Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Mar 3

Techn. Hochsch., Diss., 2003--Aachen.

Universiẗat, Diss., 2004--Kiel.

Background: COPD is associated with an increased lung macrophage burden. Whilst lung macrophages may self-renew, recruitment of peripheral blood monocytes from the systemic circulation is considered to represent their principal means of replenishment. Through modulating expression of monocytic chemokines CCL2/CCL3 and their respective receptors (CCR2/CCR1+CCR5), IL-6 could play a key role in facilitating the recruitment of monocytes to the lungs of COPD patients. COPD is associated with enhanced pulmonary and systemic IL-6 levels; concentrations of the soluble IL-6 receptor sIL-6R may be an important determinant of IL-6 signalling in COPD. Trans-signalling through sIL-6R, IL-6 may facilitate recruitment of monocytes in COPD by influencing chemokine and chemokine receptor expression. Aims: 1) To compare levels of IL-6, sIL-6R, CCL2 and CCL3 in the plasma and sputum of COPD and controls. 2) To examine of the effects of IL-6 stimulation on monocyte chemokine receptor gene expression (CCR1, CCR2 and CCR5). 3) To compare subtypes (CD14++CD16-, CD14+CD16+, CD14-CD16++) and chemokine receptor expression (CCR1, CCR2, CCR5) of monocytes in COPD (paired stable & exacerbating) and controls. 4) To compare the migratory ability of monocytes from COPD and controls. 5) To compare numbers of marginated CX3CR1+ monocytes in the pulmonary microvasculature and proliferation status (Ki67 positivity) of alveolar macrophages in COPD and controls. Methods: 1) MSD soluble marker analysis was performed on plasma and sputum supernatant. 2) Monocytes underwent stimulation with IL-6 and sIL-6R; chemokine receptor expression was determined by quantitative PCR. 3) Flow cytometry was performed on whole blood to determine monocyte subtype and chemokine receptor expression. 4) Monocyte migration towards sputum supernatant was assessed using a transwell system incorporating fluorescence based detection of DNA from migrated cells. 5) Immunofluorescence and immunohistochemistry was performed on lung tissue (obtained from patients undergoing surgical resection of lung carcinoma) to identify marginated (CX3CR1+CD14+, CX3CR1+CD16+) monocytes and proliferating alveolar macrophages (Ki67) respectively. Results and Conclusion: Levels of sIL-6R were increased in the lungs and systemic circulation of COPD patients implying potential for enhanced IL-6 trans-signalling: monocytes cultured in the presence of IL-6+sIL-6R upregulated expression of the CCR5 gene. A greater proportion of circulating COPD CD14++CD16- and CD14+CD16+ monocytes were demonstrated to express CCR5 compared to controls indicating that CCR5 ligands may have an important influence over monocyte migration in COPD. Levels of CCR5 ligand CCL3 were significantly elevated in COPD sputum supernatant; IL-6 levels were positively associated with CCL3 indicating that IL-6 trans-signalling may mediate lung chemokine expression. Nevertheless, COPD monocytes demonstrated impaired migration towards sputum supernatant and reduced margination to pulmonary microvessels. Despite this, the number of alveolar macrophages in COPD was increased; however this was not likely to be related to self-replication owing to low alveolar macrophage Ki67 expression.

Indiana University-Purdue University Indianapolis (IUPUI) / Type 1 and Type 2 diabetes (T1D/T2D) share a common etiology that involves an increase in oxidative stress that leads to dysfunction and subsequent β cell death. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids to form lipid metabolites involved in a variety of biological functions including cellular oxidative stress response. On the other hand, Interleukin 6 (IL-6) signaling has been demonstrated to be protective in islets. In this study, we explored the effect of lipoxygenase enzymes 12-Lipoxygenase, 12/15 Lipoxygenase and IL-6 on β cell function and survival in mice using both STZ and high-fat diet (HFD) models of diabetes. Alox12-/- mice showed greater impairment in glucose tolerance following STZ and HFD compared to wild-type mice (WT), whereas Alox15-/- were protected against dysglycemia. These findings were accompanied by evidence of islet oxidative stress in Alox12-/- mice and reduced oxidative stress in Alox15-/- mice, consistent with alterations in the expression of antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12-/- mice showed a compensatory increase in Alox15 gene expression and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. IL-6 was able to significantly attenuate the generation of reactive oxygen species by proinflammatory cytokines in human pancreatic islets. Furthermore, we find that IL-6 regulates the master antioxidant response protein NRF2. Collectively these results show that loss of Alox12 activates a compensatory increase in Alox15 that sensitizes β cells to oxidative stress and signaling by IL-6 is required for maximal antioxidant response under conditions of increased ROS formation, such as obesity.

Die Interleukin-6-Zytokinfamilie vermittelt pleiotrope physiologische und pathologische Wirkungen. Einerseits sind die Zytokine bei inflammatorischen Prozessen involviert, an-dererseits spielen sie auch eine Rolle in der Entwicklung und Differenzierung verschie-dener Zelltypen. Zusätzlich wirken einige von ihnen, vor allem das Interleukin-6 selbst, durch Induktion der Akute-Phase-Reaktion auf die Leber. Gemeinsames Kennzeichen der Familie der Interleukin-6-Zytokine ist die Wirkung über Rezeptorkomplexe, die als signaltranszudierende Untereinheit das Glykoprotein 130 enthalten. Ein Schwerpunkt dieser Arbeit liegt darauf, wie verschiedene toxische und inflammatorische Schädigun-gen von Zellen die Endozytose des Glykoproteins 130 beeinflussen. Es sind wichtige Bereiche in der Aminosäuresequenz des Proteins beschrieben, die eine zentrale Rolle in den Abläufen der Internalisierung via Clathrin-vermittelter Endozytose einnehmen. Re-sultate dieser Arbeit legen nahe, dass die Aktivierung der p38-Kinase durch schädigende Stimuli eine mögliche Reduzierung der Zelloberflächenexpression des Glykoproteins 130 induzieren kann. Durch Versuche mit gp130-mutanten Zellen kann zudem die es-sentielle Bedeutung eines im zytoplasmatischen Bereich von gp130 lokalisierten Dileu-cin-Motivs für die Zelloberflächenexpression weiter bestätigt werden. Auch der Einfluss des Zytoskeletts auf eine der Situation angepasste Endozytose wird durch den Einsatz verschiedener Zytoskelettinhibitoren nahegelegt. Der zweite Schwerpunkt dieser Arbeit ist die Untersuchung der Prozesse während der hepatischen Ischemia-Reperfusion Injury. Die hepatische Ischemia-Reperfusion Injury ist ein hochkomplexer Prozess aus dem Zusammenspielen sowohl physio- als auch pa-thologischer Reaktionen des Organismus. Im Rahmen dieses Prozesses ist eine leber-schützende Wirkung des Interleukin-6 über Vermittlung des JAK/STAT-Signalwegs und der Induktion von Akute-Phase-Proteinen in der Literatur vorbeschreiben. In Versuchen dieser Arbeit mit murinen Leberproben, kann gezeigt werden, dass durch die Ischämie und die anschließende Reperfusion eine gesteigerte RNA-Expression des Interleukin-6, des gp130s sowie der Akutphase-Proteine induziert wird. Jedoch ist dieser Anstieg auch für scheinoperierte Tiere nachweisbar, sodass die hier gezeigten Ergebnisse klar zeigen, dass für weiterführende Versuche zur IRI in der Zukunft spätere Zeitpunkte nach dem Abklingen der durch die OP-bedingten Akutphase gewählt werden müssen. / The interleukin-6 family cytokines mediate pleiotropic physiological and pathological effects. On the one hand, the cytokines are involved in inflammatory processes, on the other hand, they also play a role in the development and differentiation of various cell types. In addition, some of them, especially interleukin-6 itself, act on the liver by inducing the acute phase reaction. A main feature of these cytokines is their effect on the orga-nism via receptor complexes that contain glycoprotein 130 as a signal-transducing sub-unit. One focus of this work is on how various toxic and inflammatory damages of cells influence the endocytosis of glycoprotein 130. There are several important regions in the amino acid sequence of the protein described that are involved in the processes of inter-nalization via clathrin-mediated endocytosis. Results of this work suggest that activation of p38 kinase by damaging stimuli can induce a possible reduction in cell surface ex-pression of glycoprotein 130. In addition, experiments with gp130 mutant cells further confirm the importance of a dileucine motif localized in the cytoplasmic part of gp130 for the expression on the cell surface. The influence of the cytoskeleton on endocytosis adapted to the situation is also suggested by the use of various cytoskeletal inhibitors. The second focus of this work is the investigation of the processes during hepatic ische-mia-reperfusion injury. Hepatic ischemia-reperfusion injury is a highly complex process resulting from the interplay of both physiological and pathological reactions of the orga-nism. Within this process, a hepatoprotective effect of interleukin-6 via mediation of the JAK/STAT signaling pathway and induction of acute phase proteins has been described in the literature. In experiments of this work with murine liver samples, it can be shown that an increased RNA expression of interleukin-6, gp130 and acute phase proteins is induced by ischemia and subsequent reperfusion. However, this increase is also de-tectable in sham-operated animals so that the results shown here clearly indicate that for further IRI experiments in the future, later time points after the acute phase caused by the surgery has subsided must be selected.

Adipöse Patienten und Typ-2-Diabetiker zeigen ein erhöhtes Risiko für Harnwegsinfekte. Die Ursache der höheren Prävalenz ist noch nicht nachhaltig geklärt. Bekannt ist, dass Typ-2-Diabetiker erhöhte Konzentrationen freier Fettsäuren im Blut aufweisen. Der veränderte Fettstoffwechsel könnte neben bakteriellen Ursachen ein möglicher Grund für abakterielle Entzündungsreaktionen der Harnblase sein. Zur Prüfung dieser Hypothese wurden zeit- und konzentrationsabhängig kultivierte humane Detrusormyozyten im Vergleich zur Lipopolysaccharid (LPS) induzierten Entzündungsreaktion mit Palmitat stimuliert. Es wurde geprüft, ob eine autokrine und/oder endokrine Regulation des IL-6-Signalwegs vorliegt. Im Fokus standen insbesondere die IL-6- und MCP-1-Expression und deren möglichen regulatorischen Proteine gp80, gp130, NF-κB, STAT3, SOCS3 und MEK1. Die Stimulationsversuche mit LPS und Palmitat zeigen einen differenten zeit- und konzentrationsabhängigen Effekt auf die IL-6- und MCP-1-Expression in den humanen Detrusormyozyten. LPS und Palmitat induzieren eine zeitabhängige autokrine Regulation der IL-6-Signalkaskade über phosphoryliertes STAT3 und Feedback-mechanismen via SOCS3. Sowohl LPS als auch Palmitat bewirken über 48h eine mögliche endokrine Regulation des IL-6-Signalwegs. Zusammenfassend zeigt die Palmitatstimulation zeit- und konzentrationsabhängig einen stärkeren Effekt auf die IL-6-Signalwirkung als die Stimulation mit LPS. / Background: Urinary tract infections (UTI) are more frequent in type-2 diabetes mellitus patients than in subjects with normal glucose metabolism. The mechanisms underlying this higher prevalence of UTI are unknown. However, cytokine levels are altered in diabetic patients and may thus contribute to the development of UTI. Increased levels of free fatty acids (FFA), as observed in obese patients, can induce IL-6 production in various cell types. Therefore we studied the effects of the free fatty acid palmitate and bacterial lipopolysaccharide (LPS) on interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) expression and secretion in cultured human bladder smooth muscle cells (hBSMC). Methodology/Principal Findings: Biopsies were taken from patients undergoing cystectomy due to bladder cancer. Palmitate or LPS stimulated hBSMC were analysed for the production and secretion of the IL-6, gp80, gp80soluble, gp130, MCP-1, pSTAT3, SOCS3, NF-kB and SHP2 by quantitative PCR, ELISA, Western blotting, and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-kB and MEK1 in IL-6 and MCP-1 regulation. Palmitate upregulates IL-6 mRNA expression and secretion via NF-kB dependent pathways in a concentration- and timedependent manner. MCP-1 was moderately upregulated by palmitate but was strongly upregulated by LPS involving NF-kB and MEK1 dependent pathways. Soluble IL-6 receptor (gp80soluble) was downregulated by palmitate and LPS, while membrane-bound gp80 was moderately upregulated. LPS increased SOCS3 and SHP2, whereas palmitate only induced SOCS3. Secondary finding: most of the IL-6 is secreted. Conclusions/Significance: Bacterial infection (LPS) or metabolic alterations (palmitate) have distinct effects on IL-6 expression in hBSMC, (i) short term LPS induced autocrine JAK/STAT signaling and (ii) long-term endocrine regulation of IL-6 by palmitate. Induction of IL-6 in human bladder smooth muscle cells by fatty acids may represent a pathogenetic factor underlying the higher frequency and persistence of urinary tract infections in patients with metabolic diseases.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

No description available.

In vitro systems like those used for in vitro embryo production are invaluable for our understanding of embryogenesis and the processes that regulate it. However, extensive research has also highlighted that in vitro produced embryos negatively differ from their in vivo counterparts in various ways. Not surprisingly, there is ~20% decrease in pregnancy success from pregnancies established using in vitro produced embryos. Therefore, much research has relied on attempting to produce a better in vitro embryo that more closely resembles their in vivo counterparts. Our laboratory has investigated this by supplementing a cytokine, interleukin-6 (IL6), during in vitro embryo culture. My dissertation work expands upon those initial efforts by answering more detailed questions related to the biological role of IL6 during cattle embryogenesis. In the work presented herein, IL6 supplementation during in vitro culture was able to transform the transcriptome of resulting conceptuses post embryo transfer. The transcriptome of these conceptuses included an abundance of genes associated with survival. Indeed, we witnessed IL6-treated conceptuses resulted in a 20% increased survival rate and were longer than their non-treated counterparts. In the second research project, we employed CRISPR-Cas9 genome editing technology to understand the embryo phenotype after part of the IL6 receptor responsible for signal transduction, interleukin-6 signal transducer (IL6ST), is disrupted. We discovered that IL6ST is required for development before the blastocyst stage. In addition, IL6ST disrupted blastocysts, presumed to contain wildtype, presented with severe, abnormal morphology. Not only did this group of embryos have decreased ICM and TE cell numbers, but they also had an increased occurrence of cells within the TE region that were negative for its traditional marker, CDX2. This suggests IL6ST is likely involved in a pathway responsible for determining cell fate identity at the blastocyst stage. Collectively, IL6 in cooperation with IL6ST, is a key controller of embryogenesis in cattle. / Doctor of Philosophy / There are major events that an embryo must successfully advance from to continue development to form into an organism capable of survival after birth. Over 30% of pregnancies in cattle and humans will fail within the first 30 days of gestation. This time period coincides with several key developmental events that ultimately modify the morphology of the growing embryo. Our laboratory primarily focuses on embryo development around the blastocyst stage. If an embryo advances to this stage, it has a greater likelihood of maintaining viability. Therefore, my dissertation research has focused on early embryonic development from the time of first cleavage (~day 2 of gestation) through embryo elongation (~day 15 of gestation), which encompasses the blastocyst stage. Within this time frame, I have been investigating embryonic effects after supplementation of a protein, interleukin-6 (IL6). Previously, our laboratory has identified IL6 to cause favorable impacts on the developing embryo, but its mode of action was unknown. Therefore, my dissertation research has investigated the mechanistic actions of IL6, and its beta receptor subunit, interleukin-6 signal transducer (IL6ST). In my first research project, we discovered that supplementing IL6 during in vitro embryo culture resulted in increased embryo elongation and survival. In my second research project, we found IL6ST is an absolute requirement for embryo survival to the blastocyst stage. Together, these results indicate IL6 is a very important protein needed for sustained pregnancy viability.