Theodor Kocher Institute |
Ruth Lyck, PhD
Group Members:
Ruth Lyck, PhD
Mark Liebi, lab technician
Oliver Steiner, PhD Student
Downstream signalling of endothelial ICAM-1 during leukocyte transendothelial migration
Expressed on the cellular surface of endothelial cells intercellular adhesion molecule 1 (ICAM-1) plays an essential role in supporting the trans-endothelial migration (TEM) of T-lymphocytes. We have shown that endothelial cells lacking ICAM-1 and ICAM-2 are unable to support adhesion and TEM of lymphocytes. The processes of adhesion and TEM can be separated from each other by the deletion of the cytoplasmic tail of ICAM-1: Endothelial cells expressing a cytoplasmic deletion mutant of ICAM-1 support T-cell adhesion close to wild type level but show significantly reduced T-cell migration across the endothelium. Therefore we conclude that the cytoplasmic tail of ICAM-1 is dispensable for T-cell adhesion to the endothelium but fulfils an essential function during TEM.
Our research focuses on direct defining the downstream signals mediated by endothelial ICAM-1, which allow diapedesis of lymphocytes across an endothelial barrier. Currently we perform Two-Hybrid screens using the cytoplasmic tail of ICAM-1 as bait and different mouse cDNA libraries. We further investigate potential candidates by in vitro and in vivo protein-protein interaction studies. Selected ICAM-1 interaction partners will be evaluated for their functional importance in the process of T-cell TEM. For this purpose different approaches using our powerful technique of static in vitro TEM assay combined with siRNA knock down of gene expression or expression of dominant negative mutants are available. In parallel, biochemical and microscopical techniques will be used to characterize the function of ICAM-1 interaction partners.
Molecular and cellular pathways of immune cell migration across the blood-brain barrier
Interaction of circulating leukocytes with the blood brain barrier (BBB) endothelium and their subsequent diapedesis across these highly specialized endothelial cells is a critical step in the pathogenesis of CNS inflammation. Whereas the molecular mechanisms involved in tethering, rolling and firm adhesion of leukocytes to the endothelium have been characterized, little is known about the last step, the migration of leukocytes across the BBB.
BBB characteristics as tight junctions (TJ), high electrical resistance and low pinocytotic activity are not an intrinsic feature of CNS endothelium but depend on yet undefined signals provided by the CNS microenvironment. Therefore, cultured brain endothelium readily looses many barrier characteristics and in vitro investigations using brain endothelium can never fully account for the in vivo situation.
Only recently an in vitro mouse BBB model was developed where primary mouse brain capillary endothelial cells (MBCECs) are co-cultured with mouse glial cells, thus mimicking the microenvironment encountered in brain tissue (Coisne et al., 2005). In this model MBCECs retain endothelial markers and numerous phenotypic properties of in vivo cerebral endothelium such as TJ protein expression and low permeability. Compared to other in vitro BBB models of bovine, rat or porcine brain endothelium the mouse model is of special importance as mice constitute the prime model system for vascular biology and immunology. Only this system allows the use of genetically modified mouse strains in combination with the great variety of already established reagents available for mice.
We plan to adapt the novel mouse BBB model to study the cellular and molecular pathway of leukocyte diapedesis across the BBB in vitro. All methods and equipment that are important for the development and evaluation of an in vitro BBB leukocyte diapedesis system are well established at our institute. Employing the in vitro BBB leukocyte diapedesis model, we will ascertain whether T-cells migrate directly through the endothelial cell or between endothelial cell-cell-junctions. On the molecular level, we will define the endothelial adhesion and/or TJ molecules involved in T-cell diapedesis. Finally, we will adapt the static in vitro system to conditions that mimic in vivo fluid dynamics and check out whether our results obtained so far will hold true under these conditions.
Taken together both establishment of the in vitro system and identification of the cellular and molecular route of leukocyte diapedesis will help to identify new therapeutic targets aiming to specifically inhibit T-cell migration into the CNS without affecting leukocyte recruitment into other tissues.
1 Lyck R, Reiss Y, Gerwin N, Greenwood J, Adamson P, Engelhardt B. T-cell interaction with ICAM-1/ICAM-2 double-deficient brain endothelium in vitro: the cytoplasmic tail of endothelial ICAM-1 is necessary for transendothelial migration of T cells. Blood 102(10), 3675-3683.
2 Greenwood J, Amos CL, Walters CE, Couraud PO, Lyck R, Engelhardt B, Adamson P. (2003). Intracellular domain of brain endothelial intercellular adhesion molecule-1 is essential for T lymphocyte-mediated signaling and migration. J Immunology 171(4), 2099-2108.
3 Bharti K, Schmidt E, Lyck R, Heerklotz D, Bublak D, Scharf KD. (2000). Isolation and characterization of HsfA3, a new heat stress transcription factor of Lycopersicon peruvianum. Plant J 22(4), 355-365.
4 Doring P, Treuter E, Kistner C, Lyck R, Chen A, Nover L. (2000). The role of AHA motifs in the activator function of tomato heat stress transcription factors HsfA1 and HsfA2. Plant Cell 12(2), 265-278.
5 Scharf KD, Heider H, Hohfeld I, Lyck R, Schmidt E, Nover L. 1998. The tomato Hsf system: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules. Mol Cell Biol 18 (4): 2240-2251.
6 Boscheinen O, Lyck R, Queitsch C, Treuter E, Zimarino V, Scharf KD. (1997). Heat stress transcription factors from tomato can functionally replace HSF1 in the yeast Saccharomyces cerevisiae. Mol Gen Genet 255(3): 322-331.
7 Lyck R, Harmening U, Hohfeld I, Treuter E, Scharf KD, Nover L. (1997). Intracellular distribution and identification of the nuclear localization signals of two plant heat-stress transcription factors. Planta 202(1), 117-125.
8 Sen-Gupta M, Lyck R, Fleig U, Niedenthal RK, Hegemann JH. (1996). The sequence of a 24152 bp segment from the left arm of chromosome XIV from Saccharomyces cerevisiae between the BN11 and the POL2 genes. Yeast 12(5), 505-514.