Abstract Early in the evolution of the vertebrates it is thought that two genomic duplications occurred, providing a basis for the evolution in body plan and neural crest of very early vertebrates and substantive material for further evolution of various gene families such as those making up a number of components of the adaptive vertebrate immune system. While the bony fish possibly had another, genome duplications are not generally a feature of vertebrate evolution and indeed the appearance of an antigen-adaptive immune recognition system may have served to limit the size that various vertebrate genomes, including that of the human, can in fact achieve. This initial step in vertebrate immune evolution, the establishment of recognition of non-self against the unique set of 'self' epitopes for an individual, provided an immensely powerful weapon in immune function with the ability to tailor a defense against as-yet-unseen dangers at any time albeit with the pitfall of autoimmune disease. As the recognition sites of the antigen receptor molecules such as TcR are produced by clonal modification of the segments provided in the germline and are thus not in the genome itself, pathogens have not been able to hijack this one component of the immune system in the way so many other components have been put to use throughout evolution, nor do these components necessarily reveal themselves as associated with disease through genome screens. Importantly, overall immune function is determined not just by the potential repertoire of recognition receptors but also by the ability of immunocompetent cells to migrate in a tissue specific fashion through the use of various chemokines and their receptors. Typical of the hijacking of an immune system component by a pathogen is the use of a chemokine ligand gene in the viral ancestor to SIV and HIV, allowing for virus binding to immunocompetent cells as is seen in the use of the CCR5 chemokine receptor by macrophage-tropic HIV strains. This thesis describes the allele and genotype frequencies for several TcR beta-chain variable segment polymorphisms in a population of MS patients compared with controls before and after stratification for HLA-DR15, polymorphism in the Apo-1 / Fas promoter, the DRB1 Val86/Val86 genotype, CCR5-delta32 and the HLA-DRA promoter. The thesis continues with CCR5-delta32 genotyping in IDDM, MS and SLE cohorts and then examines the question of the population of origin of the delta-32 allele of the CCR5 receptor for chemokine. Here, a case / control comparison of 122 RR-MS patients with 96 normal individuals was made for allele and genotype frequencies and for haplotypes formed by pairs of TCRB markers. Further analysis was made after HLA-DR15 stratification. Linkage disequilibrium was found between pairs of alleles of bv8s1, bv10s1, bv15s1 and bv3s1 loci in both patients and controls. In the RR-MS cohort, an increase in the allele frequency of bv8s1*2 was seen (p = 0.03) and the haplotype bv8s1*2 / bv3s1*1 was increased (p = 0.006), and both were found to be statistically significant. In the DR15-positive group, association between MS and TCRB was seen with the bv8s1*2 allele (p = 0.05) and the bv8s1*2 / bv10s1 haplotypes (p = 0.048), while the haplotype associations seen among the DR15-negative patients included the bv3s1*1 allele (bv10s1*1 / bv3s1*1, p = 0.022; bv8s1*2 / bv3s1*1, p = 0.048). While no associations were found after stratification for SDF1-3'A, Apo-1 / Fas or DRB1 there were modest interactions between bv3s1, bv10s1 and bv15s1 and the HLA-DRA promoter. These results support the involvement of the TCRB region in MS susceptibility. The further study of autoimmune disease here includes genotype analysis of CCR5-delta32 in type 1 diabetes (IDDM) and SLE. CCR5 is the major co-receptor for viral entry used by macrophage-tropic HIV strains and protection from infection is seen in homozygotes for CCR5-delta32. In diabetes, infiltration of pancreatic tissue by autoreactive T-cells involves secretion of multiple cytokines and chemokine receptor expression. Variation in the chemokine receptor CCR5 may result in differences in inflammatory cell migration in response to relevant chemokines. Adolescents with type 1 diabetes were genotyped for CCR5-delta32 (n = 626). The allele frequency was compared with that of 253 non-diabetic adolescents and with that of 92 adults with SLE. A reduced allele frequency was seen in type 1 diabetes compared with controls (0.092 vs 0.123, p = 0.05). This difference was not seen for the cohort of patients with SLE (freq = 0.114). A reduction in the number of CCR5-delta32/delta32 homozygotes, who lack CCR5, in the type 1 diabetes cohort was also seen and while not statistically significant (2 observed compared to 5.25 expected; p = 0.12) is interesting. These results suggest a partial protection from type 1 diabetes for CCR5-delta32 homozygous individuals is possible and that CCR5 has a potential role in the pathogenesis of type 1 diabetes. Global surveys of the CCR5-delta32 allele have confirmed a single mutation event in a Northeastern European population as the source of this allele. Here, Australian Ashkenazi Jews (n = 807) were found to have a CCR5-delta32 allele frequency of 14.6% while Australian Sephardic Jews (n = 35) had a frequency of 5.7% and non-Jewish Australian controls (n = 311) had an allele frequency of 11.25%. Data on birthplace of grandparents showed a gradient with highest CCR5-delta32 frequencies from Eastern European Ashkenazim (~19.5% for those whose four grandparents come only from Russia, Poland, Hungary, Austria and Czechoslovakia; n = 197) which differs significantly from the frequency seen in Ashkenazi Jews from Western Europe (n = 101, p = 0.001). Homozygotes for CCR5-delta32 were genotyped with 3p21 region microsatellites. This has defined an ancestral haplotype on which the mutation first occurred and helped to date this event to between 40 and 50 generations ago or just over a thousand years ago. The population gradient, combined with the dating of the mutation by microsatellite allele frequencies, suggests an origin for the CCR5-delta32 allele in a population ancestral to the Ashkenazim. The distribution in non-Jewish populations in northern Europe has led others to postulate spread of the mutation by Vikings. It is hypothesised here that the link between the two populations could be the kingdom of Khazaria with subsequent admixture into both Swedish Vikings and Ashkenazi Jews. The basic driving force of evolution is through selection and the immune system has a role which, through the survival pressure exerted by viruses and other pathogens, has the potential to exert a great deal of selective force on the various components of this system. The effects of this pronounced selection on an immune system component can be seen for example in the increase of the CCR5-delta32 allele over the last thousand years to the current frequency. As mentioned, some immune system components are not affected by such straightforward selection. In the case of the TCRBV segments, effects on the immune repertoire can occur through MHC interaction at the point of thymic entry and in the effects of various superantigens, but the actual binding pockets that recognise antigen are themselves unable to be selected for (or against). The findings presented in this thesis provide support for the association of TCRBV gene segments with multiple sclerosis and also provide support for the further study of the role of the CCR5-delta32 allele in type 1 diabetes. Furthermore, data presented here suggests that the CCR5-delta32 allele had an origin in the Khazar Kingdom just over a thousand years ago, accounting for the allele frequencies in both the Ashkenazi Jews and in lands frequented by the Vikings. The definition of an extended ancestral haplotype for the CCR5-delta32 allele shows how the effect of selection of an allele of one gene can carry with it specific alleles of a large number of other genes as well.

<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>

Chemokines are structurally and functionally related 8-10 kDa proteins defined by four conserved cysteine residues. They consist of a superfamily of proinflammatory mediators that promote the recruitment of various kinds of leukocytes and other cell types through binding to their respective chemokine receptor, a member of the GPCR family. Abnormal control of this system results in various diseases including tumorigenesis and cancer metastasis. Deregulation can occur when constitutively active mutant (CAM) chemokine receptors are locked in the “on” position. This can lead to cellular transformation/tumorigenesis. A viral CAM receptor, ORF74, that can cause tumors in humans, also has homology to human CXC chemokine receptor 2 (CXCR2), which is a G-protein-coupled receptor (GPCR) expressed on neutrophils, some monocytes, endothelial cells, and some epithelial cells. CXCR2 activation with ELR+ CXC chemokines induces leukocyte migration, trafficking, cellular differentiation, angiogenesis and cellular transformation. Using a high throughput yeast screen we identified a novel point mutation, D9H, in CXCR2, which leads to constitutive activation (CA). Generation of positively charged substitutions, D9K and D9R, and D143V as a positive control resulted in CA CXCR2 with differential levels of cellular transformation. To further investigate how D9 mutations lead to differential CA, we used inhibitors of known signal transduction pathways. Pertusiss toxin (PTX) sensitivity in foci formation assays demonstrated that D9R uses the Gi subunit like WTCXCR2 and D143V, while D9H and D9K do not. All CA receptors use the JAK pathway based on sensitivity to the inhibitor, AG490. Phosphorylation of PLC-beta 3 and sensitivity to the PLC-beta 3 inhibitor, U73122, implicates that mutant receptors such as D143V, D9H, D9K, and D9R utilize the Gq/11 subunit. Interestingly, D9R use both Gi and Gq/11 subunits. All of the CA receptors induced phosphorylation of the epidermal growth factor receptor (EGFR) indicating a transactivation between CXCR2 and EGFR. These data describe two novel and important findings. First, N-terminal CXCR2 controls activation and signaling using multiple G protein subunits to elicit downstream signaling. Second, our work supports the “functional selectivity” model for GPCR activation. That is, mimicking agonist activation, CA CXCR2 receptors have multiple conformational states that lead to differential activation.

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.

Traumatic brain injury (TBI) is the most common cause of mortality and disability in the younger (&lt;50 years) Swedish population with an incidence rate of 20,000 cases per year. This thesis aims to increase the understanding of brain injury mechanisms, especially in a molecular and cellular context. Bone morphogenetic protein (BMP) signalling was examined in three genetically modified mice (two “loss-of-function”, one “gain-of-function”) exposed to TBI (controlled cortical impact, CCI) with CaMKII used as promoter for Cre-driven recombination in postnatal forebrain neurons. The mice survived, developed normally and did not show any obvious phenotypes except for an upregulation in Mtap2 mRNA in mice with impaired BMP signalling. Reactive Gfap and Timp1 mRNA expression measured using quantitative RT-PCR (qRT-PCR) was reduced in the mice overexpressing BMP signals. The BMP signalling pathway was further studied in cultured PC12 cells with BMP4 and NGF added. Egr3 expression was substantially increased by these growth factors. Blocking Egr or Junb functions reduced neurite outgrowth. TBI-induced mRNA expression changes in 100 selected genes in C57BL/6J mouse neocortex and hippocampus were measured using qRT-PCR at different time points post-injury. Several distinct gene clusters with similar expression patterns were identified. GeneChip analysis (Affymetrix) of the injured mouse neocortex at three days revealed 146 transcripts significantly upregulated, confirming and extending the qRT-PCR results. The findings demonstrate marked increases after injury among chemokine transcripts and activation of many genes involved in inflammation. In conclusion, the present study has revealed transcriptional changes in specific signalling pathways after brain injury. The results may help to identify novel targets for neuroprotective interventions after traumatic brain injury.

Schwannzellen sind die myelinisierenden Zellen des peripheren Nervensystems, die auch eine Rolle bei Entzündungs- und Regenerationsprozessen spielen. Diese Arbeit beschäftigt sich mit der Bedeutung der Chemokinrezeptoren CXCR4 und CXCR7 bei der SDF-1-abhängigen Signalübermittlung in der Schwannzelllinie RN22. Mittels PCR, Western-Blotting und FACS erfolgte zunächst ein Expressionsnachweis der Chemokinrezeptoren. Anschließend wurde mittels Western-Blotting festgestellt, dass eine Behandlung mit SDF-1 zu einer Aktivierung der intrazellulären Signalkinasen Erk1/2, p38 und Akt führt. Für PKC ζ/λ wurde kein Effekt beobachtet. Durch Einsatz der spezifischen Antagonisten für CXCR4 (AMD3100) und CXCR7 (CCX733) konnten diese Effekte blockiert werden. Die Ergebnisse legen nahe, dass beide Rezeptoren ihren Liganden binden müssen, damit ein intrazellulärer Effekt auftritt. Ähnliches gilt auch für die SDF-1-abhängige Migration von RN22-Zellen, die ebenfalls bereits durch einen der beiden Antagonisten unterbunden werden kann.

Das Chemokin SDF-1 spielt eine wichtige Rolle bei der Hämatopoese, bei Immunreaktionen sowie bei der Entwicklung des Herzens, der Extremitätenmuskulatur und des zentralen und peripheren Nervensystems. Lange Zeit galt CXCR4 als der einzige Chemokinrezeptor für SDF-1, bis vor wenigen Jahren CXCR7 als ein alternativer Rezeptor für SDF-1 identifiziert wurde. Da alle Zelltypen des zentralen Nervensystems (ZNS) sensitiv für SDF-1 sind, sollte in dieser Arbeit die Funktion der beiden Rezeptoren in primärer Mikroglia und primären Astrozyten untersucht werden. Bisher konnte CXCR7 nur als Scavenger-Rezeptor für SDF-1 oder als atypischer Chemokinrezeptor nachgewiesen werden. Die Untersuchungen ergaben einen mitogenen und chemotaktischen Effekt von SDF-1 auf primäre Mikroglia, wobei sowohl CXCR4 als auch CXCR7 für das SDF-1-Signalverhalten essentiell sind. Nach Aktivierung von Mikroglia in vitro und in vivo wurden beide Rezeptoren verstärkt expremiert. In primären Astrozyten ergab sich ein ligandenabhängiges Signalverhalten von CXCR7. So führte die Bindung von SDF-1 an CXCR7 zu einer Aktivierung von G-Proteinen, während die Kopplung von interferon-inducible T cell alpha chemoattractant (I-TAC), als zweiten Liganden von CXCR7, eine Signalweiterleitung über ß-Arrestin2 zur Folge hatte. Zudem konnte die G-Protein-gekoppelte Rezeptorkinase (Grk)2 als ein positiver Regulator des SDF-1-CXCR7-Signalverhaltens in Astrozyten identifiziert werden.

<p>Lymph nodes are organs of efficiency. Once activated, they essentially function to optimize and accelerate the production of the adaptive immune response, which has the potential to determine survival of the host during an initial infection and protect against repeated infections, should specific and appropriate immunological memory be sufficiently induced. We now have an understanding of the fundamental structure of lymph nodes and many of the interactions that occur within them throughout this process. Yet, lymph nodes are dynamic and malleable organs and much remains to be investigated with regards to their responses to various types of challenges. In this work, we examined multiple inflammatory scenarios and sought to understand the complex ways that lymph nodes can be externally targeted to impact immunity. First, we outline a novel mechanism of cellular communication, where cytokine messages from the periphery are delivered to draining lymph nodes during inflammation. These signals are sent as particles, released by mast cells, and demonstrate the ability of the infected tissue to communicate to lymph nodes and shape their responses. Based on these interactions, we also explored the ability to therapeutically or prophylactically modulate lymph node function, using bioengineered particles based on mast cell granules, containing encapsulated cytokines. When we used these particles as a vaccine adjuvant, we were able to polarize adaptive immune responses, such as to promote a Th1 phenotype, or enhance a specific attribute of the immune response, such as the production of high avidity antibodies. We then explore three examples of lymph node-targeting pathogens: Salmonella typhimurium, Yersinia pestis and Dengue virus. Each of these pathogens has a well-characterized lifecycle including colonization of draining lymph node tissue. In the case of S. typhimurim, we report that the virulence this pathogen depends on a specific shut down of the chemotactic signals in the lymph node that are required to maintain appropriate cellular localization within it. Our results demonstrate that these architecture changes allow S. typhimurim to target the adaptive immune process in lymph nodes and contribute to its spread in vivo and lethality to the host. With Y. pestis, similar targeting of cellular trafficking pathways occurs through the modulation of chemokine expression. Y. pestis appears to use the host's cellular trafficking pathways to spread to lymph nodes in two distinct waves, first exploiting dendritic cell movement to lymph nodes and then enhancing monocyte chemoattractants to replicate within monocytes in draining lymph nodes. These processes also promote bacterial spread in vivo and we further demonstrate that blocking monocyte chemotaxis can prolong the host's survival. In the third example of pathogen challenge, we report for the first time that mast cells can contribute functionally to immunosurveillance for viral pathogen, here, promoting cellular trafficking of innate immune cells, including NK cells, and limiting the spread of virus to draining lymph nodes. For each of these three examples of lymph node targeting by microbial pathogens, we provide data that modulation of cellular trafficking to and within lymph nodes can drastically influence the nature of the adaptive immune response and, therefore, the appropriateness of that response for meeting a unique infectious challenge. Cumulatively this work highlights that a balance exists between host and pathogen-driven modulation of lymph nodes, a key aspect of which is movement of cells within and into this organ. Cytokine and chemokine pathways are an area of vulnerability for the host when faced with host-adapted pathogens, yet the lymph node's underlying plasticity and the observation that slight modulations can be beneficial or detrimental to immunity also suggests the targeting of these pathways with therapeutic intentions and during vaccine design.</p> / Dissertation

Kawasaki disease (KD) is characterized by a systemic vasculitis of unknown etiology. More research indicates that KD is related to genetic. In 2003, Sekiya et al. studied the correlation of Th2-related genes and the KD in Japan. They found out that -431T allele would increase the concentration of Thymus and activation-regulated chemokine (TARC)/ CCL17 protein in serum by single nucleotide polymorphism (SNP) -431 C>T of chemokine TARC/ CCL17 operon 5¡¦-flanking region , which suggests that SNP has functionality. Therefore, this study explored the polymorphism and relationship between the regulation of chemokine of TARC/ CCL17 and KD. Firstly, we performed polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) to detect TARC/CCL17 -431 C>T genotype. Then enzyme immunoassay was used to detect TARC/CCL17 chemokine¡¦s expression. The results showed that the performance of TARC -431 C/T SNP, the alleles from KD patients with -431 T, were significantly less than the non-KD control group. It was observed that the -431 T alleles had a lower chance to occur in KD with aneurysms, but independent with coronary artery lesions (CAL). In addition, the acute stage of KD has a higher TARC protein expression, which gradually decreases during IVIG treatment period. However, the up-regulation of TARC protein may not be the direct consequence caused by the single nucleotide polymorphism of TARC -431 C>T.

Chemokines and their receptors are involved in many aspects of immunity. Chemokine CX3CL1, acting via its receptor CX3CR1, regulates monocyte migration and macrophage differentiation as well as T cell-dependent inflammation. Two common, nonsynonymous polymorphisms in CX3CR1 have previously been shown to alter the function of the CX3CL1/CX3CR1 pathway and were suggested to modify the risk for asthma. Using matrix-assisted laser desorption/ionization time-of-flight technology, we genotyped polymorphisms Val249Ile and Thr280Met in a cross-sectional population of German children from Munich (n = 1,159) and Dresden (n = 1,940). For 249Ile an odds ratio of 0.77 (95% confidence interval 0.63–0.96; p = 0.017) and for 280Met an odds ratio of 0.71 (95% confidence interval 0.56–0.89; p = 0.004) were found with atopy in Dresden but not in Munich. Neither polymorphism was associated with asthma. Thus, amino acid changes in CX3CR1 may influence the development of atopy but not asthma in German children. Potentially, other factors such as environmental effects may modify the role of CX3CR1 polymorphisms. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.