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Chemokines are cytokines with chemoattractant ability, and comprise one of the major groups of molecules in immune system. These are small, secreted proteins cause the migration of leukocytes to the sites of injury. Over 40 mammalian chemokines have been identified to date, and they have been implicated in a number of immune mediated processes, including regulation of inflammation, antigen presentation, blood cell development, metastasis, viral infection and wound healing. In rainbow trout, there have been fewer chemokines reported and only one functional study has been published. Rainbow trout chemokine 2 (CK-2) is the only known CC chemokine with a mucin stalk, which has the potential for extensive <em>O</em>-glycosylation. However, no functional characterization has been performed on this molecule yet. CK-2 shares the presence of a mucin stalk with the mammalian chemokines, fractalkine (CX₃CL1), lymphotactin (XCL1), and CXCL16. Another related trout CC chemokine sequence, CK-2. 1, has been discovered recently, which has 98% nucleotide sequence identity with CK-2. CK-2. 1 was believed to be a separate gene due to its apparent differential regulation in challenged rainbow trout. The question remained, however, whether or not CK-2. 1 was a separate gene or an allele of CK-2. The goal of this project was to further characterize both CK-2 and CK-2. 1. <br /><br /> Through genomic PCR on several outbred individuals it was shown that CK-2. 1 is an allele of CK-2 but not a separate gene. Reverse transcriptase (RT) PCR analysis revealed an increased level of transcript both CK-2 and CK-2. 1 in response to phytohaemagglutinin (PHA) stimulation of head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL) collected from fish with different allelic distributions. Similar results were also observed in the rainbow trout macrophage/monocyte cell line, RTS11. Moreover, an anti-CK-2 antiserum was developed in rabbits, which cross-reacted with CK-2. 1. This newly produced antibody was used to determine the protein expression levels in PHA stimulated rainbow trout tissues. RT-PCR was also performed on the same tissues in order to examine the transcript expression. Rainbow trout with both CK-2 and CK-2. 1 were used for this experiment. An overall decreasing pattern of transcript (both CK-2 and CK-2. 1) was observed in brain and HK over 24 hours, while protein was still detected at 24 hours post stimulation. However, in spleen the CK-2 transcript showed a slight upregulation at 4 hours post stimulation along with a very little or no CK-2. 1 expression, although no protein was detected in spleen. Liver showed a very low level of CK-2 and CK-2. 1 transcript at 8 hours post stimulation; while protein was again detected at 24 hours post stimulation. In addition, the sizes of the proteins found in different tissues were larger than expected (&le;30 kDa for CK-2 or &le;35 for CK-2. 1), perhaps due to the presence of extensive <em>O</em>-glycosylation at the mucin stalk of the protein. <br /><br /> A chemotaxis assay was carried out, which is the definitive assay for chemokine activity. This assay showed migration of peripheral blood leukocytes across a membrane with 5??m pores toward CK-2 at an optimal concentration of 500ng/ml (17nm). Moreover, by pre-treating the recombinant chemokine with the polyclonal antisera, it was shown that the chemokine was actually causing the chemotactic activity. Pre-treatment of the cells with pertussis toxin, an inhibitor of G-protein signalling inhibited the migration of PBLs, established the fact that CK-2 caused chemotaxis by binding to a 7 transmembrane, G-coupled receptor just like all other known chemokines. Interestingly, CK-2 was also shown to attract RTS-11 cells. <br /><br /> Overall, the above findings indicate that CK-2 is functionally a chemokine with two very different alleles in rainbow trout. It is probably heavily <em>O</em>-glycosylated and different tissues express different sizes of the protein. This is only the second functional study of a fish chemokine.

Für das Verständnis der Pathogenese entzündlicher Hauterkrankungen ist die Zusammensetzung des zellulären Entzündungsinfiltrates und die Verteilung der Entzündungszellen von wesentlicher Bedeutung. In Anbetracht der chemotaktischen Funktion der Chemokine liegt die Annahme nahe, dass das zelluläre Infiltrationsmuster in entzündlichen Hauterkrankungen das Expressionsmuster von Chemokinen und umgekehrt widerspiegelt. Die Infiltrationsroute der Leukozyten in die Haut erfolgt immer vom Lumen dermaler Gefäße in das dermale Milieu und ggf. weiter in das epidermale Kompartiment (sog. Epidermotropismus). Die Migration inflammatorischer Zellen über die Grenzen unterschiedlicher Hautkompartimente hinweg ist einzigartig und präsentiert ein ideales Modell, um die chemotaktischen Cytokin- bzw. Chemokinfunktionen zu evaluieren. Anhand verschiedener ausgewählter Hautdermatosen (Wundheilung, Psoriasis, Alopecia areata) wurden die unterschiedlichen Expressionsmuster einer Auswahl von Chemokinen untersucht. Dabei nehmen Chemokine, die von Endothelzellen exprimiert bzw. sezerniert werden, eine zentrale Rolle ein, da sie eine „Pförtnerfunktion“ ausführen. Diese Funktion ist entscheidend bei der Rekrutierung und Akkumulation der für das Erkrankungsbild und bei reparativen Vorgängen der Wundheilung spezifischen Leukozytensubpopulation ins dermale bzw. epidermale Gewebe / The composition of the cellular inflammatory infiltrates and the distribution of inflammatory cells is essential for the understanding of the pathogenesis of inflammatory skin diseases. In view of the chemotactic function of chemokines we assume that the cellular infiltration pattern in inflammatory skin diseases reflects the expression patterns of chemokines and vice versa. The route of leukocyte infiltration into the skin is always directed from the lumen of dermal vessels in the dermal milieu and in some cases further into the epidermal compartment (so-called epidermotropism). The migration of inflammatory cells across the borders of different skin compartments is unique and represents an ideal model to evaluate the chemotactic function of cytokines or chemokines. In various representative dermatoses (wound healing, psoriasis, alopecia areata) we investigated the different expression patterns of selected chemokines. Chemokines, expressed or secreted by endothelial cells, play an important role because they exert a "gatekeeper function". This is crucial in the recruitment and accumulation pattern of the disease and repair processes of wound-specific leukocyte subpopulation, which invade the dermal and epidermal compartment.

Nachdem sich in den verschiedenen Rio („Rimonabant in obesity“) -Studien bereits die Wirksamkeit des Cannabinoid-1-Rezeptorantagonisten Rimonabant durch Gewichtsreduktion und einer Verminderung des kardiovaskulären Risikos bei den untersuchten Patienten gezeigt hatte, stellte sich die Frage nach dem genauen Wirkprinzip. In unserer Arbeit konnten wir anhand von Versuchen an Ratten mit genetisch induzierter Glukosetoleranzstörung nachweisen, dass Rimonabant nicht nur eine Gewichtsreduktion, sondern auch eine Verbesserung des Lipidprofils bewirkt. Konkret fanden sich bei den behandelten Tieren nach zwei Wochen die atherogenetischen Triglyceride vermindert und das atheroprotektive HDL-Cholesterin im Vergleich zu den Kontrolltieren erhöht. Weiterhin konnten verminderte Mengen an Leukozyten, insbesondere der Neutrophilen und der Monozyten, als Zeugnis einer anti-inflammatorischen Wirkung nachgewiesen werden. Des Weiteren zeigte sich eine geminderte thrombozytäre Aktivität, verdeutlicht durch die reduzierte thrombozytäre Aktivierbarkeit durch Thrombin und die abgeschwächte Adhäsion an Fibrinogen-beschichteten Membranen. Auch lies sich eine Zunahme der VASP-Phosphorylierung als Marker einer gesteigerten Thrombozyten-Inhibition erkennen. Keine signifikanten Wirkungen fanden sich dagegen hinsichtlich des Blutglukosespiegels, des Gesamtcholesterins, der Gesamtzahl der Thrombozyten, und der pro-atherosklerotisch wirkenden Chemokine MCP-1 und RANTES. Schlussfolgernd lässt sich sagen, dass der selektive Cannabinoid-1-Rezeptorantagonist Rimonabant einen viel versprechenden Ansatzpunkt in der Behandlung von übergewichtigen Patienten mit Diabetes mellitus darstellt. Durch die Verbesserung des Lipidprofils, die anti-inflammatorische Wirkung und durch die reduzierte Thrombozytenaktivität, trägt es maßgeblich dazu bei, das kardiovaskuläre Risiko bei dieser Patientengruppe zu senken. / We investigated the effect of rimonabant, an cannabinoid receptor-1 antagonist, on inflammation and enhanced platelet activity in type 2 diabetic zucker rats, an experimental model of impaired glucose tolerance and the metabolic syndrome. Rimonabant was fed for 2 weeks to 3-month-old male obese zucker rats. We found, that rimonabant slowed weight gain in in rats with the metabolic syndrome and neutrophiles and monocytes were lowered by rimonabant. Platelet-bound-fibrinogen was also reduced by rimonabant. Platelets from obese rats were more sensitive to thrombin-induced aggregation and adhesion to fibrinogen, wich were both attenuated by rimonabnt theraby. But rimonabant shows no effect on the pro-inflammatory chemokines RANTES and MCP-1. In conclusion, rimonabant demonstrates positive modulation of circulating neutrophil and monocyte numbers and reduces platelet activation, wich may potentially contribute to a reduction of cardiovascular risk.

Dendritische Zellen stellen ein essentielles Bindeglied zwischen angeborener und adaptiver Immunität dar. Sie stammen aus dem Knochenmark und bilden ein Netzwerk von heterogenen Zellpopulationen. In peripheren Geweben liegen sie als unreife Zellen mit einem hohen Potential zur Aufnahme und Prozessierung von Mikroorganismen vor. Nach der Aufnahme von eindringenden Mikroorganismen beginnen dendritische Zellen jedoch, sich von einem prozessierenden in ein präsentierendes Stadium zu differenzieren, und wandern zu den sekundären lymphatischen Organen, um dort den naïven T-Zellen die mikrobiellen Antigene zu präsentieren. Die gerichtete Wanderung von dendritischen Zellen ist hierbei ein zentraler Bestandteil der immunstimulatorischen und -modulatorischen Funktion dieser Zellen. Eine essentielle Rolle bei diesem Migrationsverhalten spielen chemotaktische Zytokine (Chemokine). Chemokine sind Proteine mit einem niedermolekularen Gewicht (8-10 kDa), welche anhand ihres strukturellen Cysteinmotifs in vier Gruppen unterteilt und entweder als CXC-, CC-, C- und CX3C- Chemokine oder als &#945;-, &#946;-, &#948;- und &#947;-Chemokine bezeichnet werden. Arbeiten der eigenen Arbeitsgruppe am Modell der kutanen Leishmaniose haben jedoch gezeigt, dass die Funktion von Chemokinen weitaus mehr umfasst, als lediglich die zielgerichtete Steuerung der Migration immunologisch wichtiger Zellen. So konnte in diesen Studien nicht nur gezeigt werden, dass das Muster der Expression von Chemokinen mit der Schwere des Krankheitsbildes korreliert, sondern auch, dass das Chemokin CCL2/MCP-1 in der Lage ist, einen direkten Einfluss auf die intrazelluläre Erregerabwehr zu nehmen. Diese Arbeiten bezogen sich jedoch auf humane Hautbiopsien und aus Humanblut isolierten Zellen. Eine detaillierte Analyse des Infektionsverlaufes unter definierten Bedingungen (konstante Infektionsdosis und -art, kontrollierte Infektionsdauer, Verwendung von klonierten Parasiten, einheitlicher genetischer Hintergrund des Wirts) ist bei Patienten jedoch nicht möglich. Deshalb wurde die experimentelle Leishmanieninfektion von Inzuchtmäusen (suszeptible BALB/c- und resistente C57BL/6-Mäuse) herangezogen, um die Kinetik der Chemokinexpression und deren Korrelation mit dem Verlauf der kutanen Leishmaniose zu bestimmen. Die Arbeiten dieser Doktorarbeit zeigen erstmals, dass ebenso wie im humanen System bei der experimentellen Leishmaniose in der Maus die Fähigkeit zur Abwehr dieses Erregers mit einer verstärkten Expression von CCL2/MCP-1 in der infizierten Haut korreliert. Des Weiteren konnte zwar eine CCL2/MCP-1-induzierte leishmanizide Wirkung in murinen Makrophagen festgestellt werden, ein vergleichbarer Effekt blieb bei Langerhans-Zellen, den dendritischen Zellen der Haut, jedoch aus. Weiterhin sollte der Einfluss einer Leishmanieninfektion auf das CCL2/MCP-1- induzierte Wanderungsverhalten von Langerhans-Zellen untersucht werden, da aus der Literatur bekannt ist, dass das Chemokin CCL2/MCP-1 die Migration dendritischer Zellen induziert. Die Studien der Doktorarbeit ergaben hierbei, dass eine Infektion mit Leishmania major zu einer signifikanten Verminderung der durch CL2/MCP-1 oder CCL3/MIP-1&#945; induzierten Migration von Langerhans-Zellen führt. Eine mögliche Ursache für dieses Resultat war hierbei in dem Einfluss einer Infektion mit Leishmanien auf die Expression von Chemokinrezeptoren in dendritischen Zellen zu finden. Anschließende Untersuchungen der mRNA-Expression dieser Rezeptoren konnten diese Vermutung bestätigen. So wurde nach einer Infektion von dendritischen Zellen mit L. major eine Reduktion der mRNA-Expression der Chemokinrezeptoren CCR2 und CCR5 festgestellt. Anschließende FACS-Analysen und Studien mit Hilfe des konfokalen Lasermikroskops führten zu einem ähnlichen Resultat. Interessanterweise bewirkt die Infektion mit L. major andererseits eine Hochregulation der mRNA-Expression von CCR7 in dendritischen Zellen. Von diesem Rezeptor ist bekannt, dass er die Chemokine CCL19/MIP-3&#946; und CCL21/6Ckine, welche im Lymphknoten konstitutiv exprimiert werden, mit großer Affinität bindet und für die zielgerichtete Migration dendritischer Zellen in dieses Organ essentiell ist. Weitere Versuche ergaben zudem eine verstärkte CXCL10/IP-10-mRNA-Expression in dendritischen Zellen aus L. major-resistenten C57BL/6-Mäusen, welche bei dendritischen Zellen aus BALB/c-Mäusen nicht festgestellt worden ist. Zusammenfassend konnte in dieser Doktorarbeit gezeigt werden, dass eine Infektion dendritischer Zellen mit L. major sowohl bei suszeptiblen als auch bei resistenten Mäusen zu einer Modulation der Expression von Chemokinrezeptoren führt, die sowohl für die Lokalisation der Zellen in den entzündeten Hautarealen verantwortlich sind (CCR2 und CCR5), als auch ihre Wanderung in die Lymphknoten steuern CCR7). Darüber hinaus lässt die wirtsspezifische Modulation des Chemokins CXCL10/IP-10 in resistenten Tieren vermuten, dass sie zur Kontrolle der Infektion beiträgt. / Dendritic cells (DC) represent an essential link between innate and adaptive immunity. DC are bone marrow-derived and form a network of heterogeneous cell populations. In peripheral tissue they are in an immature state, with a high potential for taking up and processing microorganisms. After taking up invading pathogens, DC differentiate from a “processing” into a “presenting” stage, while migrating to the secondary lymphoid organs in order to present microbial antigens to naïve T cells. The directed migration of dendritic cells is a central component in the immunostimulatory and modulatory function of these cells. Chemotactic cytokines (chemokines) are critical for these migratory pathways. Chemokines are proteins of low molecular weight (8-10 kDa) and can be classified into four groups on the basis of a cysteine structural motif, known as either the CXC, CC, C and CX3C or the &#945;, &#946;, &#948; and &#947; subfamilies. Studies using the model of cutaneous leishmaniasis have shown, however, that the function of chemokines is much wider than only to control the migratory pathway of immunologically relevant cells. These previous studies demonstrated not only the correlation between the chemokine expression pattern and the course of the infection, but also the direct influence of the chemokine CCL2/MCP-1 on the intracellular defence of pathogens. However, these studies were based on skin lesions from patients and human monocytes. A more detailed analysis of the infection process under more defined conditions (constant infection dose, defined duration of infection, use of cloned parasites, uniform genetic background of the host) is not possible with human material. Therefore, the experimental Leishmania model with inbred mice (susceptible BALB/c and resistant C57BL/6 mice) was used to determine the kinetics of the chemokine expression and their correlation with the course of cutaneous leishmaniasis. The studies of this work demonstrated for the first time that the expression of CCL2/MCP-1 during experimental infection with Leishmania major in mice correlates with the capability to control this pathogen. Moreover, a CCL2/MCP-1 induced leishmanicidal effect on murine macrophages could be determined, but a comparable effect was not observed with Langerhans cells, the dendritic cells of the skin. Furthermore, in this work the influence of an infection with Leishmania on the CCL2/MCP-1-induced migration of Langerhans cells were examined, since it is known from the literature that CCL2/MCP-1 induces dendritic cell migration. The studies of this work demonstrated that an infection of Langerhans cells with L. major leads to a significant reduction of the CCL2/MCP-1- or CCL3/MIP-1&#945;-induced migration rate of these cells. A possible reason for this result may be the influence of an infection with Leishmania on the chemokine receptor expression by dendritic cells. Further investigations of the mRNA expression of these receptors could confirm this assumption. They demonstrated reductions in the mRNA expression of the chemokine receptors CCR2 and CCR5 after an infection of dendritic cells with L. major. Moreover, FACS analysis and studies using confocal laser microscopy led to similar results and showed that the L. major-induced alterations in mRNA expression correlate with those at the protein level. Interestingly, a strong upregulation of CCR7 mRNA expression could be detected after infection of dendritic cells. This receptor is well known for binding the chemokines CCL19/MIP-3&#946; and CCL21/6Ckine, which both are constitutively expressed on the lymph node, with high affinity. Moreover, this receptor is essential for a directed movement of dendritic cells to that organ. Further studies also showed an upregulation of the CXCL10/IP-10-mRNA expression in dendritic cells from L. major-resistant C57BL/6 mice, whereas it could not be detected in dendritic cells from susceptible BALB/c mice. In summary, this work demonstrated that an L. major-infection of dendritic cells from resistant mice as well as susceptible mice leads to a modulation in the expression of chemokine receptors that are responsible for the localisation of those cells into the inflammatory tissue (CCR2 and CCR5) and the migration of dendritic cells to the lymph node (CCR7). Moreover, the host-specific modulation of the chemokine CXCL10/IP-10 in resistant animals seems to play a role in the control of the infection.

ABSTRACT Introduction Circulating levels, and the ex vivo production, of the chemokines CCL3, CCL4, CCL5, CXCL8 and CXCL12 (known to play an important role in the pathogenesis of either human immunodeficiency virus type 1 (HIV-1) or tuberculosis (TB)) were examined in the context of both single infections with HIV-1 or Mycobacterium tuberculosis (Mtb) and coinfection with both organisms. We hypothesised that CCL3L1 gene copy number (known to affect CCL3 production, associated with susceptibility to and disease progression of HIV-1) would be associated with mother-to-child transmission (MTCT) of HIV-1, and that the IL8-251T→A single nucleotide polymorphism (SNP) (associated with enhanced CXCL8 production and susceptibility to TB in African Americans) would be highly represented in the South African Black population. Methods Samples used included (i) plasma, DNA samples and cell culture supernatants from control, HIV-1, TB and HIV-1/TB groups, (ii) DNA samples from mothers and their infants (grouped as HIV-1 exposed-uninfected, infected in utero, or infected intrapartum), and (iii) DNA samples from a populationbased study cohort. Chemokines were quantified by enzyme-linked immunosorbent assay (ELISA), CCL3L1 gene copy numbers were determined by real-time polymerase chain reaction (PCR), and a real-time PCR method was developed for identification of the IL8-251T→A SNP. DNA sequencing was used for confirmation. Results We found reduced ex vivo chemokine production in response to phytohaemagglutinin (PHA) together with increased plasma levels of chemokines in HIV-1 and TB patients. In contrast to that seen in Caucasians (median CCL3L1 copy number of 2), in Black individuals (median CCL3L1 copy number of 5) circulating levels of CCL3 did not correlate with CCL3L1 gene copy number; in addition, a high proportion of Black individuals were found to have CCL3L1 copy numbers below their population-specific median. Using MTCT as a model for studying HIV-1 transmission, infants who became infected with HIV-1 had significantly reduced CCL3L1 gene copy numbers. IL8-251A allele frequencies were found to be 0.41 for Caucasian groups, and 0.85 for Black groups; due to study limitations, the possible association of IL8-251T→A with TB susceptibility could not be addressed. Discussion The increased plasma levels of chemokines seen in HIV-1 and TB, likely due to chronic immune activation in vivo, may result in T cell anergy which in turn might be the cause of reduced PHA-stimulated ex vivo chemokine production. Our results suggest that Black South Africans may be at particularly high risk for acquiring HIV-1 (at least with respect to CCL3L1 gene copy number), and further imply the presence of other genetic polymorphisms which may influence plasma CCL3 levels. In addition, the high IL8-251A allele frequency (if indeed associated with TB in South African populations) in Black individuals suggests a greater risk for infection with Mtb. It will be important, in larger studies, to gain a more in-depth understanding of the relationships between host genotype and chemokine production phenotype, and to relate these measures to infection outcomes. Conclusions Together, these results highlight the importance of gaining an understanding of the effects of host genotype on the development of innate and acquired immunity to HIV-1 and TB, which will be key in the design of efficient therapies and prevention strategies.

The chemokine receptor CXCR3 and its agonists CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC are involved in a variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. CXCL11 has also been reported to bind to an additional receptor, namely CXCR7, which also interacts with CXCL12. Two alternatively spliced variants of the human CXCR3 receptor have been described, namely CXCR3-B and CXCR3-alt. The human CXCR3-B has been found to bind CXCL9, CXCL10, CXCL11 as well as an additional agonist CXCL4/PF4. In contrast, CXCR3-alt only binds CXCL11. This work demonstrates that CXCL4 like the original CXCR3 agonists is capable of inducing biochemical signalling, namely intra-cellular calcium elevation, and activation of p44/p42 MAPK and PI3K/Akt pathways in activated human T lymphocytes. Phosphorylation of p44/p42 MAPK and Akt was inhibited by pertussis toxin, suggesting coupling to Gi protein. In contrast CXCR3 antagonists blocked CXCR3 agonists but not CXCL4-mediated responses. Surprisingly, stimulation of T cells with CXCL4 failed to elicit migratory responses of these cells and did not lead to loss of surface CXCR3 expression. Collectively our evidence shows that although CXCL4 is coupled to downstream biochemical machinery, its function in T cells is distinct from the function of CXCR3 agonists. The work presented in this thesis also indicates that despite considerably lower surface expression in comparison to the full length CXCR3, CXCR3-B and CXCR3-alt transduce biochemical signals in response to CXCL11 in transfected cells. According to previous reports the role of CXCR7 in signalling and chemotaxis in T cells could not be detected. In T cells and transfected cells system CXCR7 was localised at the plasma membrane and was efficiently internalized in response to CXCL11 and CXCL12. Studies of the involvement of methylation in T cell chemotaxis suggest that this modification may be required in this process as it was partially inhibited by methylation inhibitor- MTA. Moreover T cell co-stimulation caused increased levels of arginine mono-methylated proteins suggesting the importance of methylation in T lymphocyte signalling.

The migration of T lymphocytes is a vital component of the immune system, with roles in immunosurveillance and inflammation. The role of Phosphoinositide 3-kinase within T lymphocyte migration is unclear, with some evidence that it may be a disposable signal. Here, using Staphylococcal Enterotoxin B activated peripheral blood mononuclear cells and the T cell line CEM cells, the role of Phosphoinositide 3-kinase and its downstream kinases was investigated. CCL22 mediated CEM cell migration and CXCL12 mediated peripheral blood mononuclear cell migration were shown to be independent of Phosphoinositide 3-kinase using several different broad-spectrum Phosphoinositide 3-kinase inhibitors. However, these cells were Akt-dependent, as demonstrated by incubation with the Akt inhibitor Akti-1/2. Differences in the effect of the inhibitors on Akt activity were discovered, indicating that either Akt can be activated in the absence of Phosphoinositide 3-kinase, or differences exist regarding the relative abundance of each protein within the cell. Th17 cells are a subtype of the T helper cell family and have been shown to be involved in inflammation and immune diseases. Mouse splenocytes were polarised to a Th17 phenotype and analysed for the surface expression of chemokine receptors. CCR2, CCR6 and CCR9 were shown to be expressed on Th17 cells and upregulated under Th17 polarising conditions. However, only CCR2 and CCR6 induced migration of Th17 cells. This migration was sensitive to Phosphoinositide 3-kinase and Akt inhibitors. This data reveals a model for the migration of Th17 cells to areas of inflammation, and sheds light on the role of Phosphoinositide 3-kinase during this process.

Glu-Leu-Arg (ELR)-CXC chemokine-mediated neutrophil migration and activation plays a key role in many inflammatory diseases. Dysregulated neutrophil activation often leads to inflammatory responses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).<p> Previously, we generated a bovine drug (i.e., bovine CXCL8(3-74)K11R/G31P, bG31P) by mutating the first two amino acids at the beginning of the N-terminus of bovine CXCL8/IL-8 and later substituting Arg for Lys11 and Pro for Gly31. Bovine G31P was shown to be a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle & guinea pigs, but a human equivalent thereof would be of significantly more use in human medicine. Published studies on the structure and function of human CXCL8 suggest that human CXCL8(3-72)K11R/G31P (i.e., hG31P) would not be a particularly effective chemokine antagonist. Thus, development of a humanized form of bG31P became a primary goal. I first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P and generated a human-bovine chimeric G31P (hbG31P; i.e., bCXCL8(3-44)K11R/G31P-hCXCL8(45-72)). I also made substitutions at each remaining human-discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T) within the 5 half of the hbG31P cDNA. The results showed that hbG31P and its analogues blocked CXCL8-induced human neutrophil chemotactic responses, reactive oxygen intermediate (ROI) release, and intracellular calcium flux. Humanized bovine G31P was also shown to significantly block pulmonary neutrophilic pathology in a guinea pig model of airway endotoxemia.<p> As bG31P, hbG31P and its further humanized forms showed essentially equivalent ELR-CXC chemokine antagonist activity, Dr. Fang Li, Ms Jennifer Town and I then generated a fully human form of bG31P, hG31P. <i>In vitro</i>, hG31P was shown to effectively inhibit CXCL-1-, -5-, and -8-induced neutrophil chemotactic responses, intracellular Ca2+ flux, and ROI release. Human G31P also desensitized heterologous G protein-coupled receptors (GPCR) including bacterial peptides (e.g., N-formyl-methionine-leucine-phenylalanine, fMLP), anaphylatoxin (e.g., complement 5a, C5a), lipid mediators (e.g., leukotriene B4, LTB4; platelet-activating factor, PAF) receptors. Moreover, hG31P, in a dose-dependent manner suppressed CXCL1 and CXCL8 expression by LPS-challenged airway epithelial cells and reversed the anti-apoptotic influence of ELR-CXC chemokines on neutrophils. <i>In vivo</i>, hG31P was significantly effective in blocking the pathology associated with airway endotoxemia, aspiration pneumonia, and intestinal ischemia and reperfusion injury, including neutrophil recruitment (70-95% reduction) into, and activation within, the airways or gut, chemokine or cytokine expression, and pulmonary vascular complications. The blockade of neutrophil recruitment by hG31P in aspiration pneumonia animals did not increase airway bacterial growth. The G31P treatment was protective in both mesenteric (i.e., local) and remote organ injury. These findings suggest that hG31P is not only a potent neutrophil antagonist, but an effective blocker of other inflammatory responses. These comprehensive anti-inflammatory effects indicate that hG31P could potentially provide a viable therapeutic approach for inflammatory diseases such as ALI /ARDS.

Chemokines are cytokines with chemoattractant ability, and comprise one of the major groups of molecules in immune system. These are small, secreted proteins cause the migration of leukocytes to the sites of injury. Over 40 mammalian chemokines have been identified to date, and they have been implicated in a number of immune mediated processes, including regulation of inflammation, antigen presentation, blood cell development, metastasis, viral infection and wound healing. In rainbow trout, there have been fewer chemokines reported and only one functional study has been published. Rainbow trout chemokine 2 (CK-2) is the only known CC chemokine with a mucin stalk, which has the potential for extensive <em>O</em>-glycosylation. However, no functional characterization has been performed on this molecule yet. CK-2 shares the presence of a mucin stalk with the mammalian chemokines, fractalkine (CX₃CL1), lymphotactin (XCL1), and CXCL16. Another related trout CC chemokine sequence, CK-2. 1, has been discovered recently, which has 98% nucleotide sequence identity with CK-2. CK-2. 1 was believed to be a separate gene due to its apparent differential regulation in challenged rainbow trout. The question remained, however, whether or not CK-2. 1 was a separate gene or an allele of CK-2. The goal of this project was to further characterize both CK-2 and CK-2. 1. <br /><br /> Through genomic PCR on several outbred individuals it was shown that CK-2. 1 is an allele of CK-2 but not a separate gene. Reverse transcriptase (RT) PCR analysis revealed an increased level of transcript both CK-2 and CK-2. 1 in response to phytohaemagglutinin (PHA) stimulation of head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL) collected from fish with different allelic distributions. Similar results were also observed in the rainbow trout macrophage/monocyte cell line, RTS11. Moreover, an anti-CK-2 antiserum was developed in rabbits, which cross-reacted with CK-2. 1. This newly produced antibody was used to determine the protein expression levels in PHA stimulated rainbow trout tissues. RT-PCR was also performed on the same tissues in order to examine the transcript expression. Rainbow trout with both CK-2 and CK-2. 1 were used for this experiment. An overall decreasing pattern of transcript (both CK-2 and CK-2. 1) was observed in brain and HK over 24 hours, while protein was still detected at 24 hours post stimulation. However, in spleen the CK-2 transcript showed a slight upregulation at 4 hours post stimulation along with a very little or no CK-2. 1 expression, although no protein was detected in spleen. Liver showed a very low level of CK-2 and CK-2. 1 transcript at 8 hours post stimulation; while protein was again detected at 24 hours post stimulation. In addition, the sizes of the proteins found in different tissues were larger than expected (&le;30 kDa for CK-2 or &le;35 for CK-2. 1), perhaps due to the presence of extensive <em>O</em>-glycosylation at the mucin stalk of the protein. <br /><br /> A chemotaxis assay was carried out, which is the definitive assay for chemokine activity. This assay showed migration of peripheral blood leukocytes across a membrane with 5µm pores toward CK-2 at an optimal concentration of 500ng/ml (17nm). Moreover, by pre-treating the recombinant chemokine with the polyclonal antisera, it was shown that the chemokine was actually causing the chemotactic activity. Pre-treatment of the cells with pertussis toxin, an inhibitor of G-protein signalling inhibited the migration of PBLs, established the fact that CK-2 caused chemotaxis by binding to a 7 transmembrane, G-coupled receptor just like all other known chemokines. Interestingly, CK-2 was also shown to attract RTS-11 cells. <br /><br /> Overall, the above findings indicate that CK-2 is functionally a chemokine with two very different alleles in rainbow trout. It is probably heavily <em>O</em>-glycosylated and different tissues express different sizes of the protein. This is only the second functional study of a fish chemokine.