Research

1) Leukocyte migration into lymphatic vessels

In contrast to the migration of leukocytes out of blood vessels into the tissue, the migration of leukocytes from the tissue into lymphatic vessels is much less well understood. This, for instance, occurs when antigen-presenting dendritic cells (DCs) migrate from the tissue into draining lymph nodes, during the initiation of an adaptive immune response.

We have recently set up a mouse model to image by confocal intravital microscopy how DCs migrate into and within lymphatic capillaries. This migratory process is only now beginning to be explored by in vivo imaging techniques. For our experiments, we employ transgenic mice expressing a red fluorescent protein in both lymphatic vessels and blood vessels. Lymphatic vessels are easily distinguishable from blood vessels by means of their different morphology

Besides intravital microscopy, we also perform other in vivo experiments to study leukocytes migration to draining lymph nodes at the cell population level. These experiments involve the adoptive transfer of fluorescently labeled leukocytes (in presence/ absence of blocking antibodies or small-molecule inhibitors) into skin and subsequent FACS-based quantification of leukocyte numbers in the draining lymph nodes. In vivo experiments are complemented by in vitro experiments, where we, for example, analyze leukocyte chemotaxis and interactions with in vitro cultured lymphatic endothelial cells.

2) Characterizing the inflammatory response of lymphatic endothelium

Tissue inflammation is generally thought to enhance dendritic cell (DC) migration either directly, by stimulating DCs, or indirectly, by up-regulating adhesion molecules and chemokines in lymphatic endothelial cells (LECs). Furthermore, inflammatory signals eventually induce a lymphangiogenic response in LECs. However, to date, the response of LECs to tissue inflammation and its impact on DC migration and on inflammation-induced lymphangiogenesis have only been marginally studied.

Isolation of LECs (CD45-CD31+podoplanin+) from from enzymatically digested ear single cell suspensions by FACS sorting for subsequent gene expression profiling. Adapted from Vigl. et al., Blood 2011.

We have recently performed a comprehensive microarray-based gene expression analysis of LECs isolated by FACS sorting from inflamed and resting murine skin. Our technically demanding approach represents the first description of the in vivo inflammatory response in LECs. In this study, we found that the gene expression profile induced in LECs by tissue inflammation strikingly depended on the nature of inflammatory stimulus. Similarly, also the extent of inflammation-induced DC migration to draining lymph nodes highly varied between different inflammatory stimuli (see Vigl. et al., Blood 2011).

We are currently working on the characterization of various genes that we found to be induced in LECs during inflammation. Of particular interest to us are LEC-expressed adhesion molecules and chemokines, which could be involved in leukocyte recruitment into lymphatic vessels. Furthermore, we have become interested in LEC-expressed receptors of cytokines and growth factors and of matrix remodeling enzymes, which could be involved in inflammation-induced vascular remodeling and lymphangiogenesis.

To study the potential role of these molecules in lymphatic endothelial cell biology, we perform in vitro experiments with cultured human and murine lymphatic endothelial cells and primary leukocytes (isolated from murine tissues, or isolated from human blood), as well as in vivo functional studies in gene-targeted mice or wild-type mice treated with blocking antibodies or small-molecule in inhibitors.

3) Chronic inflammation and its effects on lymphatic vessel function and adaptive T cell immunity

Chronic inflammation, as it for instance occurs in psoriasis or rheumatoid arthritis, is accompanied by the proliferative expansion and remodeling of lymphatic vessels. However, it is currently not well understood how such changes impact lymphatic vessel function - namely, fluid transport and DC migration to draining lymph nodes - and thereby affect the course of the inflammatory and/or autoimmune response. During chronic inflammation the concentrations of inflammatory mediators rise not only in the inflamed tissue but also in draining lymph nodes. Surprisingly, little is known to date on how the lymph node microenvironment affects the induction of adaptive T cell responses in the context of inflammation. To investigate how chronic inflammation affects lymphatic vessel function or the induction of adaptive immune responses, our group works with different mouse models of the chronic inflammatory skin disease psoriasis (see Halin et al., Blood 2007 & Halin et al., Am. J. Pathol. 2008).

The lymphatic vessel network is markedly expanded in chronically inflamed skin and draining lymph nodes. Immunofluorescence was performed on inflamed and control mouse ear and lymph node sections by staining for MECA-32 (blood vessels) and LYVE-1 (lymphatic vessels).