Zelluläre Interaktionen während der plazentaren Vaskularisation : Modell der plazentaren Vaskulogenese unter besonderer Berücksichtigung der Rolle des Trophoblasten

Die Entwicklung und die uneingeschränkte Funktion des plazentaren Gefäßsystems sind für die normale Embryonalentwicklung und das fetale Wachstum von entscheidender Bedeutung. Die plazentare Gefäßbildung beginnt zwischen dem 21. und 32. Tag p.c. und wird durch die Differenzierung von Angioblasten und den Aufbau des primitiven Gefäßnetzwerkes charakterisiert. Durch die Expression zahlreicher angiogen wirkender Substanzen scheint der Trophoblast bei der Initialisierung der Angioblasten essenziell zu sein. Es wird postuliert, dass der Trophoblast über Wechselwirkungen mit den Vorläuferzellen (Angioblasten) an der Entstehung des plazentaren Gefäßsystems ursächlich beteiligt ist. Die Herkunft der plazentaren Angioblasten ist umstritten. Als mögliche Quellen werden sowohl das Zottenmesenchym, als auch die hämatopoetischen Organe genannt. Das Nabelschnurblut enthält große Mengen von Vorläuferzellen, die sowohl zur hämatopoetischen, als auch zu endothelialen Zellen differenzieren können. Im Rahmen der vorliegenden Arbeit wurden Interaktionen zwischen Trophoblasten und CD133+-Zellen untersucht. Um diese Untersuchungen zu ermöglichen, wurden Methoden zur primären Isolierung von Vorläuferzellen aus Nabelschnurblut und Trophoblasten aus frühem Plazentagewebe etabliert. Es konnte gezeigt werden, dass CD133+-Zellen wichtige Pluripotenzmarker wie Oct-4, SOX-1, SOX-2, FGF-4 und REX-1 exprimieren und die Fähigkeit besitzen sich in endotheliale Zellen zu differenzieren. Darüber hinaus exprimieren diese Zellen ähnliche Marker wie die villösen Angioblasten und konnten als plazentare Angioblasten betrachten werden. Der Einfluss von Trophoblasten auf die funktionellen Eigenschaften und Differenzierungsprozesse der CD133+-Zellen wurde durch die Kultivierung der Zellen in konditioniertem Trophoblastenmedium untersucht. Es konnte gezeigt werden, dass die Migration der CD133+-Zellen unter dem Einfluss von konditioniertem Trophoblastemedium stark erhöht war. Konditioniertes Trophoblastenmedium induzierte zudem konzentrationsabhängig die Proliferation der CD133+-Zellen und wies eine antiapoptotische Wirkung auf. Durch die Kultivierung der CD133+-Zellen in konditioniertem Trophoblastenmedium konnte festgestellt werden, dass CD133+-Zellen zu Zellen mit monozyten/makrophagen-spezifischen Markern differenzieren. Die Zellen zeigten eine negative Regulation von Stammzell spezifischen Markern, aber eine positive Regulation von CD11b- und CD14-Rezeptoren. Die unter dem Einfluss von Trophoblastenmedium differenzierten CD11b+CD14+-Zellen zeigten eine gleichzeitige Expression des CD31-Rezeptors. Die langzeitige Kultivierung der CD133+-Zellen im Trophoblastenmedium führte zu der Expression von CD68 und VEGFR-3. Die unter dem Einfluss von Trophoblasten entstandenen Zellen, konnten in die von HUVEC gebildeten Kapillarnetze eingebaut werden. Es kann angenommen werden, dass Stammzellen durch den Kontakt mit von Trophoblasten freigesetzten Faktoren die Fähigkeit erwerben, sich an der Vaskulogenese zu beteiligen. Da hCG ein trophoblastspezifischer Faktor ist, sollte seine Wirkung auf CD133+-Zellen untersucht werden. Es konnte gezeigt werden, dass der hCG-Rezeptor von CD133+-Zellen exprimiert wird. Durch die Zugabe von hCG wurde die Proliferation der CD133+-Zellen dosisabhängig induziert. Ein Einfluss von hCG auf Apoptose und Migration der CD133+-Zellen konnte nicht beobachtet werden. Bei den Prozessen der Blutgefäßentwicklung spielen nicht nur humorale Faktoren eine wichtige Rolle, es sind auch die direkten Zell-Zell-Interaktionen zwischen Angioblasten und den benachbarten Zellen von entscheidender Bedeutung. Im Rahmen der vorliegenden Arbeit wurde ein komplexes dreidimensionales Sphäroid-Modell zur in vitro-Untersuchung der plazentaren Vaskulogenese, unter besonderer Berücksichtigung der Rolle des Trophoblasten etabliert. Die Ähnlichkeit des Modells zur in vivo-Situation ermöglicht die Erforschung der plazentaren Gefäßentwicklung auf einem physiologischen Niveau.Development of a functional placental vascular system of the placental vascular system is essential for normal embryonal and fetal growth. Placental vascular development begins between day 21 and 32 p.c. and is characterised by differentiation of angioblasts and construction of a primitive vascular network. The trophoblast, which expresses numerous substances, seems to be essential for the initialisation of angioblasts. It is postulated, trophoblast interact with precursor cells (angioblasts) and that this interaction leads to development of the placental vascular system. The origin of placental angioblasts is a controversial issue; possible sources are mesenchymal stroma of placental villi and hematopoetic organs. Cord blood contains a large number of precursor cells, which can differentiate either into hematopoetic or into endothelial cells. Cells expressing the CD133 antigen were isolated with magnetic beads. In this work the interaction of trophoblasts and CD133+cells was investigated. To enable this investigation, methods for isolating precursor cells from cord blood and trophoblasts from early placenta tissue were established. It was shown that CD133+cells express important markers of pluripotency such as Oct-4, SOX-1, SOX-2, FGF-4 and REX-1. CD133+cells were able to differentiate into various cell types including endothelial cells or neural cells. This leads to the assumption, that CD133+cells are potential placental angioblasts. The influence of trophoblast on functional properties and differentiation of CD133+cells was investigated by cultivation of CD133+cells in trophoblast conditioned medium. A strong increase of CD133+cells migration in trophoblast conditioned medium was shown. Furthermore, trophoblast conditioned medium dose dependently increased CD133+cells proliferation and inhibited apoptosis. Cultivation in trophoblast conditioned medium lead to differentiation of CD133+cells. Stem cell specific markers (e.g. CD133, CD117) were downregulated. The cells showed a positive expression of monocytic receptors (CD11b, CD14). Long term cultivation of CD133+cells in trophoblast conditioned medium lead to the expression of CD68 and VEGFR-3. Cells cultured under the influence of trophoblasts incorporate into capillary networks generated by HUVEC. It can be assumed, that factors released by trophoblasts enable stem cells to take part in vasculogenesis. hCG is one of the trophoblast specific angiogenic factors. CD133+cells express the hCG receptor. hCG dosedependently influences the proliferation of CD133+cells. Influence of hCG on apoptosis and migration was not observed. In vascular development, not only humoral factors, but also cell-cell-interactions between neighbouring cells are of critical importance. In this work a complex three dimensional spheroid model for in vitro investigation of placental vasculogenesis with regard to trophoblasts was established. The similarity of this model to the in vivosituation allowes investigation of placental vascular development under more physiological conditions

Skin TLR7 triggering promotes accumulation of respiratory dendritic cells and natural killer cells.

The TLR7 agonist imiquimod has been used successfully as adjuvant for skin treatment of virus-associated warts and basal cell carcinoma. The effects of skin TLR7 triggering on respiratory leukocyte populations are unknown. In a placebo-controlled experimental animal study we have used multicolour flow cytometry to systematically analyze the modulation of respiratory leukocyte subsets after skin administration of imiquimod. Compared to placebo, skin administration of imiquimod significantly increased respiratory dendritic cells (DC) and natural killer cells, whereas total respiratory leukocyte, alveolar macrophages, classical CD4+ T helper and CD8+ T killer cell numbers were not or only moderately affected. DC subpopulation analyses revealed that elevation of respiratory DC was caused by an increase of respiratory monocytic DC and CD11b(hi) DC subsets. Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a significant reduction of B lymphocytes. Analysis of cytokine responses of respiratory leukocytes after stimulation with Klebsiella pneumonia indicated reduced IFN-γ and TNF-α expression and increased IL-10 and IL-12p70 production after 7 day low dose skin TLR7 triggering. Additionally, respiratory NK cytotoxic activity was increased after 7d skin TLR7 triggering. In contrast, lung histology and bronchoalveolar cell counts were not affected suggesting that skin TLR7 stimulation modulated respiratory leukocyte composition without inducing overt pulmonary inflammation. These data suggest the possibility to modulate respiratory leukocyte composition and respiratory cytokine responses against pathogens like Klebsiella pneumonia through skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel, noninvasive means to modulate respiratory immunity

Prospectively defined murine mesenchymal stem cells inhibit Klebsiella pneumoniae-induced acute lung injury and improve pneumonia survival

Background: Numerous studies have described the immunosuppressive capacity of mesenchymal stem cells (MSC) but these studies use mixtures of heterogeneous progenitor cells for in vitro expansion. Recently, multipotent MSC have been prospectively identified in murine bone marrow (BM) on the basis of PDFGRa+ SCA1+ CD45- TER119- (PaS) expression but the immunomodulatory capacity of these MSC is unknown. Methods: We isolated PaS MSC by high-purity FACS sorting of murine BM and after in vitro expansion we analyzed the in vivo immunomodulatory activity during acute pneumonia. PaS MSC (1?×?106) were applied intratracheally 4 h after acute respiratory Klebsiella pneumoniae induced infection. Results: PaS MSC treatment resulted in significantly reduced alveolitis and protein leakage in comparison to mock-treated controls. PaS MSC-treated mice exhibited significantly reduced alveolar TNF-a and IL-12p70 expression, while IL-10 expression was unaffected. Dissection of respiratory dendritic cell (DC) subsets by multiparameter flow cytometry revealed significantly reduced lung DC infiltration and significantly reduced CD86 costimulatory expression on lung CD103+ DC in PaS MSC-treated mice. In the post-acute phase of pneumonia, PaS MSC-treated animals exhibited significantly reduced respiratory IL-17+ CD4+ T cells and IFN-gamma+ CD4+ T cells. Moreover, PaS MSC treatment significantly improved overall pneumonia survival and did not increase bacterial load. Conclusion: In this study we demonstrated for the first time the feasibility and in vivo immunomodulatory capacity of prospectively defined MSC in pneumonia

In vitro analysis of anti-HPA-1a dependent platelet phagocytosis and its inhibition using a new whole blood phagocytosis assay (WHOPPA)

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a serious bleeding condition mostly caused by the reaction between maternal anti-HPA-1a antibodies and fetal platelets. This reaction leads to Fc-dependent platelet phagocytosis. Although several serological methods have been developed to identify maternal antibodies, a reliable laboratory parameter as a prognostic tool for FNAIT severity is still lacking. In this study, we developed whole blood platelet phagocytosis assay (WHOPPA), a flow cytometry-based phagocytosis assay that uses a pH-sensitive fluorescent dye (pHrodo-SE) to analyze anti-HPA-1a-dependent platelet phagocytosis in whole blood. WHOPPA revealed a high phagocytosis rate for the anti-HPA-1a opsonized platelets by monocytes but not by neutrophils. Analysis of different monocyte populations showed that all monocyte subsets, including classical (CD14++CD16−), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes, were able to engulf opsonized platelets. A unique monocyte subset, termed shifted monocytes (CD14+CD16−), showed the highest phagocytosis rate and was detected after platelet engulfment. FcγR inhibition tests revealed that except for FcγRIIa, FcγRI and FcγRIII on monocytes were responsible for the phagocytosis of anti-HPA-1a opsonized platelets. Analysis of anti-HPA-1a antibodies from FNAIT cases (n = 7) showed the phagocytosis of HPA-1aa but not of HPA-1bb platelets by monocytes. The phagocytosis rate was highly correlated with bound antibodies measured by flow cytometry (p < 0001; r = 0.9214) and MAIPA assay (p < 0.001; r = 0.7692). The phagocytosis rates were equal for type I and II anti-HPA-1a antibodies recognizing the plexin–semaphoring–integrin (PSI) domain and PSI/epidermal growth factor 1 domain of β3 integrin, respectively. By contrast, type III anti-HPA-1a antibodies reacting with αvβ3 integrin did not induce platelet phagocytosis. Furthermore, effector-silenced mAbs against HPA-1a inhibited the phagocytosis of anti-HPA-1a opsonized platelets. In conclusion, WHOPPA is a reliable in vitro platelet phagocytosis assay that mimics the phagocytosis of anti-HPA-1a opsonized platelets in whole blood. This assay allows to prove platelet phagocytosis ex vivo and evaluate the inhibitory capacity of different inhibitors as therapeutically strategies for the prevention of fetal thrombocytopenia in FNAIT in the future

The Cyclophilin-Binding Agent Sanglifehrin A Is a Dendritic Cell Chemokine and Migration Inhibitor

Sanglifehrin A (SFA) is a cyclophilin-binding immunosuppressant but the immunobiology of action is poorly understood. We and others have reported that SFA inhibits IL-12 production and antigen uptake in dendritic cells (DC) and exhibits lower activity against lymphocytes. Here we show that SFA suppresses DC chemokine production and migration. Gene expression analysis and subsequent protein level confirmation revealed that SFA suppressed CCL5, CCL17, CCL19, CXCL9 and CXCL10 expression in human monocyte-derived DC (moDC). A systems biology analysis, Onto Express, confirmed that SFA interferes with chemokine-chemokine receptor gene expression with the highest impact. Direct comparison with the related agent cyclosporine A (CsA) and dexamethasone indicated that SFA uniquely suppresses moDC chemokine expression. Competitive experiments with a 100-fold molar excess of CsA and with N-Methyl-Val-4-cyclosporin, representing a nonimmunosuppressive derivative of CsA indicated chemokine suppression through a cyclophilin-A independent pathway. Functional assays confirmed reduced migration of CD4+ Tcells and moDCs to supernatant of SFA-exposed moDCs. Vice versa, SFA-exposed moDC exhibited reduced migration against CCL19. Moreover, SFA suppressed expression of the ectoenzyme CD38 that was reported to regulate DC migration and cytokine production. These results identify SFA as a DC chemokine and migration inhibitor and provide novel insight into the immunobiology of SFA

Protein Tyrosine Phosphatase Interacting Protein 51 (PTPIP51) mRNA Expression and Localization and Its In Vitro Interacting Partner Protein Tyrosine Phosphatase 1B (PTP1B) in Human Placenta of the First, Second, and Third Trimester

The cellular localization of protein tyrosine phosphatase 51 (PTPIP51) and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) was studied in human placentae of different gestational stages. The expression of PTPIP51 protein and mRNA was observed in the syncytiotrophoblast and cytotrophoblast layer of placentae from the first, second, and third trimesters. In contrast, PTP1B expression was restricted to the syncytiotrophoblast during all gestational stages. Cells of the cytotrophoblasts and parts of the syncytiotrophoblasts expressing high amounts of PTPIP51 were found to execute apoptosis as shown by TdT-mediated dUTP-biotin nick end labeling assay, cytokeratin 18f, and caspase 3 expression. PTPIP51 could also be traced in the endothelium and smooth muscle cells of placental arterial and venous vessels, identified by double immunostainings with antibodies directed against van Willebrand factor and α-smooth muscle actin. Some of these cells showing a high PTPIP51 reactivity were Ki67 positive, indicating proliferation. Additionally, a small population of placental CD14-positive macrophages and mesenchymal cells within the villous stroma were detected as PTPIP51 positive. Our data suggest that both proteins, PTPIP51 and PTP1B, play a role in differentiation and apoptosis of the cytotrophoblast and syncytiotrophoblast, respectively. Moreover, PTPIP51 may also serve as a cellular signaling partner in angiogenesis and vascular remodeling. (J Histochem Cytochem 57:143–153, 2009

In vitro analysis of anti-HPA-1a dependent platelet phagocytosis and its inhibition using a new whole blood phagocytosis assay (WHOPPA)

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a serious bleeding condition mostly caused by the reaction between maternal anti-HPA-1a antibodies and fetal platelets. This reaction leads to Fc-dependent platelet phagocytosis. Although several serological methods have been developed to identify maternal antibodies, a reliable laboratory parameter as a prognostic tool for FNAIT severity is still lacking. In this study, we developed whole blood platelet phagocytosis assay (WHOPPA), a flow cytometry-based phagocytosis assay that uses a pH-sensitive fluorescent dye (pHrodo-SE) to analyze anti-HPA-1a-dependent platelet phagocytosis in whole blood. WHOPPA revealed a high phagocytosis rate for the anti-HPA-1a opsonized platelets by monocytes but not by neutrophils. Analysis of different monocyte populations showed that all monocyte subsets, including classical (CD14++CD16−), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes, were able to engulf opsonized platelets. A unique monocyte subset, termed shifted monocytes (CD14+CD16−), showed the highest phagocytosis rate and was detected after platelet engulfment. FcγR inhibition tests revealed that except for FcγRIIa, FcγRI and FcγRIII on monocytes were responsible for the phagocytosis of anti-HPA-1a opsonized platelets. Analysis of anti-HPA-1a antibodies from FNAIT cases (n = 7) showed the phagocytosis of HPA-1aa but not of HPA-1bb platelets by monocytes. The phagocytosis rate was highly correlated with bound antibodies measured by flow cytometry (p < 0001; r = 0.9214) and MAIPA assay (p < 0.001; r = 0.7692). The phagocytosis rates were equal for type I and II anti-HPA-1a antibodies recognizing the plexin–semaphoring–integrin (PSI) domain and PSI/epidermal growth factor 1 domain of β3 integrin, respectively. By contrast, type III anti-HPA-1a antibodies reacting with αvβ3 integrin did not induce platelet phagocytosis. Furthermore, effector-silenced mAbs against HPA-1a inhibited the phagocytosis of anti-HPA-1a opsonized platelets. In conclusion, WHOPPA is a reliable in vitro platelet phagocytosis assay that mimics the phagocytosis of anti-HPA-1a opsonized platelets in whole blood. This assay allows to prove platelet phagocytosis ex vivo and evaluate the inhibitory capacity of different inhibitors as therapeutically strategies for the prevention of fetal thrombocytopenia in FNAIT in the future

Supernatant of SFA-exposed moDC induces reduced migration of activated moDC and CD4+ T cells.

<p>moDCs were generated in the presence of GM-CSF and IL-4 and activated for 12 h with LPS. CD4 T cells were isolated by microbead-sorting and activated for 16 h with CD3/CD28 mAbs. DC supernatant from SFA- or vehicle-exposed moDCs was harvested 12 h after LPS activation and added to the lower chamber of the transwell. The “SFA carry over control” consisted of supernatant of control-treated moDCs+1 µM SFA. Migration of cells was quantitated by flow cytometry. (A) Activated moDCs were inserted in the upper chamber of the transwell and migration was analysed after 4 h. (B) Activated CD4⁺-T cells were set in the upper chamber of the transwell and migration was analysed after 4 h. The spontaneous migration of cells was subtracted from the results (mean DCs: 1673; T cells: 8676). The results are representative for n = 9 (A) and n = 5 (B) independent experiments. Mean (± SEM) **p<0.01, *p<0.05 versus vehicle.</p

Dose-dependent suppression of CCL5, CCL17 and CCL19 in moDCs by SFA.

<p>Human moDC were exposed on day 5 with 10, 50 100, 250 and 500 nM of SFA and 4 hours later stimulated with 100 ng/mL LPS. CCL5 (A), CCL7 (B) and CCL19 (C) production were analyzed after 12 h stimulation by ELISA. Mean (± SEM) of n = 3 (A) and n = 4 (B, C) independent experiments.</p