<p>Mast cells are very potent multifunctional effector cells of the immune system normally distributed throughout connective tissues. An accumulation of mast cells has been described in several pathological conditions such as allergic- and autoimmune inflammations and in certain tumours. This necessitates two different processes: 1) Recruitment of mast cell progenitors from peripheral blood; 2) Accretion of mature mast cells at sites of inflammation and tumour areas. Both processes are depending on the local production of chemotactic factors. The aim of this study was to investigate the role of chemokines and their corresponding receptors in mast cell chemotaxis. </p><p>By cloning and mRNA-screening of cord blood derived mast cells several chemokine receptors were found to be expressed. Functional expression was confirmed of chemokine receptors CXCR4, CCR1 and CCR4. CXCL12, the only known ligand for CXCR4, acted as a mast cell chemotaxin and induced migration of progenitor cells with capacity to differentiate into mast cells. Of several ligands known to bind CCR1 and CCR4, only CCL5 induced migration of mast cells. The migration to CCL5 was mediated through both CCR1 and CCR4. In contrast, the ligands to CCR4, CCL17 and CCL22, could inhibit CCL5-induced migration. Expression of CCR1 and CCR4 could also be confirmed on mast cells in lung from asthmatic patients. Furthermore, we could demonstrate that mast cells were attracted by CCL5 produced by tumour cells in Hodgkin´s lymphoma.</p><p>In conclusion, the work in this thesis has identified two chemokines that regulates mast cell migration. This knowledge helps us understand the mechanisms behind homing of mast cell progenitors from the blood into the tissue and the accumulation of mature mast cells at sites of inflammation and tumourigenesis.</p>

Mast cells are very potent multifunctional effector cells of the immune system normally distributed throughout connective tissues. An accumulation of mast cells has been described in several pathological conditions such as allergic- and autoimmune inflammations and in certain tumours. This necessitates two different processes: 1) Recruitment of mast cell progenitors from peripheral blood; 2) Accretion of mature mast cells at sites of inflammation and tumour areas. Both processes are depending on the local production of chemotactic factors. The aim of this study was to investigate the role of chemokines and their corresponding receptors in mast cell chemotaxis. By cloning and mRNA-screening of cord blood derived mast cells several chemokine receptors were found to be expressed. Functional expression was confirmed of chemokine receptors CXCR4, CCR1 and CCR4. CXCL12, the only known ligand for CXCR4, acted as a mast cell chemotaxin and induced migration of progenitor cells with capacity to differentiate into mast cells. Of several ligands known to bind CCR1 and CCR4, only CCL5 induced migration of mast cells. The migration to CCL5 was mediated through both CCR1 and CCR4. In contrast, the ligands to CCR4, CCL17 and CCL22, could inhibit CCL5-induced migration. Expression of CCR1 and CCR4 could also be confirmed on mast cells in lung from asthmatic patients. Furthermore, we could demonstrate that mast cells were attracted by CCL5 produced by tumour cells in Hodgkin´s lymphoma. In conclusion, the work in this thesis has identified two chemokines that regulates mast cell migration. This knowledge helps us understand the mechanisms behind homing of mast cell progenitors from the blood into the tissue and the accumulation of mature mast cells at sites of inflammation and tumourigenesis.

Chemokines are chemotactic cytokines, which mediate cell trafficking and play a key role in mobilisation of leukocytes. More recently, chemokines and their cognate receptors have been described as key players in different aspects of cancer biology contributing to proliferation, angiogenesis and metastasis. In particular, chemokines CCL19 and CCL21 acting on their associated receptor CCR7 are postulated to be key drivers of lymph node metastasis in a number of malignancies including breast, colon, gastric, & thyroid cancers. It has been reported that the cleavage of the pre-cysteine bridge N-terminal residues of CCL21 (SDGGAQD) and of CCL19 (GTNDAED) renders both peptides incapable of fully activating CCR7. However, little is known about the nature of the interactions that occur between the N-terminal residues of CCL19 or CCL21 and the CCR7 receptor, or the role they have in activation of CCR7. The aim of this study is to investigate the role of the residues in the N-terminus of CCL19 and in particular CCL21 in the context of CCR7 activation and to use this information in the discovery of novel CCR7 antagonists or agonists. To achieve this, we synthesised a number of short (three to seven amino acids) peptides and peptidomimetics inspired by the seven N-terminal amino acid residues of CCL19 and CCL21 and pharmacologically characterised their ability to activate CCR7 or block the activation of CCR7 using a number of in vitro assays such as calcium flux, trans-well (Boyden chamber), and Western blotting. We also carried out computational studies to better understand and predict the activity of these peptides. Our results demonstrate that some of these peptides are indeed capable of acting as agonists or antagonists of CCR7. / Kuwait Health Ministry and Kuwait Civil Services Commission

Metastasis is a major cause of death in cancer patients but currently there are no drugs available for its treatment. Hence there is an urgent clinical need for identifying and developing anti-metastatic drugs. The activation of CC chemokine receptor 7 (CCR7) and C-X-C chemokine receptor type 4 (CXCR4) plays an important role in lymph node metastasis in a variety of cancers. Indeed, in patients with tumours which are positive for CCR7 and/or CXCR4 expression, prognosis and survival are poorer than those whose tumours are negative for these receptors. CCR7 and/or CXCR4 activation, in addition to being involved in inducing invasive phenotypes in cancer cells, promotes tumour cell growth and survival. Our group has previously identified a series of sulfonamides as CCR7 antagonists. This project aims to extend on those studies and to develop a dual CCR7 and CXCR4 antagonist to reduce metastasis in cancer. Novel potent biaryl sulfonamide CCR7 antagonists were synthesised and assessed by calcium flux assay. Several potential dual CCR7 and CXCR4 biaryl sulfonamide antagonists have been synthesised, these are hybrid compounds incorporating features from CCR7 antagonists of this project, and from known sulfonamide CXCR4 antagonists. The most potent of such compound was able to inhibit CCR7 activation in calcium flux assay (95% inhibition at 1 µM), however, the relative potency of these compounds as CXCR4 antagonists was low. Molecular docking was used to investigate the binding mode of the synthesised compounds in CCR7 and CXCR4. The generated docking poses were able to rationalise some of the calcium flux assay results.

No description available.

Abstract : Long-term survival of T lymphocytes in a quiescent state is essential to maintain their cell numbers in secondary lymphoid organs. Interaction of the T cell antigen receptor (TCR) with self-peptide/MHC synergizes with IL-7-induced anti-apoptotic signals to promote T cell survival. These extrinsic stimuli are also implicated in T cell metabolism and survival by regulating several signaling pathways including the phosphatidyl-inositol-3 kinase (PI3K)/Akt pathway. In mice and in rats, loss of functional GTPase of the immune associated nucleotide binding protein 5 (GIMAP5) causes peripheral T lymphopenia due to spontaneous death of T cells. The underlying mechanisms responsible for the pro-survival function of GIMAP5 in T lymphocytes remain largely unknown. Previous work from my laboratory has shown that T cells from GIMAP5-deficient rats show reduced influx of calcium (Ca[superscript 2+]) from the extracellular milieu following stimulation of the TCR complex. In this thesis, I characterized the mechanism by which GIMAP5 regulates Ca[superscript 2+] homeostasis, and elucidated the signaling pathways modulated by GIMAP5 to facilitate the survival of T cells. Firstly, I investigated if GIMAP5 prevents apoptotic death of T lymphocytes by affecting the Ca[superscript 2+] buffering capacity of mitochondria, which is required for sustained Ca[superscript 2+] influx via the plasma membrane channels. I observed that mitochondrial Ca[superscript 2+] accumulation following capacitative Ca[superscript 2+] entry is defective in T cells from Gimap5 deficient rats. Disruption of microtubules, but not the actin cytoskeleton, abrogated Ca[superscript 2+] sequestration by mitochondria in T cells from control but not Gimap5 deficient mice. Similarly, mice lacking functional GIMAP5 displayed defective T cell development and Ca[superscript 2+] influx. Furthermore, I observed that the proximal signaling events following TCR stimulation was reduced and was accompanied by defective proliferation in T cells from Gimap5 deficient mice. Additionally, IL-7-induced STAT5 phosphorylation was decreased in CD4[superscript +] T cells from Gimap5 deficient mice. I also showed that loss of functional Gimap5 results in increased basal activation of mammalian target of rapamycin (mTOR), independent of protein phosphatase 2A (PP2A) or AMP-activated protein kinase (AMPK). Instead, the constitutive activation the PI3K pathway contributed to the spontaneous high mTOR activation. Collectively, my observations suggest that the pro-survival function of GIMAP5 in T-lymphocytes may be linked to the regulation of diverse signaling pathways in a context dependent manner. GIMAP5 also facilitates microtubule-dependent mitochondrial buffering of Ca[superscript 2+] following capacitative entry. GIMAP5 is required to integrate the survival signals generated following activation through TCR and IL-7R. / Résumé : La survie à long terme des lymphocytes T en état de repos est essentielle pour maintenir leurs nombres dans les organes lymphoïdes secondaires. Le récepteur antigénique des cellules T (TCR) en contact avec les peptides du soi / CMH et en synergie avec l'IL-7 induit des signaux anti-apoptotiques pour favoriser la survie des cellules T. Ces stimuli extrinsèques sont également impliqués dans le métabolisme et la survie des cellules T grâce à la régulation de plusieurs voies de signalisation dont la voie phosphatidyl-inositol-3 kinase (PI3K) /AKT. Chez la souris et chez le rat, la perte de l’activité de GIMAP5 (GTPase of Immune Associated Nucleotide Binding Protein 5), provoque une lymphopénie T périphérique en raison de la mort spontanée des cellules T. Le mécanisme sous-jacent responsable de la fonction de survie de GIMAP5 dans les lymphocytes T reste largement inconnu. Nous avons observé que les cellules de rats déficients en GIMAP5, après stimulation par complexe TCR, montrent un afflux de calcium (Ca[indice supérieur 2+]) réduit provenant du milieu extracellulaire. Dans cette thèse, J’ai caractérisé le mécanisme d’action de GIMAP5 dans la régulation de l'homéostasie du Ca[indice supérieur 2+], ainsi que les voies de signalisation modulées par GIMAP5 pour faciliter la survie des cellules T. Tout d'abord, j’ai étudié si GIMAP5 empêche l’apoptose des lymphocytes T en affectant la capacité des mitochondries à réguler la concentration du Ca[indice supérieur 2+], ce qui est nécessaire pour soutenir l’influx de Ca[indice supérieur 2+]. J’ai trouvé que l’accumulation du Ca[indice supérieur 2+] mitochondrial après l’entrée capacitive de Ca[indice supérieur 2+] est défectueuse dans les cellules T de rat déficientes en Gimap5. La disruption des microtubules, mais pas du cytosquelette d'actine, abroge la séquestration du Ca[indice supérieur 2+] mitochondrial dans les cellules T primaires de rat, mais pas dans les cellules T déficientes en Gimap5. J’ai observé que les cellules T provenant de souris déficientes en Gimap5 démontrent une diminution de l’entrée de Ca[indice supérieur 2+]. De plus, la prolifération des cellules T déficientes en Gimap5 est diminuée suite à la stimulation du TCR. En outre, la phosphorylation de STAT5 induit par l'IL-7 est diminuée dans les cellules T CD4[indice supérieur +] de souris déficientes en Gimap5. Également, la perte de Gimap5 aboutit à une activation accrue de la cible mammalienne de la rapamycine (mTOR), indépendamment de la protéine phosphatase 2A (PP2A) ou de la protéine kinase activée par l'AMP (AMPK). Au lieu de cela, l'activation constitutive de la voie PI3K contribue à une forte activation spontanée de mTOR. Collectivement, la fonction de survie de GIMAP5 dans les lymphocytes T peut être liée à la régulation de différentes voies de signalisation. GIMAP5 facilite la fonction, microtubule dépendant, des mitochondries dans leurs actions de régulation du Ca[indice supérieur 2+] après l’entrée capacitive de Ca[indice supérieur 2+]. GIMAP5 est nécessaire pour intégrer les signaux de survie produits suite à l'activation du TCR et de l’IL-7R, qui pourrait être associée à la régulation de l'activité PI3K / AKT / mTOR.

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.