Das humorale Immungedächtnis wird von reifen Plasmazellen des Knochenmarks vermittelt, welche bei Immunreaktionen aus aktivierten B-Lymphozyten gebildet werden. Dabei sind im Blut Plasmablasten als unmittelbare Vorläufer der Plasmazellen nachweisbar, die von dort aus in das Knochenmark einwandern. Anhand der durchflusszytometrischen Detektion spezifischer Plasmablasten gelang es hier, das simultane Auftauchen von Wellen neu generierter, migratorischer Plasmablasten und reifer, nicht-migratorischer Plasmazellen im Blut eine Woche nach einer Tetanusimpfung nachzuweisen. Plasmablasten und Plasmazellen lagen stets im Gleichgewicht vor, wodurch auf die stöchiometrische Mobilisierung reifer Plasmazellen des Knochenmarks durch systemisch induzierte Plasmablasten geschlossen wurde. Ein solcher Verdrängungsmechanismus wird hier erstmalig als Anpassungsmechanismus des humoralen Immungedächtnisses dargestellt, der die Aufnahme neuer Spezifitäten in das Gedächtnis unter Wahrung der Stabilität präexistierender Spezifitäten erlaubt. Anders als systemisch induzierte Plasmablasten, weisen Plasmablasten, die im immunologischen Ruhephase zirkulieren, Kennzeichen mukosaler Immunreaktionen auf: sie exprimieren IgA sowie die mukosalen Zellmigrationsrezeptoren alpha4beta7-Integrin und CCR10. Wahrscheinlich wandern sie in mukosale Plasmazelldepots ein und interferieren nicht mit den Plasmazellen des Knochenmarks, sodass die Stabilität des humoralen Gedächtnisses in der Ruhephase gewahrt bleibt. Eine Anpassung des humoralen Gedächtnisses findet somit nur im Rahmen systemischer Immunreaktionen statt. Bei splenektomierten Patienten und unter der B-Zell-Depletionstherapie bei Rheumapatienten bleiben mukosale Plasmablasten im Blut nachweisbar. Dies belegt deren autonome Bildung aus mukosalen, therapie-refraktären B-Zellen. Insgesamt wird hier eine bisher unbeachtete Komplexität menschlicher peripherer Plasmablasten und Plasmazellen und ihren Beziehungen zum humoralen Immungedächtnis dargestellt. / Humoral memory, i.e. persistence of specific antibody titers, is provided by plasma cells in the bone marrow, which are generated from activated B cells during immune responses. At this, immediate plasma cell precursors, the plasmablasts, migrate via the blood to the bone marrow. Using cytometric detection of antigen-specific plasmablasts, synchronous circulation of waves of recently generated, migratory plasmablasts and non migratory plasma cells with a mature phenotype was demonstrated one week after tetanus vaccination. Circulating plasmablast and plasma cell numbers were always in homeostasis, so that the stoichiometric mobilization of old bone marrow plasma cells by recently generated plasmablasts was hypothesized. This plasma cell replacement mechanism is herein described for the first time as an adaption mechanism of the humoral memory that allows incorporation of new antibody specificities while maintaining pre-existing ones. In immunological steady state, very low numbers of plasmablasts are detectable in any donor. These express IgA and receptors for mucosal homing, alpha4beta7 integrin and CCR10, and therefore most likely migrate into mucosal plasma cell depots and do not interfere with plasma cells of the bone marrow, preserving the stability of humoral memory during steady state. Hence, adaption of humoral memory is only possible during systemic immune reactions. Circulating mucosal plasmablasts produced during steady state remain detectable in patients with rheumatoid arthritis during B cell depletion therapy as well as in asplenic patients. Hence, this type of plasmablasts is self-sufficiently generated from mucosal B cells that are refractory to B cell depletion therapy. This work demonstrates a hitherto disregarded complexity of peripheral plasmablast and plasma cell subsets in healthy humans, with implications for the regulation of induction and maintenance of humoral memory.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.

Einführung - Periphere Blutmonozyten spielen eine Rolle in der Pathogenese der Arteriosklerose. Bei spontan hypertensiven Ratten wurden im Vergleich zu normotonen Wistar-Kyoto-Ratten signifikant erhöhte Zahlen aktivierter Monozyten beobachtet [Liu 1996]. Wir untersuchten Monozyten von Patienten mit essentieller Hypertonie und von gesunden Probanden hinsichtlich möglicher Unterschiede in der Aktivierung anhand ihrer Adhäsion an von uns kultivierte (HAEC) und isolierte (HSVEC, HUVEC, HUAEC) humane Endothelzellen. Wir bestimmten die Adhäsionsmoleküle ICAM-1, VCAM-1 sowie E-Selektin und analysierten die Superoxidfreisetzung von humanem Endothel und Monozyten. Methoden - Humane periphere Blutmonozyten wurden mittels Dichtegradienten-zentrifugation und Plastikadhärenz isoliert und mit LPS, Angiotensin II (Ang II), und Ang II nach Vorinkubation mit dem AT1-Antagonisten Eprosartan stimuliert. Die Monozyten wurden auf einschichtigem Endothel ausgesät und die Adhäsion als Prozentsatz der initial gesäten Zellen erfasst. Die durch PMA induzierte Superoxidfreisetzung des Endothels oder der Monozyten wurde mittels Chemilumineszenz bestimmt. Ergebnisse - Monozyten von Patienten mit essentieller Hypertonie hefteten sich im Vergleich zu gesunden Probanden spontan und nach Stimulation mit Ang II signifikant verstärkt an HAEC und HUVEC-Monolayer an. Die Spiegel von ICAM-1 und VCAM-1 waren bei Patienten mit arterieller Hypertonie im Vergleich zu den gesunden Kontrollpersonen signifikant erhöht. Die Chemilumineszenzaktivität postkonfluenter Endothelzellen erhöhte sich nach Stimulation mit Ang II im Vergleich zur Messung ohne vorherige Stimulierung. Nach Stimulation mit PMA oder mit Ang II wurden bei Hypertonikern signifikant höhere Werte für die Chemilumineszenzaktivität der Monozyten gemessen als bei gesunden Kontrollpersonen. Schluss - Mit diesen Versuchen an humanen Monozyten und Endothelzellen wurde ein weiterer Beweis für die Aktivierung der Monozyten von Patienten mit essentieller Hypertonie erbracht. Meine Ergebnisse unterstützen die Sicht einer Monozytenbeteiligung an der Pathogenese atherosklerotischer Läsionen, die mit arterieller Hypertonie in Zusammenhang stehen. / Introduction - Peripheral blood monocytes are involved in the pathogenesis of atherosclerosis. Significantly elevated numbers of activated monocytes were observed in spontaneously hypertensive rats compared to those in normotensive Wistar-Kyoto rats [Liu 1996]. We isolated and cultivated human endothelial cells and examined monocytes from patients with arterial hypertension and healthy volunteers to identify possible differences in their adhesion behavior to human endothelial cells (HAEC, HSVEC, HUVEC, HUAEC). We determined the levels of ICAM-1, VCAM-1 and E-selectin, and we analyzed superoxide release by human endothelium and human monocytes. Methods - Peripheral blood monocytes were isolated by density gradient centrifugation and plastic adherence. Subsets of the samples were stimulated with LPS, Angiotensin II, Angiotensin II following preincubation with the AT1-antagonist eprosartan or left without a stimulant. After incubation, monocytes were seeded onto confluent monolayers of human aortic endothelial cells and the adhesion was determined as the percentage of the initially seeded cells. Oxygen species release induced by PMA was analyzed for endothelium and monocytes in suspension by chemiluminescence. Results - Peripheral blood monocytes of patients with essential hypertension performed a significantly increased spontaneous adhesion and adhesion following stimulation with Angiotensin II to HAEC- and HUVEC-monolayers. Levels of human soluble adhesion molecules ICAM-1 and VCAM-1 were significantly raised in hypertensive patients. Chemiluminescence activity of post confluent endothelial cells was increased after stimulation with Angiotensin II compared to the measurement before stimulation. Following stimulation with PMA or Angiotensin II, significantly higher chem-iluminescence levels were measured in hypertensive patients compared to healthy volunteers. Conclusion - These data indicate that monocytes of patients with essential hypertension may be preactivated. My results support the view of a monocyte involvement in the pathogenesis of atherosclerotic lesions that are related to arterial hypertension.