Elevated eosinophil counts in the circulation and/or tissues is considered a clinical feature and biomarker of several chronic airway diseases including asthma. As such, many therapeutic biologics for asthma developed within the past decade target eosinophil recruitment to and accumulation in the airways to mixed success. Although the nature of adhesive interactions and directional migration of eosinophils has been well studied, there remains a lack of comprehensive understanding regarding the components which modulate eosinophil movement from the blood into respiratory tissues that impacts the efficacy of these clinical studies; therefore, continued research in this area may reveal novel therapeutic targets and ultimately improve clinical outcomes of patients with eosinophilia-mediated diseases. The Janssen lab serendipitously discovered that the mere perfusion of standard media without pharmacological additives over human eosinophils in vitro induced the release of intracellular calcium (Ca2+) reminiscent of chemokine-induced Ca2+ release well documented in the literature. The central focus of my doctoral research was to characterize this novel phenomenon of the perfusion-induced calcium response (PICR), and to determine its physiological role in the eosinophil extravasation process to inflamed tissue sites. In our first research objective, we optimized a protocol of eosinophil isolation directly from whole blood with emphases on maximizing population purity and yield efficiency while minimizing cell activation that could potentially interfere with secondary functional assays. For our latter two studies, we utilized real-time fluorescent confocal microscopy and immunofluorescence staining to investigate the PICR. We observed that the latency to the PICR post-perfusion was significantly shorter in eosinophils subjected to physiological rates of shear stress, suggesting a temporal-regulatory function of eosinophil mechanosensitivity. Furthermore, the disruption of the PICR via pharmacological inhibitors significantly reduced eosinophil motility by increasing the latency to cytoskeletal rearrangements (flattening onto substrate-coated surfaces, formation of membrane protrusions that explore the environment) necessary for cell migration out of the vasculature. Detailing the role of eosinophil sensitivity to the mechanical trigger of fluid shear stress expands upon the current paradigm of eosinophil recruitment and will contribute to the development of clinical strategies. / Dissertation / Candidate in Philosophy

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24838号 / 医博第5006号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 大森, 孝一, 教授 中山, 健夫, 教授 後藤, 慎平 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

abstract: Eosinophils are innate immune cells that are most commonly associated with parasite infection and allergic responses. Recent studies, though, have identified eosinophils as cells with diverse effector functions at baseline and in disease. Eosinophils in specific tissue immune environments are proposed to promote unique and specific effector functions, suggesting these cells have the capacity to differentiate into unique subtypes. The studies here focus on defining these subtypes using functional, molecular, and genetic analysis as well as using novel techniques to image these subtypes in situ. To characterized these subtypes, an in vitro cytokine induced type 1 (E1) and type 2 (E2) eosinophil model was developed that display features and functions of eosinophils found in vivo. For example, E1 eosinophils secrete type 1 mediators (e.g., IL-12, CXCL9 and CXCL10), express iNOS and express increased levels of the surface molecules PDL1 and MHC-I. Conversely, E2 eosinophils release type 2 mediators (e.g., IL4, IL13, CCL17, and CCL22), degranulate and express increased surface molecules CD11b, ST2 and Siglec-F. Completion of differential expression analysis of RNAseq on these subtypes revealed 500 and 655 unique genes were upregulated in E1 and E2 eosinophils, respectively. Functional enrichment studies showed interferon regulatory factor (IRF) transcription factors were uniquely regulated in both mouse and human E1 and E2 eosinophils. These subtypes are sensitive to their environment, modulating their IRF and cell surface expression when stimulated with opposing cytokines, suggesting plasticity. To identify and study these subtypes in situ, chromogenic and fluorescent eosinophil-specific immunostaining protocols were developed. Methods were created and optimized, here, to identify eosinophils by their granule proteins in formalin fixed mouse tissues. Yet, eosinophil-specific antibodies alone are not enough to identify and study the complex interactions eosinophil subtypes perform within a tissue. Therefore, as part of this thesis, a novel highly-multiplexed immunohistochemistry technique was developed utilizing cleavable linkers to address these concerns. This technique is capable of analyzing up to 22 markers within a single biopsy with single-cell resolution. With this approach, eosinophil subtypes can be studied in situ in routine patient biopsies. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

Orientador: Gilberto De Nucci / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T16:13:57Z (GMT). No. of bitstreams: 1 Maziero_AlineMendes_D.pdf: 2175096 bytes, checksum: b3e712ed8baf6a7f7d4ef12131b9e85a (MD5) Previous issue date: 2014 / Resumo: Os eosinófilos participam de processos inflamatórios e alérgicos. Estando relacionados com o sistema de imunidade inata do organismo, eles representam uma linha fundamental de defesa contra invasão microbiana e, quando ativados, produzem uma série de mediadores solúveis que atuam nas respostas inflamatórias e alérgicas. A relação entre a atividade dos eosinófilos e plaquetas foi observada nas últimas décadas por muitos cientistas. Estas observações incluem o aumento do número de eosinófilos associados a desordens plaquetárias, incluindo alterações na cascata de coagulação e agregação plaquetária. Com base nessas observações, a interação entre os eosinófilos e plaquetas foram analisadas na agregação plaquetária. Plaquetas humanas foram incubadas com a fração citosólica de eosinófilos, linhagem celular promielocítica humana HL-60 clone 15 e proteína catiônica do eosinófilo (ECP). A agregação em plasma rico em plaquetas (PRP) foi induzida por difosfato de adenosina, fator de ativação plaquetária, ácido araquidônico e colágeno, e as plaquetas lavadas (PL) foram ativadas por trombina. A agregação induzida por todos os agonistas foi inibida de maneira concentração de células dependente pela fração citosólica de eosinófilos. Esta inibição foi apenas parcialmente revertida pela prévia incubação dos eosinófilos com L-Nitro-Arginina-metil-éster (L-NAME). A prévia incubação com indometacina não impediu a inibição induzida pela fração citosólica. A separação da fração citosólica de eosinófilos por gel filtração em Sephadex G-75 mostrou que a atividade inibitória foi concentrada na fração de peso molecular mais baixo. As células HL -60 clone 15 diferenciadas em eosinófilos por 5 e 7 dias foram capazes de inibir a agregação plaquetária. A proteína de ECP inibiu a agregação plaquetária em PRP e PL. Esta inibição foi mais evidente em PL, e o ensaio de citotoxicidade com MTT demonstrou a viabilidade de plaquetas testadas, indicando que a inibição observada pela proteína ECP não ocorre simplesmente pela morte celular. A proteína EDN, clonada e expressa em sistema eucarioto, também apresentou efeito de inibição sobre a agregação plaquetária em PRP, enquanto que a proteína MBP não apresentou efeito de inibição da agregação plaquetária significativo. Os nossos resultados indicam que os eosinófilos desempenham um papel fundamental na inibição da agregação plaquetária / Abstract: Eosinophils participate in allergic and inflammatory processes, being related to innate immunity system of the body, they represent a fundamental line of defense against microbial invasion and when activated produce a number of soluble mediators that act in inflammatory and allergic responses. The relationship between the activity of eosinophils and platelets has been observed in recent decades by many scientists. These observations include increased numbers of eosinophils associated with platelet disorders, including changes in the coagulation cascade and platelet aggregation. Based on these observations, the interaction between eosinophils and platelets in platelet aggregation was analyze. Human platelets were incubated with eosinophil cytosolic fraction, promyelocytic human HL-60 clone 15 cell lineage, and eosinophil cationic protein (ECP). Platelet rich plasma (PRP) aggregation was induced by adenosine diphosphate, platelet activating factor, arachidonic acid, and collagen, and washed platelets (WP) were activated by thrombin. Aggregation induced by all agonists was dose dependently inhibited by eosinophil cytosolic fraction. This inhibition was only partially reversed by previous incubation of the eosinophils with L-Nitro-Arginine-Methyl-Ester (L-NAME). Previous incubation with indomethacin did not prevent the cytosolic fraction induced inhibition. The separation of eosinophil cytosolic fraction by gel filtration on Sephadex G-75 showed that the inhibitory activity was concentrated in the lower molecular weight fraction. HL-60 clone 15 cells differentiated into eosinophils for 5 and 7 day were able to inhibit platelet aggregation. The ECP protein inhibited the platelet aggregation on PRP and WP. This inhibition was more evident in WP, and the citotoxicity MTT assay proved the viability of tested platelets, showing that the observed inhibition by the ECP protein does not occur simply by cell death. The EDN protein, cloned and expressed in eukaryotic system, also showed inhibitory effect on platelet aggregation in PRP, whereas the protein MBP had no effect significative inhibiting platelet aggregation. Our results indicate that eosinophils play a fundamental role in platelet aggregation inhibition / Doutorado / Farmacologia / Doutora em Farmacologia

Le parasitisme est un des principaux problèmes dans les élevages des ruminants. Les nématodes parasites du tractus digestif des ovins et caprins sont responsables d’importantes baisses de rendement. La maîtrise de ces parasitoses a été longtemps basée sur l’utilisation de molécules anthelminthiques. Cependant, l’efficacité des traitements est fréquemment remise en cause par l’émergence d’isolats résistants à une ou plusieurs de ces molécules. Dans ce contexte, une meilleure connaissance des mécanismes impliqués dans l’installation et la survie des parasites dans leur hôte est essentielle pour le développement de méthodes de lutte efficaces. Les P-glycoprotéines sont des pompes membranaires de la superfamille des transporteurs ABC. Ces pompes transportent des molécules très variées qui ont en commun leur caractère hydrophobe. Nous avons émis l’hypothèse de l’implication de ces transporteurs dans l’interaction hôte-parasite. Dans le contexte de ce travail nous avons identifié des séquences partielles ou complètes d’ADNc de 9 Pgps du nématode parasite Haemonchus contortus. Une forte activation des Pgps des nématodes en présence des produits de dégranulation des éosinophiles de l’hôte a été observée, démontrant ainsi l’interaction entre les Pgps des nématodes et les produits issus de l’hôte. De plus, l’exposition in vitro des nématodes parasites aux produits de l’hôte montrent après analyse par PCR quantitative une induction significative de l’expression de deux Pgps (Hco-pgp-3, et Hco-pgp-16). Chez le nématode murin Heligmosomoides polygyrus bakeri 5 Pgps ont été identifiés. D’autre part, l’analyse du niveau d’expression des Pgps d’H. bakeri a permis de montrer que le gène Hba-pgp-2 est exprimé uniquement chez les stades en contact avec les produits de l’hôte (oeufs, L4 et adultes). De plus, une induction spécifique d’Hba-pgp-2 par le cholestérol a été observée suggérant ainsi l’implication d’Hba-pgp-2 dans la capture et/ou la distribution des stérols des cellules de l’hôte indispensable aux nématodes. Ce travail constitue la première mise en évidence de l’interaction entre les Pgps des nématodes parasites et des produits issus de leur hôte. Ces résultats constituent une base solide pour le développement d’une méthode efficace permettant de bloquer ces transporteurs et d’éliminer les nématodes parasites. / Gastrointestinal nematodes cause significant economic loses in goat and sheep livestocks. Control of these parasites is mainly based on anthelmintic treatments. However, the efficacy of these molecules is questioned by the emergence of isolates resistant to one or several antiparasitic drugs. In this context, a better understanding of the mechanisms involved in the nematode parasites establishment and survival in the host is essential for the development of an effective control methods. P-glycoproteins are membrane pumps belonging to the ABC transporter family. These pumps transport a wide range of hydrophobic molecules. In the present study, we hypothesized that in addition to their critical role in xenobiotic resistance, helminth ABC transporters such as P-glycoproteins (Pgps) may also be involved in the transport of host products. Using the sheep parasitic nematode Haemonchus contortus, we investigated the modulation and expression of parasite Pgps activity in response to host eosinophil granule products. These works allowed to identify nine partial or complete H. contortus Pgps. Using a rhodamine efflux assay, we provided functional evidence that host eosinophil granule products can activate Pgps from the parasite suggesting that granule products could act as potential modulators of the ABC transporters activity. We showed by quantitative RT-PCR that among nine different H. contortus Pgp genes; Hco-pgp-3, Hco-pgp-9.2, Hco-pgp-11 and, Hco-pgp-16 were specifically up-regulated in parasitical life stages suggesting a potential involvement of these Pgps during the host-parasite interaction. Using exsheated L3 larvae, we demonstrated that eosinophil granules induced in a dose response manner an overexpression of Hco-pgp-3 and the closely related Hco-pgp-16 gene highlighting the possible involvement of these Pgps in host product transport. . The mice parasitic nematode Heligmosomoides polygyrus bakeri was used for studying the involvement of Pgps in the cholesterol transport. These works allowed identifying five Pgps in H. bakeri. The analysis of the mRNA expression level of H. bakeri Pgps has shown that Hba-pgp-2 gene is expressed only in stages in contact with host products. In addition, a specific induction of Hba-pgp-2 by cholesterol was observed suggesting the involvement of Hba-pgp-2 in the capture and / or distribution of cholesterol from host cells. Taken together, our results provide the first evidence that a subset of helminth Pgps could be involved in the transport of host products. This opens the way for further studies aiming to explore the function of helminth Pgps in host-parasite interactions including host immune response evasion.

Regulation of the inflammatory response is essential for the successful resolution of inflammation, and restoration of normal tissue homeostasis. Eosinophils are granulocytic cells of the innate immune system historically considered to be primarily involved in the defence against parasitic infection. Eosinophils are also key effector cells in the allergic inflammatory response, initiation of which is associated with the recruitment and activation of eosinophils culminating in the release of their intracellular granule contents. Eosinophil granules contain a range of cytotoxic proteins (major basic protein, eosinophil cationic protein and eosinophil peroxidase) that act to destroy infectious and parasitic organisms. However, these cytotoxic proteins can also cause damage to surrounding host tissue cells. The resolution of the inflammatory response acts to limit the extent of eosinophil-mediated tissue damage. Programmed cell death (apoptosis) of eosinophils represents an important component of this resolution process, limiting release of granule contents and triggering efferocytosis (the removal of apoptotic cells by phagocytes). Apoptosis is initiated by the activation of intracellular caspases, a family of cysteine proteases. Caspase activation primarily occurs as a result of changes in the balance of intracellular pro- and anti-apoptotic Bcl-2 family proteins. Mcl-1, an anti-apoptotic Bcl-2 protein has been shown to play a pivotal role in the regulation of neutrophil apoptosis. Pharmacological down-regulation of Mcl-1 initiates apoptosis and promotes the resolution of neutrophil-dominant inflammation. The importance of Mcl-1 in the regulation of apoptosis was shown using cyclin-dependent kinase inhibitors (CDKis), where induction of neutrophil apoptosis by CDKis was due to down-regulation of intracellular Mcl-1. Apoptotic cells display distinct surface molecules known as ‘eat-me’ signals that identify them for phagocytosis by macrophages and other phagocytes. One key receptor involved in the removal of apoptotic cells from tissue is the receptor tyrosine kinase Mer, a member of the Tyro3/Axl/Mer (TAM) family, which recognises the ‘eat me’ signal phosphatidylserine expressed on apoptotic cells. In the absence of Mer expression, clearance of apoptotic cells is compromised delaying the resolution of neutrophil-dominant inflammation. However, the roles of Mcl-1 and Mer in eosinophil apoptosis and clearance, respectively, and the resolution of allergic inflammation are not known. Asthma is a chronic inflammatory lung disease characterised by shortness of breath, airway obstruction, wheeze, non-specific bronchial hyper-responsiveness, excessive airway mucus production and an eosinophil dominant inflammatory infiltrate. The persistent presence of eosinophils in the lung, in chronic asthma, is likely due to a combination of excessive eosinophil recruitment and activation together with impaired eosinophil apoptosis. Investigation into the underlying mechanisms of these processes in allergic airway disease is of critical importance, as blocking eosinophil recruitment and/or promoting eosinophil apoptosis could provide a therapeutic approach to reduce associated eosinophil-mediated tissue damage. Understanding the regulation of eosinophil apoptosis and phagocytic clearance may identify novel pharmacological targets to enhance the resolution of allergic inflammation. We hypothesise that Mcl-1 and Mer play vital roles in the successful resolution of allergic airway inflammation. To investigate this hypothesis, we have used pharmacological and genetic manipulation of intracellular eosinophil Mcl-1 levels, and phagocyte Mer expression, to determine the role they play in the regulation of eosinophil apoptosis and phagocytic clearance of apoptotic eosinophils, respectively. Human and mouse eosinophils were cultured, and rates of constitutive and CDKi-induced apoptosis were determined, to investigate eosinophil apoptosis in vitro. Mice expressing human Mcl-1 (hMcl-1) were used to determine the effect of over-expression of Mcl-1 on eosinophil viability in vitro. The effect of hMcl-1 on eosinophil viability and disease severity in vivo was determined using an ovalbumin-induced model of allergic airway inflammation, which mimicked the symptoms of human asthma. Apoptotic eosinophils were co-incubated with macrophages in vitro to investigate the capacity for phagocytosis by different macrophage populations. Apoptotic cell clearance was further investigated using a Mer-kinase-dead mouse, which lacked Mer expression, to determine the role of Mer-dependent phagocytosis on the process of resolution of inflammation in vivo. Over-expression of Mcl-1 in eosinophils significantly delayed both constitutive and CDKi-induced apoptosis in vitro. In vivo in the ovalbumin-induced model of allergic airway inflammation, over-expression of Mcl-1 resulted in a significantly increased number of eosinophils in the lung and delayed rate of resolution of allergic airway inflammation. Alveolar and bone marrow-derived macrophages exhibited Mer-dependent phagocytosis of eosinophils, which was significantly reduced by an inhibitor of Mer kinase activity (BMS777607) or lack of macrophage Mer expression. The absence of Mer expression resulted in a significant increase in the number of apoptotic eosinophils in the lung together with a delayed rate of resolution of allergic airway inflammation in vivo. Together this work has shown that delayed rates of eosinophil apoptosis and impaired phagocytic clearance both delayed the resolution of allergic airway inflammation. These data suggest that both Mcl-1 and Mer are pivotal for the successful regulation of eosinophil apoptosis and phagocytic clearance of apoptotic eosinophils in asthma and may provide attractive novel therapeutic targets.

Myocardial infarction (MI) occurs following acute thrombotic occlusion of a coronary artery, and triggers a robust inflammatory response. Within hours, neutrophils are recruited to the infarcted myocardium followed by the infiltration of pro-inflammatory Ly6Chi monocytes. Transition from the pro-inflammatory macrophage phenotype (M1) to an anti-inflammatory, pro-resolution phenotype (M2-like) is critical to successful infarct healing. Interventions that polarize macrophages towards an anti-inflammatory 'M2-like' phenotype improve infarct healing in the experimental MI mouse model and reduce subsequent adverse remodelling of the myocardium, but the endogenous mechanisms that regulate repair are not well understood. Furthermore, differences in the resolution of inflammation in C57BL/6 and BALB/c mice, which are two of the commonly used wild-type mouse strains in experimental MI have not been characterised. We previously found that low peripheral blood eosinophil count is associated with increased short-term risk of mortality in low-intermediate risk patients with ischaemic heart disease. This suggests that eosinophils may have a role in the successful remodelling and repair of the heart following myocardial infarction. Eosinophils express a number of immuno-modulating cytokines and lipid mediators implicated in the resolution of inflammation. Increasingly prominent is interleukin-4 (IL-4), a cytokine that has been found to maintain the anti-inflammatory M2-like phenotype in macrophages. We therefore hypothesised that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are key in regulating the subsequent inflammatory response and scar tissue formation during infarct repair and cardiac remodelling. Experimental MI was induced by permanent left anterior descending artery ligation in isofluorane anaesthetized 12-15 week-old male wild-type (WT) BALB/c, WT C57BL/6, IL4Rα-/-, IL-4Rαflox/-, IL-4Rαflox/-LysMCre mice and eosinophil-deficient ΔdblGATA mice. Cardiac function was characterised by high-resolution ultrasound and immune cell infiltration by flow cytometry of single cell infarct and remote zone tissue digests. Blood eosinophil count and 6-month all-cause mortality were assessed in 732 consecutive patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction (STEMI). The rate of mortality due to cardiac rupture was significantly higher in C57BL/6 mice in comparison with BALB/c mice at Day 7 post-MI. This was associated with a higher proportion of pro-inflammatory Ly-6Chi macrophages infiltrating the infarct zone tissue of C57BL/6 mice following MI. An accompanying reduction in the number of splenic Ly-6Chi monocytes post-MI, suggestive of splenic monocyte mobilisation, was seen in C57BL/6 mice but not found in BALB/c mice. Furthermore, C57BL/6 mice had a delayed transition in macrophage polarisation towards an anti-inflammatory phenotype. Disruption of IL4Rα signalling, in mice null for the IL4Rα gene, resulted in increased F4/80+ macrophage and pro-inflammatory Ly6Chi macrophage infiltration of the infarct zone and reduced expression of the anti-inflammatory macrophage marker CD206, compared to wild-type controls. Furthermore, expression of GATA3 and ST2, both associated with the immunosuppressive function of (CD4+ Foxp3+) regulatory T cells, was reduced in infarct zone regulatory T cells from IL4Rα-/- mice. These findings were associated with defective wound healing with impaired angiogenesis, increased scar size, disarrayed infarct zone collagen deposition, accompanied by modified expression of plod2 that encodes the collagen cross-linking enzyme lysyl hydroxylase 2. Resulting in greater left ventricular dilatation and loss of cardiac function, as well as a higher 7- day mortality due to cardiac rupture in IL4Rα-/- mice. This indicates that successful infarct repair requires the engagement of IL-4Rα signalling to facilitate the accumulation of anti-inflammatory macrophages and highly immunosuppressive ST2+ regulatory T cells in the heart following MI. Resident cardiac macrophages from naïve hearts of IL-4Rαflox/-LysMCre mice failed to undergo LysMCre-mediated deletion of the IL-4Rα gene, potentially because low or absent expression of Lyz2 (encoding lysozyme M). In both ST-elevation MI (STEMI) patients and mice after acute MI, there was a decline in peripheral blood eosinophil count, with activated eosinophils being recruited to the infarct zone and paracardial adipose tissue of mice. The transcription factors GATA-1 plays a role in the differentiation of eosinophils from eosinophil progenitor cells. Deletion of GATA-1 results in loss of the eosinophil lineage and has been exploited to develop the eosinophil-deficient ΔdblGATA mouse. ΔdblGATA mice were used to address the role of eosinophils in cardiac remodelling following MI. ΔdblGATA mice had increased left ventricular dilatation and reduced ejection fraction after induction of MI, relative to wild-type mice. ΔdblGATA mice had increased scar size with disarrayed infarct zone collagen deposition, accompanied by modified expression of the genes plod2 and lox, which are associated with collagen cross-linking. The proportion of CD206+ anti-inflammatory macrophages was less in the infarct zone of ΔdblGATA mice, but was restored by adoptive transfer of eosinophils from WT mice. Furthermore, adverse cardiac remodelling in eosinophil-deficient ΔdblGATA mice was rescued by provision of IL-4 complex following MI. In conclusion, an enhanced inflammatory response following MI underlies the increased risk of cardiac rupture seen with WT C57BL/6 mice in comparison to WT BALB/c mice. WT BALB/c mice are protected from cardiac rupture, which was associated with an absence of splenic monocyte mobilisation following ischaemic injury. The resolution of inflammation was found to be dependent on IL4Rα signalling which is crucial for cardiac repair and remodelling, through modulating inflammatory cell recruitment and phenotype, as well as scar formation. Eosinophils are recruited to the heart post-MI and are essential for regulating cardiac repair and remodelling, likely through provision of IL-4. Therefore, we were able to show that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are both key in regulating the subsequent inflammatory response and scar tissue formation during infarct healing and cardiac remodelling.

Approximately two billion people are infected with helminths worldwide. In order to develop a vaccine against these pathogens, more needs to be known about the immune response to helminths. Eosinophils are important for resistance to some helminth species and their recruitment to infected tissues, attachment to parasites and degranulation may all be critical processes for immunity. Complement may contribute to these processes via generation of chemotactic factors (C3a and C5a) or opsonisation of the parasite with C3b/iC3b. The importance of complement during helminth infection is unclear, though complement does promote leukocyte-mediated killing of several helminth species in vitro. The aim of the present study was to investigate the role of complement in immunity of mice to Nippostrongylus brasiliensis, with a focus on whether complement facilitates eosinophildependent resistance to this parasite. A new fluorescence-based method for quantifying in vitro complement deposition and leukocyte adherence on N. brasiliensis was developed. C3 from human serum was deposited on infective-stage L3 via the classical or lectin complement pathways. In contrast, the alternative complement pathway mediated binding of mouse C3 and eosinophil-rich mouse peritoneal leukocytes to L3. Interestingly, the ability of complement and leukocytes to bind to the parasite changed as it matured. Larvae recovered from the skin 30 min post-injection (p.i.) were coated with C3, however those harvested 150 min p.i. exhibited reduced C3 binding capacity. Binding of C3 and eosinophils to larvae recovered from the lungs 24-48 h p.i. (L4) was also diminished compared to that seen on L3. Adult intestinal worms bound C3 and leukocytes only when treated ex vivo with serum and cells. Mice lacking in classical (C1q-deficient), alternative (factor B-deficient) or all complement pathways (C3-deficient) were then employed to determine if complement was important for resistance of mice to N. brasiliensis. IL-5 Tg mice deficient in individual complement genes were generated to assess whether complement contributed to eosinophildependent resistance to the parasite. Factor B deficient mice exhibited impaired C3 deposition on larvae, eosinophil recruitment, eosinophil degranulation and larval aggregation in the skin 30 min p.i. Eosinophil recruitment was similarly abolished by treatment of mice with the C5aR inhibitor PMX53. However at 150 min p.i., larval aggregation, eosinophil and neutrophil recruitment, leukocyte adherence and eosinophil degranulation were largely complement-independent. Ablation of factor B or C3 caused minor but significant increases in lung-larval burden during primary, but not in secondary, infections. Critically, a lack of C3 or factor B in IL-5 Tg mice failed to greatly impair the strong innate anti-parasite resistance typical of these animals, suggesting that eosinophils can provide immunity to N. brasiliensis infection in the absence of complement. This was unexpected, given the evidence from this and previous studies which suggested that in vitro, complement is important for promoting eosinophil-dependent killing of N. brasiliensis and other helminth species. The mechanism(s) by which eosinophils kill N. brasiliensis remain unknown, but may involve the coordination of the complement system with complement-independent factors that act in the early stages of infection. Critically, the influence of complement is limited, because soon after entry into the host, the parasite develops the ability to resist complement activation. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311182 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Airway epithelial damage is commonly found in asthma patients. Epithelial damage was investigated with special reference to contacts between epithelial cells. Eosinophils, common in allergic asthma, secrete cationic proteins, particularly major basic protein (MBP). The effect of poly-L-arginine, an analogue of MBP, on airway epithelial cells was investigated. Poly-L-arginine induced membrane damage, resulting in increased permeability, loss of cell-cell contracts (tight junctions and desmosomes) and generalized cell damage. Adhesion molecules on airway epithelial cells may be important in recruiting leukocytes. Interferon (IFN)-γ increased intracellular adhesion molecule-1 expression in airway epithelial cell lines. A combination of interleukin-4 and IFN-γ opened the tight junctions. Epithelial damage in asthma was studied at the ultrastructural level in bronchial biopsies from patients with atopic or non-atopic asthma, and healthy controls. Epithelial damage was extensive in both asthma groups. In basal and columnar cells, relative desmosome length was reduced by 30-40%. In columnar cells, half-desmosomes were noticed. Changes tended to be more extensive in atopic asthma, but there was no significant difference between the two groups. Reduced desmosomal contact may be important in the epithelial shedding observed in asthma. The contact area between columnar cells and basal lamina is relatively small in the human airway. Attachment of columnar cells to the basal lamina occurs indirectly, via desmosomal attachment to basal cells. Direct attachment of columnar cells to the basal lamina is weakened in asthmatics. Nasal polyposis is a chronic inflammatory disease often associated with asthma. An ultrastructural study showed that epithelial damage of columnar cells is more pronounced in allergic patients. The length of columnar cell desmosomes was significantly reduced in asthmatics vs. non-asthmatics, and in allergics vs. non-allergics. Cell contacts in airway epithelium in asthmatics are weakened, which may be an intrinsic feature or due to the presence of eosinophils producing toxic proteins.