Legler, Daniel F.
Angiogenesis and lymphangiogenesis are downregulated in primary breast cancer
2009, Boneberg, Eva-Maria, Legler, Daniel F., Hoefer, Melanie M., Öhlschlegel, Christian, Steininger, Helmuth, Füzesi, Laszlo, Beer, Gertrude M., Dupont-Lampert, Véronique, Otto, Florian, Fürstenberger, Gregor
Background: Angiogenesis and lymphangiogenesis are considered to play key roles in tumour growth, progression and metastasis. However, targeting tumour angiogenesis in clinical trials showed only modest efficacy. We therefore scrutinised the concept of tumour angiogenesis and lymphangiogenesis by analysing the expression of crucial markers involved in these processes in primary breast cancer.
Methods: We analysed the expression of angiogenic, lymphangiogenic or antiangiogenic factors, their respective receptors and specific markers for endothelial and lymphendothelial cells by quantitative real-time RT-PCR in primary breast cancer and compared the expression profiles to non-cancerous, tumour-adjacent tissues and breast tissues from healthy women.
Results: We found decreased mRNA amounts of major angiogenic and lymphangiogenic factors in tumour compared to healthy tissues, whereas antiangiogenic factors were upregulated. Concomitantly, angiogenic and lymphangiogenic receptors were downregulated in breast tumours. This antiangiogenic, antilymphangiogenic microenvironment was even more pronounced in aggressive tumours and accompanied by reduced amounts of endothelial and lymphatic endothelial cell markers.
Conclusion: Primary breast tumours are not a site of highly active angiogenesis and lymphangiogenesis. Selection for tumour cells that survive with minimal vascular supply may account for this observation in clinical apparent tumours.
Prostaglandin E(2) enhances T-cell proliferation by inducing the costimulatory molecules OX40L, CD70, and 4-1BBL on dendritic cells
2009, Krause, Petra, Bruckner, Markus, Uermösi, Christina, Singer, Eva, Gröttrup, Marcus, Legler, Daniel F.
Dendritic cell (DC) based immunotherapy of malignant diseases relies on 2 critical parameters: antigen transport from the periphery to draining lymph nodes and efficient priming of primary and stimulation of secondary immune responses. Prostaglandin E2 (PGE2) signaling has been shown to be pivotal for DC migration toward lymph node derived chemokines in vitro and in vivo. Here, we demonstrate that PGE2 induced the expression of the costimulatory molecules OX40L, CD70, and 4-1BBL on human DCs. Short triggering by PGE2 early during DC maturation was sufficient to induce the costimulatory molecules. The expression of the costimulatory molecules was independent of the maturation stimulus but strictly dependent on PGE2 on both monocyte-derived (Mo) DCs and peripheral blood myeloid (PB) DCs. PGE2-matured MoDCs showed enhanced costimulatory capacities resulting in augmented antigen-specific CD4+ and CD8+ T-cell proliferation in primary and recall T-cell responses. Blocking OX40/OX40L signaling impaired the enhanced T-cell proliferation induced by PGE2-matured MoDCs. Moreover, MoDCs matured in the presence of PGE2 induced the expression of OX40, OX40L, and CD70 on T cells facilitating T-cell/T-cell interaction that warrant long-lasting costimulation. This newly identified parameter will help to further optimize DC-based immunotherapy.
Prostaglandin E2 is a key factor for monocyte-derived dendritic cell maturation : enhanced T cell stimulatory capacity despite IDO
2007-11, Krause, Petra, Singer, Eva, Darley, Paula I., Klebensberger, Janosch, Gröttrup, Marcus, Legler, Daniel F.
The exclusive ability of dendritic cells (DCs) to stimulate primary and secondary immune responses favors the use of antigen-loaded human monocyte-derived DCs (MoDCs) in vaccinations against tumors. Previous studies demonstrated that PGE(2) is fundamental during MoDC maturation to facilitate migration toward lymph node-derived chemokines. A recent study challenged the use of PGE(2), as PGE(2) induced IDO in mature MoDCs. In MoDCs compatible for clinical use, we now demonstrate that PGE(2) is responsible for IDO induction if matured by soluble CD40 ligand, LPS, or cytokines. In contrast, IDO expression in MoDCs matured by TLR3 triggering occurs independently of PGE(2). It is surprising that despite active IDO protein, MoDCs matured with PGE(2) display a greater potential to stimulate naïve CD4(+) and CD8(+) T cell proliferation, which is not increased further by IDO inhibition. Moreover, we found elevated levels of tryptophanyl-tRNA-synthetase (TTS) in T cells cultured with PGE(2)-matured MoDCs. Our data demonstrate that PGE(2) induces IDO in MoDCs but that T cell-stimulating capacities of PGE(2)-matured MoDCs overcome IDO activity, probably through TTS induction. As PGE(2) is critical for DC migration and enhances the capability of MoDCs to induce T cell proliferation, we highly recommend supplementing DC maturation stimuli with PGE(2) for use in clinical trials.
Soluble CD146 is generated by ectodomain shedding of membrane CD146 in a calcium-induced, matrix metalloprotease-dependent process
2009, Boneberg, Eva-Maria, Illges, Harald, Legler, Daniel F., Fürstenberger, Gregor
CD146 is a cell adhesion molecule localized at the endothelial junction and is involved in the control of cell-cell cohesion. In this study, we showed that calcium influx in human microvascular lung endothelial cells results in the loss of surface CD146 and the release of soluble CD146. This calcium-induced CD146 shedding could be prevented with inhibitors of matrix metalloproteases indicating a central role of matrix metalloproteases in this process. We also investigated if CD146 shedding influences vascular permeability. Endothelial cell monolayers cultured on filter membranes showed an increased permeability for albumin when stimulated with ionomycin. This calcium-induced increase in permeability was inhibited when CD146 shedding was prevented by a matrix metalloprotease inhibitor. Our data indicate that surface CD146 plays a central role in the regulation of vascular permeability and demonstrate that CD146 and matrix metalloproteases are potential targets to modify endothelial barrier function.
Increased Mobility of Major Histocompatibility Complex I-Peptide Complexes Decreases the Sensitivity of Antigen Recognition
2008-06-04, Segura, Jean-Manuel, Guillaume, Philippe, Mark, Silke, Dojcinovic, Danijel, Johannsen, Alexandre, Bosshard, Giovanna, Angelov, Georgi, Legler, Daniel F., Vogel, Horst, Luescher, Immanuel F.
CD8+ cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2Kd molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules.
Proinflammatory cytokines cause FAT10 upregulation in cancers of liver and colon
2008, Lukasiak, Sebastian, Schiller, Claudia, Oehlschläger, Peter, Schmidtke, Gunter, Krause, Petra, Legler, Daniel F., Autschbach, Frank, Schirmacher, Peter, Breuhahn, Kai, Gröttrup, Marcus
The mRNA of the ubiquitin-like modifier FAT10 has been reported to be overexpressed in 90% of hepatocellular carcinoma (HCC) and in over 80% of colon, ovary and uterus carcinomas. Elevated FAT10 expression in malignancies was attributed to transcriptional upregulation upon the loss of p53. Moreover, FAT10 induced chromosome instability in long-term in vitro culture, which led to the hypothesis that FAT10 might be involved in carcinogenesis. In this study we show that interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha synergistically upregulated FAT10 expression in liver and colon cancer cells 10- to 100-fold. Real-time RT-PCR revealed that FAT10 mRNA was significantly overexpressed in 37 of 51 (72%) of human HCC samples and in 8 of 15 (53%) of human colon carcinomas. The FAT10 cDNA sequences in HCC samples were not mutated and intact FAT10 protein was detectable. FAT10 expression in both cancer tissues correlated with expression of the IFN-gamma- and TNF-alpha-dependent proteasome subunit LMP2 strongly suggesting that proinflammatory cytokines caused the joint overexpression of FAT10 and LMP2. NIH3T3 transformation assays revealed that FAT10 had no transforming capability. Taken together, FAT10 qualifies as a marker for an interferon response in HCC and colon carcinoma but is not significantly overexpressed in cancers lacking a proinflammatory environment.
Prostaglandin E(2) at new glance : Novel insights in functional diversity offer therapeutic chances
2009, Legler, Daniel F., Bruckner, Markus, Uetz-Allmen, Edith, Krause, Petra
Prostaglandin E2 (PGE2) is the most abundant eicosanoid and a very potent lipid mediator. PGE2 is produced predominantly from arachidonic acid by its tightly regulated cyclooxygenases (COX) and prostaglandin E synthases (PGES). Secreted PGE2 acts in an autocrine or paracrine manner through its four cognate G protein coupled receptors EP1 to EP4. Under physiological conditions, PGE2 is key in many biological functions, such as regulation of immune responses, blood pressure, gastrointestinal integrity, and fertility. Deregulated PGE2 synthesis or degradation is associated with severe pathological conditions like chronic inflammation, Alzheimer's disease, or tumorigenesis. Therefore, pharmacological inhibition of COX enzymes and PGE2 receptor antagonism is of great therapeutic interest.
V domain of RAGE interacts with AGEs on prostate carcinoma cells
2008, Uetz-von Allmen, Edith, Koch, Michael, Fritz, Günter, Legler, Daniel F.
BACKGROUND: The expression of the scavenger receptor for advanced glycation end products (RAGE) and various ligands of RAGE correlate significantly with cancer progression. However, the mechanism of RAGE/sRAGE-induced cancer cell activation and ligand usage remain largely unknown.
METHODS: Androgen-independent, highly invasive, as well as androgen-dependent, non-invasive human prostate carcinoma (CaP) cells were investigated for their interaction with the soluble form of RAGE (sRAGE). Using neutralizing antibodies and soluble proteins, the ligand for RAGE was identified on CaP cells and ligand binding with sRAGE was biochemically characterized.
RESULTS: Both androgen-independent, highly invasive and androgen-dependent, non-invasive CaP cells interacted with immobilized sRAGE in a surprisingly strong manner. Using C-terminal truncation variants of RAGE we identified the V domain being responsible for the adhesion of CaP cells to sRAGE. Moreover, we demonstrate that this adhesion cannot be blocked by S100B or neutralizing antibodies against β integrins, or amphoterin. However, the CaP cell–RAGE interaction was inhibited with either AGE-modified proteins, or with neutralizing antibodies against AGE or RAGE. Despite similar binding kinetics between AGE-modified BSA and different RAGE domains, only applying an excess of sRAGE, but not the VC1 or V domain of RAGE, was able to block the CaP cell–RAGE interaction.
CONCLUSIONS: We identified AGEs as the ligand for RAGE on both invasive and non-invasive prostate cancer cells.
Distinct motifs in the chemokine receptor CCR7 regulate signal transduction, receptor trafficking and chemotaxis
2008, Otero, Carolina, Eisele, Petra S., Schauble, Karin, Gröttrup, Marcus, Legler, Daniel F.
The chemokine receptor CCR7, together with its ligands CCL19 and CCL21, is responsible for the correct homing and trafficking of dendritic cells and lymphocytes to secondary lymphoid tissues. Moreover, cancer cells can utilize CCR7 to metastasize to draining lymph nodes. However, information on CCR7 signaling leading to cell migration or receptor trafficking is sparse. Using novel CCR7 deletion mutants with successive truncations of the intracellular C-terminus and a mutant with impaired G-protein coupling, we identified distinct motifs responsible for various aspects of CCR7 signal transduction. Deleting a Ser/Thr motif at the tip of the intracellular tail of CCR7 resulted in an impaired chemokine-mediated activation of Erk1/2 kinases. Interestingly, deleting an additional adjacent motif restored the ability of CCL19-mediated Erk1/2 phosphorylation, suggesting the presence of a regulatory motif. Both the Ser/Thr and the regulatory motif are dispensable for signaling events leading to cell migration and receptor trafficking. A CCR7 mutant lacking virtually the complete C-terminus readily bound CCL19 and was internalized, but was unable to activate the G protein and to transmit signals required for cell migration, mobilization of [Ca2+](i) and Erk1/2 activation. Finally, G-protein coupling was critical for [Ca2+](i) mobilization, Erk1/2 phosphorylation and chemotaxis, but not for CCR7 trafficking.