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Characterization of tolerogenic rat bone marrow-derived dendritic cells and regulatory T cells
(2010)
Tolerogenic dendritic cells (DC) and regulatory T (Treg) cells are able to prevent destructive immune responses. There is reason to hope that it may soon be possible to use DC and Treg cells to suppress immune responses antigen-specific, not only after transplantation, but also in the case of autoimmunity and allergy. At the moment, the generation of such cell types is very time-consuming and not suitable for clinical routine. In addition, it is not yet fully understood how these cells elicit a desired protective immune response in vivo and how the risks of an excessive immune suppression can be managed. The rat is one of the most important animal models in biomedical research. It is therefore surprising that tolerogenic DC and Treg cells in particular have not been more thoroughly investigated in this model. Thus, the aim of the present study was to systematically characterize these immune cells and investigate their impact on the immune system. Tolerogenic DC were generated from bone marrow precursors cultured with GM-CSF and IL-4 (= IL-4 DC). The proportion of naturally occurring Treg cells with a CD4posCD25posFoxp3pos phenotype comprises approximately 5-8% of the peripheral CD4pos T cells. The characterization of IL-4 DC revealed an up to 26-fold reduced expression of surface molecules such as MHC class II molecules, CD80, CD86, ICAM-1 and CD25 in comparison to mature splenic DC (S-DC). This low expression did not change when the cells where stimulated with different maturation-inducing signals such as replating, LPS, TNF- α and CD40L. Thus, these cells possess a robust phenotype resistant to maturation-inducing stimuli. IL-4 DC take up antigen via endocytosis and are not able to activate naïve T cells or to restimulate antigen-specific T cells. Furthermore, they are able to inhibit and prolongate mature S-DC induced T cell proliferation as well as mature S-DC induced restimulation of antigen-specific T cells, respectively. Thereby, the T cell proliferation was reduced up to 95%. This strong inhibitory effect was mediated within 24 hours in association with a reduced cytokine production (IL-2 about 49% and IFN-γ about 92%). The inhibitory properties of IL-4 DC don´t seem to be caused exclusively by the reduced expression of co-stimulatory molecules. In this study, the detection of the inhibitory molecules PD-L1 and PD-L2 on IL-4 DC suggests they have an impact on mediating inhibitory signals to the T cells. In addition, a suppressive effect of soluble factors was shown. The supernatant of one million IL-4 DC, collected after a 24 hour culture, suppressed mature S-DC induced proliferation of naïve T cells by about 90%. TGF-β, which was detected in the supernatant (up to 300 pg/ml), appears to be the causing soluble factor for this immune inhibition. By contrast, the supernatants of mature S-DC, which did not inhibit the activation of T cells, showed a TGF-β concentration of only about 100 pg/ml. The cytotoxic nitric oxide does not contribute to the IL-4 DC-mediated inhibition of T cell proliferation. The NO synthase inhibitor NMMA reduced the amount of NO by about 50%, but the decreased NO levels did not influence T cell proliferation. Indeed, IL-4 DC are not able to induce T cell proliferation, but this doesn´t mean that there is no change on the molecular level. For instance, T cells co-cultured with IL-4 DC during a first culture are not able to proliferate in the presence of mature S-DC during a second culture. This anergic-like state, however, could be abolished by adding exogenous IL-2. In addition, T cells co-cultured with IL-4 DC are able to inhibit the activation of naïve T cells. Naïve and activated T cells were not able to inhibit the mature S-DC induced T cell proliferation. This observation suggests the induction of Treg cells and was investigated in more detail. Indeed, flow cytometric analysis showed a 1.6-fold expansion of CD4posCD25posFoxp3pos T cells from naturally occurring Treg cells in the presence of IL-4 DC. Thereby, the expansion of CD4posCD25posFoxp3pos T cells occurs independently of the maturation state of DC. Both immature IL-4 DC as well as mature S-DC were able to expand the percentage of naturally occurring Treg cells. However, Treg cells pre-incubated with mature S-DC demonstrated a diminished inhibitory effect compared to Treg cells pre-incubated with IL-4 DC. Treg cells pre-incubated with IL-4 DC were able to inhibit the activation of naïve T cells. In this study it was shown that the regulatory potential of DC cannot be deduced solely by their phenotype or maturation state. Other factors, such as functional properties, need to taken into consideration, too. The induction of Treg cells with suppressive properties induced by in vitro generated tolerogenic IL-4 DC might provide an important mechanism for the maintenance of peripheral tolerance. However, for clinical application further investigation is necessary, not only to understand the interactions between tolerogenic DC and Treg cells, but also to investigate the impact of the transfer of a larger quantity of regulatory cells on the immune system of the recipient.
Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that is engaged in regulating versatile cellular processes that are pivotal for development and homeostasis of most tissues in multicellular organisms. TGF-ß signal transduction is initially propagated by binding of TGF-ß to transmembrane serine/threonine kinase receptors, designated TßRI and TßRII. Upon activation, the receptors phosphorylate Smad proteins which serve as downstream mediators that enter the nucleus and finally trigger transcriptional responses of specific genes. During the past years, it became evident that signaling cascades do not proceed in a linear fashion but rather represent a complex network of numerous pathways that mutually influence each other. Along these lines, members of the TGF-ß superfamily are attributed to synergize with neurotrophins. Together, they mediate neurotrophic effects in different populations of the nervous system, suggesting that an interdependence exists between TGF-ßs on the one hand and neurotrophins on the other. In the present work, the crosstalk of NGF and TGF-ß/Smad signaling pathways is characterized in rat pheochromocytoma cells (PC12) which are frequently used as a model system for neuronal differentiation. PC12 cells were found to be unresponsive to TGF-ß due to limiting levels of TßRII. However, stimulation with NGF results in initiation of Smad-mediated transcription independent of TGF-ß. Binding of NGF to functional TrkA receptors triggers activation of Smad3. This NGF-dependent Smad activation occurs by a mechanism which is different from being induced by TGF-ß receptors in that it provokes a different phosphorylation pattern of R-Smads. Together with an inferior role of TßRI, Smad3 is proposed to serve as a substrate for cellular kinases other than TßRI. Based on the presented involvement of components of both, the MAPK/Erk and the TAK1/MKK6 cascade, signal mediators of these pathways rank as candidates to mediate direct activation of Smad3. Smad3 is subsequently translocated to the nucleus and activates transcription in a Smad4-dependent manner. Negative regulation is provided by Smad7 which was found to act as a potent inhibitor of Smad signaling not only in TGF-ß- but also in NGF-mediated cascades. The potential of NGF to activate the Smad pathway independent of TGF-ß might be of special importance in regulating expression of genes that are essential for the development and function of neuronal cells or of other NGF-sensitive cells, in particular those which are TGF-ß-resistant.
Die osteoinduktive Wirkung von „Bone morphogenetic protein 2“ (BMP-2) und der regulative Einfluss des Faktors „Transforming growth factor beta 1“ auf das physiologische Remodelling des Knochens sind durch zahlreiche In-vitro- und In-vivo-Untersuchungen belegt. Die gentechnische Modifikation der N-terminalen Aminosäuresequenz von rekombinantem, im bakteriellen System (E. coli) exprimiertem BMP-2 (rh-BMP-2) führt zu rh-BMP-2-Varianten mit verstärktem (T3 und T4) bzw. nicht mehr messbarem (EH-BMP) Bindungsvermögen zur extrazellulären Matrix und verändert somit die Retentionszeit dieser Morphogene im Gewebe. In der vorliegenden Arbeit sollte die osteoinduktive Potenz der genannten BMP-Varianten (T3, T4, EH-BMP) mit der ihres Wildtyps rh-BMP-2 verglichen werden. Zudem war zu untersuchen, ob ein Zusammenhang zwischen applizierter Wirkstoffmenge und dem Ausmaß der induzierten Osteogenese vorliegt, und zu zeigen, dass dieser durch gemeinsame Applikation der verwendeten Morphogene mit dem nicht osteoinduktiven Wachstumsfaktor rh-TGF-ß-1 zu steigern ist. Hierzu wurden zylinderförmige (Länge l=10mm, Durchmesser Ø=5mm), bovine EXKK-Träger (extrahiertes, xenogenes Knochen-Kollagen) mit den genannten BMPs, TGF-ß-1 und Kombinationen dieser Wachstumsfaktoren bestückt und für 28 Tage in die Oberschenkelmuskulatur von 50 Sprague-Dawley-Ratten implantiert. Jedes Tier erhielt eine mit 2µg bzw. 4µg des jeweiligen BMPs dotierte Probe und einen zusätzlich, entsprechend dem Massenverhältnis von 20:1 mit 100ng, bzw. 200ng rh-TGF-ß-1 beladenen Träger auf der kontralateralen Seite. Als Kontrolle wurden unbehandelte bzw. nur mit rh-TGF-ß-1 (10µg und 20µg) dotierte EXKK-Zylinder eingebracht. Durch am 5., 11., 17., 23. und 28. Tag post OP durchgeführte Röntgenkontrollen konnten Beginn, Verlauf und Geschwindigkeit der ablaufenden Osteoinduktionsprozesse bewertet werden. Mittels zwei- und dreidimensionaler radiologischer Methoden, labortechnischer und histologischer Auswertungsschritte wurden zudem die in den am 28. Tag post OP explantierten Zylindern vorhandenen Knochenformationen auf ihre Menge, ihre Dichte, ihren Kalziumgehalt und ihre histologische Qualität hin untersucht. Durch Röntgen der histologischen Schnitte wurde ein direkter Vergleich histologischer Strukturen mit ihrer röntgenologischen Darstellung möglich. Für die undotierten EXKK-Träger und die nur mit rh-TGF-ß-1 rekonstituierten Proben ergab sich weder nach röntgenologischer noch nach histologischer Auswertung ein Anhalt auf osteoinduktive Aktivität. Alle außer den mit EH-BMP dotierten Proben zeigten schon ab dem 11. Tag post OP signifikante Knochenneubildungen. BMP-2 und T4 führten, gefolgt von T3, insgesamt zur schnellsten und mengen-, kalzium- und dichtemäßig am stärksten ausfallenden Osteoneogenese. Bei Verwendung von 4µg Morphogen und Zusatz von rh-TGF-ß-1 erreichten rh-BMP-2 und T4 bereits am 23. Tag post OP die auch zu Versuchsende ermittelten Werte. Die Variante EH-BMP lieferte deutlich weniger Knochen und relativ unkonstante Ergebnisse. Die Erhöhung der implantierten Proteinmenge auf 4µg und der Zusatz von rh-TGF-ß-1 führten im Mittel in allen Auswertungen zur Steigerung von Menge, Dichte, Kalziumgehalt und Bildungsgeschwindigkeit des induzierten Knochens. Die osteoinduktive Ausbeute von BMP-Varianten mit verbesserter Matrixbindung und verlängerter Retentionszeit im Lagergewebe ist für klinische Belange einer einfachen Dosissteigerung der BMP-Wildtypen vorzuziehen. Die Varianten führen früher und bei Verwendung geringerer Morphogenmengen zu einer dem Wildtyp äquivalenten Knochenneubildung. Auch die mitogenen Effekte von rhTGF-ß-1 auf das durch die BMPs determinierte Gewebe haben potenzierend wirkenden Einfluss auf osteoinduktive Effekte. Das Vorhandensein weiterer, an der Regulation des Knochenmetabolismus beteiligter Faktoren deutet auf noch verborgenes therapeutisches Potential hin.