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Ákos Heinemann, MD:

Leukocyte trafficking, activation and inflammatory mediators

Otto Loewi Research Centre, Division of Pharmacology, Medical University of Graz, Universitätsplatz 4, A-8010 Graz;
phone: +43-316-380 4508, fax: +43-316-380 9645,  e-mail
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Keywords:

Inflammation, immune response, endothelium, macrophages, cytokines, prostaglandins, adhesion, chemotaxis, placenta, umbilical cord

Research interest:

Accumulation of leukocytes in tissues is a key feature of inflammation and a major determinant of tissue damage. Pregnancy is an immunological challenge for mother and fetus, and numerous immune cells take part in the development of the decidua. Particularly macrophages are thought to be crucial in maintaining an immune-tolerant environment in the semi-allogeneic setting of trophoblast invasion (1).

The main focus of our laboratory is to define the mechanisms that govern the trafficking of leukocytes from bone marrow, where they are generated, to the inflammatory site and their subsequent activation in tissue, where they may become harmful. Chemoattractants, their receptors, and adhesion molecules both on the leukocytes and the endothelial side, play crucial roles in the multi-step process of leukocyte infiltration by facilitating leukocyte locomotion and activation, and are thus considered as promising therapeutic targets in various inflammatory conditions. Conversely, several endogenous mediators exist that down-regulate the responsiveness of leukocytes and might hence exert potent anti-inflammatory effects. When supplemented pharmaceutically, these mediators might likewise open novel therapeutic avenues. Among others we have elucidated the opposing roles of two cyclooxygenase (COX) products, prostaglandin (PG) E2 and D2 in leukocyte trafficking in human and animal models, and have characterized their receptors at the molecular and pharmacological level. While we have shown that its receptor EP4 is a negative regulator of eosinophil and neutrophil trafficking, and endothelial, thrombocyte and macrophage activation (2, 3), we have also revealed a novel role for PGD2 and its receptors DP1 and DP2 as potent activators of eosinophils, basophils and macrophages (4, 5).

It has been suggested previously that PGE2 and PGD2 might play distinct roles in pathological conditions of pregnancy, but how these prostaglandins contribute to the regulation of leukocyte function in the developing decidua and, even more, how responses to PGE2 and PGD2 of immune cells in the placenta are altered in different gestational pathologies has not been addressed in detail. Currently we are elucidating the expression patterns and levels of enzymes involved in prostaglandin synthesis (COX isoforms, PGE and PGD synthases) and receptors for PGE2 and PGD2 in placental tissue. We hypothesize that an imbalance of anti-inflammatory PGE2 effects and pro-inflammatory PGD2 actions might contribute to complications in pregnancy, such as hypertension, pre-eclampsia or preterm labor. We will characterize these alterations on the cellular (e. g. endothelial cells, macrophages) and tissue level (i. e. placenta).

Illustrations:

Fig. 1: Full-term human placenta stained for D-type prostanoid (DP)1 receptor. The syncytiotrophoblast, surrounding the chorionic villi and forming part of the placental barrier shows strong staining for the DP1 receptor. The arrow indicates a Hofbauer-cell (fetal macrophage) also showing positive staining for DP1. Red asterisks show fetal capillaries inside the villus and the blue asterisk marks the intravillous space (maternal side).
Magnification 100× (A) and 600× (B).
Fig. 2: Full-term human placenta stained for D-type prostanoid (DP)2 receptor. The syncytiotrophoblast, surrounding the chorionic villi and forming part of the placental barrier shows staining for the DP2 receptor. The arrow indicates a Hofbauer-cell (fetal macrophage) and the arrowhead points to a fetal leukocyte inside a capillary, both showing positive staining for DP2. Red asterisks show fetal capillaries inside the villus and the blue asterisk marks the intravillous space filled with maternal erythrocytes (maternal side).
Magnification 100× (A) and 600× (B).

References:

  1. Svensson-Arvelund J, Ernerudh J: The role of macrophages in promoting and maintaining homeostasis at the fetal-maternal interface. Am J Reprod Immunol, 2015; 74(2):100–109.
  2. Konya V, Marsche G, Schuligoi R, Heinemann A: E-type prostanoid receptor 4 (EP4) in disease and therapy. Pharmacol Ther, 2013; 138(3):485–502.
  3. Konya V, Maric J, Jandl K, Luschnig P, Aringer I, Lanz I, Platzer W, Theiler A, Bärnthaler T, Frei R, Marsche G, Marsh LM, Olschewski A, Lippe IT, Heinemann A, Schuligoi R: Activation of EP4 receptor prevents acute lung injury in mice by enhancing the microvascular barrier function. Br J Pharmacol, 2015; 172(18):4454–4468.
  4. Schuligoi R, Sturm E, Luschnig P, Konya V, Philipose S, Sedej M, Waldhoer M, Peskar BA, Heinemann A: CRTH2 and D-type prostanoid receptor antagonists as novel therapeutic agents for inflammatory diseases. Pharmacology, 2010; 85(6):372–382.
  5. Jandl K, Stacher E, B├ílint Z, Sturm EM, Maric J, Peinhaupt M, Luschnig P, Aringer I, Fauland A, Konya V, Dahlen SE, Wheelock CE, Kratky D, Olschewski A, Marsche G, Schuligoi R, Heinemann A: Activated prostaglandin D2 receptors on macrophages enhance neutrophil recruitment into the lung. J Allergy Clin Immunol, 2016; 137(3):833–843.

Collaborations within the DP-iDP:

  • G. Desoye will teach the students how to isolate endothelial cells and macrophages from the placenta and / or umbilical cord, introduce them to the biology of angiogenesis and help with assays of 2-D network formation and tube formation.
  • G. Marsche will supervise the studies addressing lipid metabolism of placental macrophages.
  • C. Wadsack will provide clinically well-defined placenta samples and help the students with ex vivo placenta perfusion assays.
  • In studies conducted by M. van Poppel the students will learn how to measure and statistically analyze cytokine plasma profiles in normal and pathological pregnancies.

Collaborating research groups where PhD students could perform their research stay abroad:

  • S. E. Dahlen (Karolinska Institutet, Stockholm, Sweden) is an expert in prostaglandin biology and will facilitate the mass spectrometric analysis of eicosanoids and endocannabinoids generated in vivo and in vitro.
  • E. Kostenis (University of Bonn, Germany) is a molecular pharmacologist in the field of eicosanoid receptors. She will train the students in heterologous expression of prostanoid receptors and characterization of ligands in binding studies and second messenger signaling including assays of dynamic mass redistribution.
  • B. Lindenthal (Bayer Pharma, Berlin, Germany) is an industrial pharmacologist who will introduce the students to the drug development program and the methods employed by Bayer in the field of prostaglandin antagonists for the treatment of gynecological disorders.

Industrial partners:

Bayer AG, Germany; AstraZeneca AB, Sweden; Protaffin GmbH, Graz, Austria.

Know-how and infrastructure of the research group:

The laboratory of Ákos Heinemann has a long-standing expertise in the field of leukocyte biology and pharmacology, but also in hemodynamic regulation and vascular biology. Studies are routinely carried out both with primary cells isolated from humans and cells from animal sources, complemented with cell lines for transfection / silencing experiments. The group comprises two post-doctoral fellows, three technicians and five PhD students. Various techniques are being used for a detailed analysis of leukocyte and endothelial cell function, and the group has considerable experience with in vivo models of leukocyte trafficking and inflammation. All the required equipment is available at the institute, including animal and cell-culture facilities, radionuclide laboratory, flow cytometry, real-time PCR systems, fluorescence plate reader, tissue processing and fluorescence microscopy, and a microscopy system to study cell-to-cell interaction and thrombus formation under flow conditions. The group has also access to video-tracking of leukocyte locomotion and laser-scanning microscopy at the CMR.

Scientific concepts and techniques that students will learn in this laboratory:

DP-iDP students will be trained in immunology and will receive insights in the regulation of leukocyte and endothelial function, in particular trafficking and activation, and how this process impacts on inflammation and tissue damage. In addition, they will get background knowledge on current concepts of anti-inflammatory therapies and where demand for further research exists. Students will learn how to analyze signaling properties of G protein-coupled receptors and characterize respective ligands in specific pharmacological and functional immunological assays. The acquired methodologies will comprise isolation and culturing of cells from blood and tissues, flow cytometry, RNA techniques including siRNA gene knock-down, immunoprecipitation and western blot, and laser-scanning microscopy. The students will also be trained to characterize and quantitate specific lipid subclasses by mass spectroscopy (GC-MS, LC-MS).