Mast cells play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow-derived progenitors that circulate in the blood as immature precursors. Mast cell progenitors are likely to encounter the serum-borne bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), during migration to target tissues. Mast cells developed from human cord blood-derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MCT), the phenotype predominant in the lung. S1P accelerated the development of cord blood-derived mast cells (CB-MCs) and strikingly increased the numbers of mast cells expressing chymase. These mast cells have functional FcepsilonRI, and similar to skin mast cells that express both tryptase and chymase (MCTC), also express CD88, the receptor for C5a, and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL-6, a cytokine known to promote development of functionally mature MCTC, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage-depleted CB-MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP-1/CCL2), from these macrophage-depleted and purified CB-MCs.

Occupational exposure to endotoxin-rich swine barn air induces respiratory diseases and loss of lung function. Barn exposure induces recruitment of pulmonary intravascular monocytes/macrophages (PIMMs) and subsequent increased host sensitivity to <i>Escherichia coli</i> LPS challenge. Therefore, to further clarify the biology of PIMMs we examined the role of recruited PIMMs in a rat <i>Escherichia coli</i>-induced lung inflammation model. Following sepsis, lung inflammation was induced with recruitment of PIMMs and subsequently, <i>Escherichia coli</i> LPS challenge exacerbated the lung inflammation with localization of multiple inflammatory cytokines in PIMMs to suggest their possible involvement in modulating lung inflammation in this model.<p> In order to delineate mechanisms of barn air induced lung dysfunction, a rat model of occupational exposure was characterized to show that one and five exposures to the barn environment induced acute lung inflammation and increased airway hyperresponsiveness (AHR). Following 20 exposures, AHR was dampened to indicate adaptive responses. Barn air contains high levels of endotoxin which led us to investigate its role in lung inflammation and AHR. Exposure of mice with either a functional TLR4 (WT) or non-functional TLR4 (mutants) to barn air revealed dependence of lung inflammation but not AHR on a functional TLR4.<p> I investigated whether exposure to barn air alters host responses to a subsequent microbial challenge. Following one day barn exposure and <i>Escherichia coli</i> LPS challenge, lung inflammation was exacerbated with increased granulocytes and IL-1β levels compared to one day barn exposed rats without <i>Escherichia coli</i> LPS challenge. However, increased granulocytes and IL-1β levels in barn exposed and <i>Escherichia coli</i> LPS challenged rats were not different from control rats treated with <i>Escherichia coli</i> LPS indicating a lack of priming effect of barn exposure. However, above results are suggestive of an underlying risk of increased lung inflammation following secondary microbial infection in naïve barn workers.<p> Lastly, I investigated the expression and activity of novel signalling molecules called <i>N</i>-myristoyltransferase and calcineurin in barn air and <i>E. coli</i> LPS induced lung inflammation models. Following one day barn exposure, increased protein expression but not activity of <i>N</i>-myristoyltransferase and calcineurin was shown. However, there is a need to identify the specific role of these two molecules in barn air induced lung inflammation. To conclude, animal models of barn exposure are useful tools to understand mechanisms of lung inflammation and AHR. However, there is still a need to examine endotoxin-independent nature of AHR and roles of other molecules of the innate immune system in regulating barn air induced effects.

Occupational exposure to endotoxin-rich swine barn air induces respiratory diseases and loss of lung function. Barn exposure induces recruitment of pulmonary intravascular monocytes/macrophages (PIMMs) and subsequent increased host sensitivity to <i>Escherichia coli</i> LPS challenge. Therefore, to further clarify the biology of PIMMs we examined the role of recruited PIMMs in a rat <i>Escherichia coli</i>-induced lung inflammation model. Following sepsis, lung inflammation was induced with recruitment of PIMMs and subsequently, <i>Escherichia coli</i> LPS challenge exacerbated the lung inflammation with localization of multiple inflammatory cytokines in PIMMs to suggest their possible involvement in modulating lung inflammation in this model.<p> In order to delineate mechanisms of barn air induced lung dysfunction, a rat model of occupational exposure was characterized to show that one and five exposures to the barn environment induced acute lung inflammation and increased airway hyperresponsiveness (AHR). Following 20 exposures, AHR was dampened to indicate adaptive responses. Barn air contains high levels of endotoxin which led us to investigate its role in lung inflammation and AHR. Exposure of mice with either a functional TLR4 (WT) or non-functional TLR4 (mutants) to barn air revealed dependence of lung inflammation but not AHR on a functional TLR4.<p> I investigated whether exposure to barn air alters host responses to a subsequent microbial challenge. Following one day barn exposure and <i>Escherichia coli</i> LPS challenge, lung inflammation was exacerbated with increased granulocytes and IL-1β levels compared to one day barn exposed rats without <i>Escherichia coli</i> LPS challenge. However, increased granulocytes and IL-1β levels in barn exposed and <i>Escherichia coli</i> LPS challenged rats were not different from control rats treated with <i>Escherichia coli</i> LPS indicating a lack of priming effect of barn exposure. However, above results are suggestive of an underlying risk of increased lung inflammation following secondary microbial infection in naïve barn workers.<p> Lastly, I investigated the expression and activity of novel signalling molecules called <i>N</i>-myristoyltransferase and calcineurin in barn air and <i>E. coli</i> LPS induced lung inflammation models. Following one day barn exposure, increased protein expression but not activity of <i>N</i>-myristoyltransferase and calcineurin was shown. However, there is a need to identify the specific role of these two molecules in barn air induced lung inflammation. To conclude, animal models of barn exposure are useful tools to understand mechanisms of lung inflammation and AHR. However, there is still a need to examine endotoxin-independent nature of AHR and roles of other molecules of the innate immune system in regulating barn air induced effects.

Background: According to recent studies in children, IgE sensitization not only against perennial allergens, but also against food allergens, is related to asthma risk and increased airway inflammation. During the last decade, a new technique for IgE determination based on allergen components has become available, but its use in epidemiological studies has been limited. Aims: To investigate the relationship between the pattern of IgE sensitization to allergen components and the prevalence of asthma, airway inflammation and hyperresponsiveness in a population-based setting. To examine the relationship of IgE sensitization to allergen extract, and airway inflammation, airway hyperresponsiveness and blood eosinophilia in asthmatics. To examine the natural history of IgE sensitization to food allergens in adults. To compare extract-based and component-based IgE measurements in relation with new-onset respiratory disease and airway inflammation and hyperresponsiveness. Methods: The present thesis is based on cross-sectional and longitudinal analyses of the adult, the population-based study ECRHS (European Community Health Survey) and a cross-sectional, observational study of young subjects with asthma. IgE sensitization was examined by means of both extract-based and component-based tests. Airway inflammation was assessed by exhaled NO and airway hyperresponsiveness with methacholine test. Results: IgE sensitization to food allergens independently related to increased airway inflammation in both a population-based study and a study of asthmatics. Furthermore, a relation was found with increased blood eosinophils in asthmatics. The decrease in prevalence of IgE sensitization against food allergens during a 9-year follow-up was larger than the decrease of aeroallergens. Subjects with IgE sensitization to both cat extract and components showed more frequent airway inflammation, greater bronchial responsiveness and higher likelihood of developing asthma and rhinitis than subjects with IgE sensitization only to cat extract. Conclusions: The presence of IgE antibodies against food allergens was independently associated with airway and systemic inflammation. Both aeroallergens and food allergens should be examined in order to understand the signaling of local and systemic inflammation in asthma. Prevalence of IgE sensitization to food decreased in adults to a larger extent than IgE sensitization against aeroallergens. Measurement of IgE sensitization to cat allergen components appears to have a higher clinical value than extract-based measurement

<p>Asthma is a disease characterized by transient airway smooth muscle contraction leading to episodes of reversible airway narrowing. It affects over 300 million people worldwide and is implicated in over 250 000 deaths annually. The primary clinical features of asthma include airway inflammation, hyperresponsiveness, and remodeling. Generally, asthmatic patients experience exacerbations between periods of diminished symptoms. Interestingly, in addition to these above mentioned hallmarks, asthmatics have also been shown to react differently to ventilatory mechanical strain. This is most evident when assessing the effect of a deep inspiration (DI), clinically measured as a breath taken from functional residual capacity to total lung capacity, in healthy individuals <em>versus</em> asthmatics. These deep inspiratory efforts have been shown to produce a bronchodilatory response in healthy individuals, whereas in asthmatics, DIs are less effective in producing bronchodilation, can cause more rapid airway re-narrowing, and even bronchoconstriction in moderate to severe asthmatics. The mechanism by which a DI is able to cause bronchoconstriction remains ambiguous. Previous theories suggest that this phenomenon is intrinsic to airway smooth muscle (ASM) itself. However, the airway inflammation present in asthmatic airways may also add to the increased ASM contractility following stretch, by the release of mediators that can prime the contractile apparatus to react excessively in the presence of stretch.</p> <p>Thus, collectively, the studies contained in this thesis are linked to the general theme of greater characterization of the signalling mechanisms that regulate airway stretch-activated contractions using a pharmacological approach in intact bovine bronchial segments, with the hope of providing novel insights into the mechanisms that regulate the DI-induced bronchoconstriction seen in asthmatics.</p> / Doctor of Philosophy (Medical Science)

<p><strong>Introduction</strong></p> <p>Asthma is a chronic respiratory disease that is becoming more prevalent. Airway hyperresponsivness, a key feature of asthma, involves increased narrowing of the airways in response to bronchoconstricting agents. Airway smooth muscle (ASM) functioning is largely responsible for hyperresponsiveness yet the mechanisms behind excitation-contraction coupling are not fully understood. Src tyrosine kinase contributes to contraction in other smooth muscle types. Furthermore, STIM1, Orai1, IPLA₂b and RyRs play a role in ASM excitation-contraction coupling.</p> <p><strong>Aim</strong></p> <p>We sought to determine whether Src activity is involved in serotonin (5-HT)- and acetylcholine (ACh)-induced ASM contraction. We also examined whether the gene expression of molecules involved in sarcoplasmic reticulum emptying and refilling is altered during airway hyperresponsiveness.</p> <p><strong>Methods</strong></p> <p>Bovine tracheal ASM strips were pre-treated with the non-specific tyrosine kinase inhibitor genistein (10^-4M), src kinase family inhibitors PP1 (10^-5M) and PP2 (10^-5M) or vehicle and challenged with either 5-HT or ACh to determine the involvment of Src in contraction. Western blotting was used to examine Src activity following 5-HT or ACh treatment. Female BALB/c mice were exposed to an intranasal injection of [1.7mg/ml] HDM extract or saline. Real time, reverse-transcriptase polymerase chain reaction was used to examine gene expression.</p> <p><strong> </strong></p> <p><strong>Results</strong></p> <p>Genistein, PP1 and PP2 significantly reduced 5-HT-induced ASM contractions and Src activity was significantly increased in response to 5-HT. ACh-induced contractions were significantly reduced by genistein, but not PP1 and PP2. However, Src activity was significantly increased by ACh. RyR3 mRNA expression was significantly increased, Orai1 was significantly decreased, and STIM1, IPLA₂b, RyR1 and RyR2 were unchanged by the house dust mite treatment.</p> <p><strong>Conclusion</strong></p> <p>These data suggets 5-HT-induced ASM contraction involves Src activity. However, ACh-induced ASM contractions might not require Src. The changes in RyR3 and Orai1 expression might alter Ca²⁺-handling in such a way as to potentiate airway hyperresponsiveness but further investigation is required.</p> / Master of Science (MSc)

Doctor of Philosophy (PhD) / Asthma is a chronic inflammatory disease of the airways. Corticosteroid medication is the most effective currently available treatment. Complications of corticosteroid therapy are dose-dependent, however, the clinical efficacy of varying doses of inhaled corticosteroids has been studied with mixed results. A randomized, double-blind, parallel group study was used to evaluate the inhaled corticosteroid dose-response relationship for clinical endpoints and in vitro parameters of underlying airway inflammation and remodelling. The mannitol provocation test with Forced Oscillation Technique (FOT) was used to derive potential dose-differentiating endpoints. In vitro inflammatory markers were measured in alveolar macrophages from bronchoalveolar lavage. Basement membrane thickness was measured from bronchial biopsies. Eleven nonasthmatic subjects were enrolled for comparison. This thesis addresses the null hypothesis that there is no significant difference in clinical and biological effects between low dose (200mcg/day, n=11) and high dose (1000mcg/day, n=11) treatment (for 6-7 weeks) with inhaled fluticasone propionate (FP) for a range of clinical outcomes and in vitro markers of airway inflammation and remodelling. Significant changes after FP included increased FEV1, reduced airway hyperresponsiveness (AHR) (by FOT and FEV1), exhaled nitric oxide and Juniper symptom score. In addition, significant reductions occurred in expression of GM-CSF, TNF-alpha and IL-1ra in macrophages. A lower baseline FOT-derived respiratory system conductance was predictive of a greater degree of improvement in symptoms. No statistically significant differences in the changes after treatment between low and high dose FP were found in spirometry, exhaled nitric oxide, symptom scores, AHR, alveolar macrophage cytokine levels (GM-CSF, TNF-alpha, IL-1ra, IL-10) and basement membrane thickness, although there were trends towards greater improvements in many of the parameters after high dose FP. Basement membrane thickness appeared to be reduced by high dose FP, although this reduction was not statistically significant. There was a weak, but statistically significant, negative correlation between basement membrane thickness and FOT-derived conductance (r2=0.135, p=0.042). With the recognition of the limitations in the interpretation of these data, the results suggest that, in previously steroid naïve mild to moderate asthmatics, there may be only minimal benefit derived from an additional 800µg/day of inhaled fluticasone above the low dose of 200µg/day.

Typ 2-cytokiner, IL-4 och IL-13 är kända för att spela en viktig roll i airway hyperresponsiveness (AHR) eller luftvägshyperreaktivitet, där de glatta muskelcellerna (ASM) drar ihop sig för lätt och för mycket som svar på direkta eller indirekta stimuli. Detta gör AHR till en avgörande egenskap hos astmatiker. Det har gjorts studier som har visat involvering av typ 2-cytokiner i AHR, men deras specifika inflammatoriska mekanismer är ännu outforskade. Denna experimentella studie på glatta muskelceller från luftvägarna (ASM) syftade till att undersöka involveringen av typ 2-cytokin inducerad kontraktilitet genom att fokusera på receptoraktivering, receptoruttryck samt ge insikter om frisättning av proteiner relaterade till luftvägsfibros, så kallad remodelling. Dessutom studerar den också effekten av IL- 4/IL-13 receptor antagonisten Dupilumab (anti-IL4Ra) på cytokinbehandlade HASM. Efter stimulering av HASM med IL-13 under 24 timmar visade resultaten att IL-13 orsakar en ospecifik, icke-receptormedierad ökning av intracellulärt kalciumflöde som svar på kalciumjonoforen A23187. Detta skulle potentiellt kunna öka kontraktiliteten hos glatta muskelceller som svar på flera olika kontraktila stimuli. Denna forskning ger också preliminära resultat som tyder på att IL-13 och IL-4 också ökar kalciumflödet som svar på aktivering av receptorer för specifika kontraktila mediatorer (Histamin, Carbachol, Leukotriene D4 och Substance P), och att effekten förmedlas via IL-4/IL-13-receptorn vilket blockeras med dupilumab som verkade minska effekten. Närvaron av fler receptorer för kontraktila mediatorer kan också öka kontraktiliteten hos glatta muskelceller som svar på IL-4 och IL-13. Återigen sågs ökat uttryck av receptorer för kontraktila stimuli efter behandling med cytokinerna som inhiberades av dupilumab. Dessutom undersökte vi effekten av IL-13 och IL-4 på frisättning av prokollagen 1 (en prekursor för mogna kollagena former) från mänskliga glatta muskelceller i luftvägarna. Våra resultat visade inte några signifikanta effekter på frisättningen av just detta kollagenprotein. Sammantaget visar vi att typ 2 cytokiner kan på flera olika sätt öka kontraktiliteten av glatta muskelceller vilket ökar vår kunskap om mekanismerna som orsakar luftvägshyperreaktivitet hos astmatiker. / Type 2 cytokines, IL-4 and IL-13 are known to play an essential role in airway hyperresponsiveness (AHR) - in response to direct or indirect stimulus the smooth muscle cells (ASM) contract too easily and too heavily. This makes AHR a defining feature of asthma. There have been studies that have demonstrated involvement of type 2 cytokines in AHR. However, the specific mechanisms involved remain undefined. This experimental study in airway smooth muscle (ASM) was aimed to investigate the involvement of type 2 cytokine on AHR by focusing on the expression and activation of receptors for contractile mediators as well as provide insights on the release of proteins related to airway remodelling. Experiments were performed where human airway smooth muscle cells were treated with IL-4 and/or IL-13, with or without Dupilumab, an antagonist of the joint IL-4/IL-13 receptor (anti-IL4Ra). After stimulating HASMs with IL-13 for 24 hours, results showed that IL-13 caused a non-specific, non-receptor-mediated increase in intracellular calcium flux in response to the calcium ionophore A23187. This could potentially increase the contractility of smooth muscle cells in response to any contractile stimulus. This study also suggests that IL-13 and IL-4 increased calcium flux in response to activation of receptors for specific contractile mediators (Histamine, Carbachol, Leukotriene D4 and Substance P). The mechanism involved likely involves the common IL-4/IL-13 receptor as blocking this with dupilumab seemed to reduce the effect. The presence of more receptors for contractile mediators could also increase contractility of smooth muscle cells in response to IL-4 and IL-13. Preliminary results show that mRNA expression of receptors for Histamine (H1) and LTD4 (CYSLT1) were upregulated by type 2 cytokines and again, this upregulation appeared to be inhibited by dupilumab. Moreover, we examined the effect of IL-13 and IL-4 on release of procollagen 1 (a precursor of mature collagen forms) from human airway smooth muscle cells. Our results did not show any significant effects on the release of this particular collagen protein. Taken together these findings increase our understanding of the mechanisms whereby type 2 cytokines may increase the contractility of airway smooth muscle and provide a basis for follow-up investigations. Improved knowledge of the mechanisms underlying AHR could ultimately lead to improved treatment of asthma.