Eosinophil granulocytes are tissue dwelling leukocytes that are implicated in host defence, particularly against helminthic parasites; they also participate in most inflammatory disorders. Although eosinophils have important roles in host defence mechanisms, their actions can also be harmful to the host as in the allergic inflammation where lung epithelium is destructed due to the release of toxic granule proteins. The focus of the present thesis work has been to characterize the molecular and functional heterogeneity of the granule protein Eosinophil Cationic Protein (ECP). We investigated a coding ECP gene polymorphism (arg97thr) in an African population endemically exposed to the Schistosoma mansoni parasite and found a correlation between ECP genotype and disease manifestations; ECP97arg was more effective in terms of host defence against the parasite, but was also correlated to development of liver fibrosis in infected subjects. We purified ECP97arg and ECP97thr from healthy blood donors and showed that they differ in their cytotoxic activities; ECP97arg was cytotoxic whereas ECP97thr was non-cytotoxic. They did not differ in terms of RNase activity or in their ability to stimulate fibroblast-mediated collagen gel contraction. We developed a new SELDI-TOF MS assay to enable the study of the structure of ECP in more detail and showed that ECP is produced in several glycosylated forms, and that the degree of glycosylation determines the cytotoxic activity. Enzymatic deglycosylation significantly enhanced the cytotoxic activity of highly glycosylated ECP-variants. To summarize, in this thesis we demonstrated that the cytotoxic activity of ECP is dependent on both a gene polymorphism and post-translational modifications, and that the cytotoxic activity is distinct from other functions of ECP. We speculate that ECP is synthesised in heavily glycosylated variants as a means to protect the host from its harmful effects and that ECP is activated by deglycosylation when required at the site of inflammation.

Allergic diseases are chronic inflammatory conditions, characterised by eosinophil inflammation systemically and in target organs, where cytotoxic granule proteins are responsible for tissue injury. Allergic rhinitis is known to be a risk factor for the development of asthma, yet not all with rhinitis develop asthma. The overall aim was to investigate the involvement of eosinophils in allergic rhinitis and allergic asthma in vivo and in experimental settings, with a focus on differences between rhinitis and asthma. Birch pollen allergy was used as a model and patients were studied during pollen season and after nasal and bronchial allergen challenge. During pollen season and at baseline, allergic rhinitis and allergic asthma had the same degree of systemic eosinophil inflammation. Despite this, impairment in lung function during season and increased bronchial responsiveness at baseline were more common in the asthmatics. Systemic inflammation was more pronounced after seasonal exposure than after experimental challenge. Allergic rhinitis and allergic asthma had the same degree of eosinophil airway inflammation after bronchial challenge, but only the asthmatics had increased bronchial responsiveness measured as PD20 for birch allergen. Allergen primed eosinophils were investigated in vitro for C3b-induced degranulation after seasonal and experimental challenge. The released amount of eosinophil granule proteins was within the same range for all three allergen challenge models with just minor differences in propensity for degranulation between rhinitics and asthmatics. Signalling through PI3K for degranulation was studied with the specific inhibitor Wortmannin. PI3K signalling for eosinophil degranulation was clearly involved in allergic rhinitis and allergic asthma irrespective of the model for allergen exposure. Asthmatics demonstrated less inhibition of degranulation through PI3K during pollen season, indicating that other pathways contribute to eosinophil degranulation in allergic asthmatics. Conclusion: Allergic rhinitis and allergic asthma present with the same degree of systemic and local eosinophil inflammation. The eosinophils are primed for degranulation equally and follow the same pathway through PI3K for degranulation. Our data indicates that eosinophil inflammation per se is not sufficient for the development of asthma.

La liaison de CXCL12 à ses récepteurs CXCR4 et CXCR7 peut-être bloquée par un neutraligand de CXCL12, la chalcone 4, qui présente une activité anti-inflammatoire dans un modèle d’asthme chez la souris. Notre travail a consisté à proposer des stratégies permettant le développement de molécules biodisponibles et actives localement, et d’étudier le mécanisme d'action in vivo des neutraligands de CXCL12. Nous avons développé un modèle court et reproductible d'asthme allergique chez la souris, adapté à une évaluation rapide de l’activité anti-inflammatoire des nouveaux composés et un développement raisonné des stratégies envisagées. Nous avons synthétisé trois prodrogues solubles, adaptées à une administration locale, inactives et rapidement clivées en chalcone 4 active. In vivo, les prodrogues sont anti-inflammatoires à des doses susceptibles de limiter les effets indésirables. Pour favoriser davantage l'action anti-inflammatoire locale du neutraligand de CXCL12, nous avons synthétisé une ante-drogue, la carbonitrile-chalcone 4, active in vivo par administration locale et rapidement dégradée en deux composés inactifs avant sa distribution dans l'organisme. Enfin, l'étude de la chalcone 4 a mis en évidence une activité antiasthmatique significative, liée à l'élimination rapide de la chimiokine CXCL12 du poumon. La capture de CXCL12 par le neutraligand réduit la différenciation des macrophages M1 et leur libération de cytokines pro-inflammatoires, ainsi que le recrutement des éosinophiles et des lymphocytes CXCR4+. En conclusion, nos travaux ont permis de conforter les rôles de la chimiokine CXCL12 dans l'asthme et présenter deux stratégies aptes à limiter les effets indésirables. / The binding of the chemokine CXCL12 to its receptors CXCR4 and CXCR7 may be prevent by a CXCL12 neutraligand, chalcone 4, having an anti -inflammatory activity in a mouse model of allergic asthma. Our work consisted in proposing strategies for the development of active and bioavailable molecules, able to promote local action, and to study the in vivo mechanism of action of the CXCL12 neutraligands. We initially developed a short and reproducible mouse model of allergic asthma, suitable for a rapid assessment of the anti -inflammatory activity of new compounds, in order to ensure rational development of the proposed strategies. We developped three soluble prodrugs, adapted to local administration, inactive but rapidly cleaved in active chalcone 4. In vivo, the prodrugs have an anti-inflammatory activity at suitable doses to minimize side effects. To promote the benefit of local anti-inflammatory action of CXCL12 neutraligand, we synthesized an antedrug, carbonitrile-chalcone 4, active locally in vivo after local administration and rapidly degraded before its distribution in the body.Finally, the study of chalcone 4 allowed us to highlight a significant asthma activity. An activity related to the rapid elimination of the CXCL12 chemokine from the lung. The trapping of CXCL12 by the neutraligand reduced M1 macrophage activation and their release of pro inflammatory cytokines, as decreases the recruitment of CXCR4+ eosinophils and lymphocytes. In conclusion, our work provided mechanistic elements related to the roles of the chemokine CXCL12 in asthma, and present two interesting strategies adapted to local administration to limit adverse effects.

Parasitic protists of genera Trypanosoma and Leishmania are members of Trypanosomatidae family. In our studies, we investigated genetic influence on infections caused by these parasites in a mouse model. These diseases are on genetic level controlled by quantitative trait loci (QTLs), when the resulting phenotype is controlled by set of genes with small individual effect. As a mouse model for mapping of QTLs controlling these infections, we used recombinant congenic strains (RCS). Each RCS carry unique set of 12.5% of the genome from donor parental strain on genetic background of other parental strain. For mapping of QTLs controlling infections caused by Trypanosoma brucei brucei (T. b. brucei) and Leishmania tropica (L. tropica) and eosinophil infiltration into inguinal lymph nodes after Leishmania major (L. major) infection, we used RCS from CcS/Dem series, where STS is donor strain and BALB/cHeA is strain of genetic background. First, it was necessary to find suitable model strains for mapping. In all three studies, we selected RCS, which exceeded range of monitored phenotype parameters in comparison with any other tested RCS or parental strains. Mice of RCS CcS-11 showed shorter survival after T. b. brucei infection and strain CcS-9 exhibited higher eosinophil infiltration after L. major infection. For...

Mammalian heme peroxidases are fascinating due to their unique peculiarity of oxidizing (pseudo)halides under physiologically relevant conditions. These proteins are able either to incorporate oxidized halides into substrates adjacent to the active site or to generate different oxidized (pseudo)halogenated species, which can take part in multiple (pseudo)halogenation and oxidation reactions with cell and tissue constituents. The present article reviews basic biochemical and redox mechanisms of (pseudo)halogenation activity as well as the physiological role of heme peroxidases. Thyroid peroxidase and peroxidasin are key enzymes for thyroid hormone synthesis and the formation of functional cross-links in collagen IV during basement membrane formation. Special attention is directed to the properties, enzymatic mechanisms, and resulting (pseudo)halogenated products of the immunologically relevant proteins such as myeloperoxidase, eosinophil peroxidase, and lactoperoxidase. The potential role of the (pseudo)halogenated products (hypochlorous acid, hypobromous acid, hypothiocyanite, and cyanate) of these three heme peroxidases is further discussed

The formation of hypochlorous and hypobromous acids by heme peroxidases is a key property of certain immune cells. These products are not only involved in defense against pathogenic microorganisms and in regulation of inflammatory processes, but contribute also to tissue damage in certain pathologies. After a short introduction about experimental approaches for the assessment of the halogenating activity in vitro and in cell suspensions, we are focusing on novel applications of fluorescent dye systems to detect the formation of hypochlorous acid (HOCl) in leukocytes. Special attention is directed to properties and applications of the non-fluorescent dye aminophenyl fluorescein that is converted by HOCl, HOBr, and other strong oxidants to fluorescein. This dye allows the detection of the halogenating activity in samples containing free myeloperoxidase and eosinophil peroxidase as well as in intact granulocytes using fluorescence spectroscopy and flow cytometry, respectively.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.

<p>Nitric oxide (NO) is expressed in inflammatory tissues. However, NO effects are controversial in inflammation; NO is described as acting in a dose dependent manner and possess both pro-inflammatory and anti-inflammatory properties. </p><p>The present thesis explored the role of NO in relation to white blood cell (WBC) and protein system activation by foreign surfaces in simulated extracorporeal circulation (SECC) using human whole blood from volunteer donors. Three doses of NO, 40 ppm, 80 ppm and 500 ppm, were administered and an array of markers of WBC and protein activation were studied. Neutrophil degranulation was detected with myeloperoxidase (MPO), human neutrophil lipocalin (HNL) and lactoferrin (LF); eosinophil degranulation with eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO); and basophil degranulation with histamine. Furthermore, whole blood and WBC capacity to produce reactive oxygen species (ROS) were studied and cytokine release was measured with IL-1 and IL-10. Complement activation was measured with C3a and C5b-9 complex and contact system activation with FXIIa-C1INH, FXIIa-AT, FXIa-C1INH and FXIa-AT.</p><p>NO increased neutrophil degranulation at all dose levels and 80 ppm NO increased basophil degranulation; whereas, NO exerted no effect on eosinophil degranulation, WBC subset counts, cytokine release or capacity to produce ROS. In addition, while increasing both specific and azurophil degranulation with 40 ppm, 80 ppm and 500 ppm, NO reversed the classical degranulation hierarchy with 500 ppm and azurophil degranulation became predominant. Furthermore, NO effect was greater with 500 ppm than with 80 ppm, indicating a dose response effect. The lack of iNOS mRNA expression in WBC and lack of L-NAME effect on degranulation and nitrite/nitrate production, together with absent increase in nitrite/nitrate in controls, excluded autocrine or paracrine regulation of degranulation. FXIIa-AT and FXIa-AT complexes increased and became predominant during early recirculation, whereas FXIIa-C1INH and FXIa-C1INH complexes were predominant at baseline but remained unaltered, suggesting contact system inhibition predominantly via AT. C3a and C5b-C9 increased. NO had no effect on either contact or complement system activation; however, 500 ppm NO shortened active clotting time.</p><p>In conclusion, the present data suggest that NO has a direct effect on neutrophil and basophil degranulation. Recognition of NO as an enhancer of degranulation may give access to new therapeutic tools for local and systemic inflammatory therapies; whereas, the identification of increased AT mediated inhibition of FXIIa and unchanged C1INH complexes presents new possibilities for therapeutic intervention in conditions such as hereditary angioedema and heart surgery.</p>

<p>Collagenous colitis (CC) is an inflammatory bowel disease of unknown etiology. It is characterized by watery diarrhoea without blood, normal endoscopic findings but microscopically colonic mucosal inflammation and increased thickness of the subepithelial collagen band, the latter being a pathognomonic sign. The inflammatory infiltrate in the mucosa of CC contains lymphocytes, plasma cells, eosinophils, mast cells but few neutrophils. The pathophysiological roles of the thickened collagen band and the inflammatory infiltrate in CC are not fully understood. The aims of the present study were to develop a colonoscope based segmental perfusions technique and to analyze local intestinal secretion of inflammatory mediators: Eosinophilic Cationic Protein (ECP), Myeloperoxidase (MPO), Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF) and permeability marker albumin in CC patients without medication and also during steroid treatment. Furthermore, the colonic mucosal distribution of bFGF and VEGF were studied by immunohistochemical methods.</p><p>Colonoscope-based segmental perfusions were performed in totally 22 patients and the success rate was 76% in both rectal and descending colon segments. The analysis showed high intraluminal concentrations of ECP, bFGF, VEGF and albumin in ten CC patients compared to 10 control patients. Further, albumin had correlations with ECP and VEGF. However, elevated concentrations of MPO, an important feature of ulcerative colitis, were only observed in a few CC patients. Immunohistochemistry visualized bFGF and VEGF in the colonic epithelium but also deeper in the lamina propria. The steroid treatment study (including 12 patients) showed that the perfusate concentrations of ECP, bFGF and VEGF declined significantly in parallel with decreased frequency of diarrhoea. </p><p>In conclusion, a safe colonoscope-based, segmental perfusion technique was developed and perfusions of the rectum and descending colon were performed. CC patients had elevated perfusate concentrations of ECP, VEGF and bFGF. There was a marked reduction of these mediators during steroid treatment supporting the hypothesis that these inflammatory mediators separately or synergistically participate in the inflammatory reaction and tissue remodelling in CC patients. The finding of correlations between albumin and ECP or VEGF implies that permeability is increased in CC and may be triggered by ECP and VEGF. </p>

Nitric oxide (NO) is expressed in inflammatory tissues. However, NO effects are controversial in inflammation; NO is described as acting in a dose dependent manner and possess both pro-inflammatory and anti-inflammatory properties. The present thesis explored the role of NO in relation to white blood cell (WBC) and protein system activation by foreign surfaces in simulated extracorporeal circulation (SECC) using human whole blood from volunteer donors. Three doses of NO, 40 ppm, 80 ppm and 500 ppm, were administered and an array of markers of WBC and protein activation were studied. Neutrophil degranulation was detected with myeloperoxidase (MPO), human neutrophil lipocalin (HNL) and lactoferrin (LF); eosinophil degranulation with eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO); and basophil degranulation with histamine. Furthermore, whole blood and WBC capacity to produce reactive oxygen species (ROS) were studied and cytokine release was measured with IL-1 and IL-10. Complement activation was measured with C3a and C5b-9 complex and contact system activation with FXIIa-C1INH, FXIIa-AT, FXIa-C1INH and FXIa-AT. NO increased neutrophil degranulation at all dose levels and 80 ppm NO increased basophil degranulation; whereas, NO exerted no effect on eosinophil degranulation, WBC subset counts, cytokine release or capacity to produce ROS. In addition, while increasing both specific and azurophil degranulation with 40 ppm, 80 ppm and 500 ppm, NO reversed the classical degranulation hierarchy with 500 ppm and azurophil degranulation became predominant. Furthermore, NO effect was greater with 500 ppm than with 80 ppm, indicating a dose response effect. The lack of iNOS mRNA expression in WBC and lack of L-NAME effect on degranulation and nitrite/nitrate production, together with absent increase in nitrite/nitrate in controls, excluded autocrine or paracrine regulation of degranulation. FXIIa-AT and FXIa-AT complexes increased and became predominant during early recirculation, whereas FXIIa-C1INH and FXIa-C1INH complexes were predominant at baseline but remained unaltered, suggesting contact system inhibition predominantly via AT. C3a and C5b-C9 increased. NO had no effect on either contact or complement system activation; however, 500 ppm NO shortened active clotting time. In conclusion, the present data suggest that NO has a direct effect on neutrophil and basophil degranulation. Recognition of NO as an enhancer of degranulation may give access to new therapeutic tools for local and systemic inflammatory therapies; whereas, the identification of increased AT mediated inhibition of FXIIa and unchanged C1INH complexes presents new possibilities for therapeutic intervention in conditions such as hereditary angioedema and heart surgery.