fibrosis, atopic dermatitis, pheochromocytoma, myasthenia gravis, hypertension, and sepsis have all been linked to decreased beta1- / beta2-adrenergic receptor density (depending on the cell type) and / or uncoupling of the respective receptor from its effector enzyme, adenylyl cyclase. Dysfunction of the beta2-adrenergic receptor mechanism has also been described in pulmonary airway and vascular smooth muscle tissue from cattle, sheep, and rats exposed to Manheimia haemolytica, which provides insight into the pathophysiology of BRD. Despite the prominent role of the bovine neutrophil in the acute inflammatory stage of BRD, and despite the potential for dysfunction following excessive exposure to inflammatory stimuli, there are no reports that describe the presence of the beta2-adrenergic receptor on bovine neutrophils, nor function of the components responsible for its signal transduction cascade. Without complimentary work with bovine neutrophils, using data from human neutrophils to examine treatment options for the acute inflammatory stage of BRD is unrealistic. For this reason, the present dissertation proposed that 1) bovine neutrophils possess the beta2-adrenergic receptor mechanism, 2) components of the beta2-adrenergic receptor mechanism work in concert to increase bovine neutrophil adenosine 3,5-cyclic monophosphate (cAMP) levels and suppress superoxide anion production, and 3) the beta2-adrenergic receptor mechanism is dysfunctional following exposure to inflammatory stimuli. Using the nonselective beta1- / beta2-adrenergic receptor antagonist [3H]CGP-12177 we observed a maximum specific binding density (Bmax) value of 0.19 fmol per 100,000 bovine neutrophils. Although this value is approximately equal to what we observed with dairy cow neutrophils, human neutrophil Bmax values with this radioligand are anywhere from five to ten-fold greater, which suggests a significant species difference. We further defined the adrenergic receptor population on bovine neutrophils to be dominated by the beta2-subtype. Next, we characterized the function of beta2-adrenergic receptors by stimulating cAMP production with the beta2-adrenergic receptor agonist, terbutaline. The role of the beta2-subtype was confirmed when the terbutaline-mediated effect was negated by ICI-118,551, a beta2-adrenergic receptor antagonist. Also, the role of the phosphodiesterase enzyme in cAMP recycling in bovine neutrophils was illustrated, as the terbutaline-mediated rise in cAMP concentration was dependent upon phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine (IBMX). This study confirms the anti-inflammatory nature of the beta2-adrenergic receptor on bovine neutrophils by demonstrating the ability of terbutaline and IBMX to decrease superoxide anion production in a dose-dependent manner. The synthetic cAMP analog, 8-bromo-cAMP also decreased superoxide anion production, but the effect was time-dependent because of its need to diffuse across the cell membrane. Moreover, IBMX exaggerated the terbutaline-mediated effect on superoxide anion production, while cAMP exaggerated the IBMX-mediated effect on superoxide anion, demonstrating that the beta2-adrenergic receptor acts in concert with adenylyl cyclase, while the phosphodiesterase enzyme functions to decrease their signal. By increasing the dose of the inflammatory stimulant opsonized zymosan eight-fold, we were able to eliminate the ability of various concentrations of terbutaline and IBMX to reduce superoxide anion production. We sought to provide a more specific demonstration of this phenomenon by activating protein kinase C (PKC) via phorbol 12-myristate 13-acetate (PMA) administration. However, preincubation with PMA actually increased terbutaline-mediated cAMP production, in a dose and time-dependent manner. At this time, we cannot explain why increasing the dose of opsonized zymosan and PMA had opposite effects on beta2-adrenergic receptor mechanism function. The answer may reside in the many reported functions of PKC isoforms. Additional studies that identify the PKC isoform repertoire in bovine neutrophils may illustrate the potential for selective inhibition, and may lead to more specific identification and treatment of beta2-adrenergic receptor mechanism dysfunction. Also, it remains to be seen how the various components of the bovine neutrophil beta2-adrenergic receptor mechanism function in-vivo during the acute inflammatory stage of BRD. / Ph. D.