M. Gabriele Bixel

Molecular events during leukocyte diapedesis

The recruitment of leukocytes from the circulation into tissues requires leukocyte migration through the vascular endothelium. The mechanisms by which leukocytes attach and firmly adhere to the endothelial cell surface have been studied in detail. However, much less is known about the last step in this process, the diapedesis of leukocytes through the vascular endothelium. This minireview focuses on the interactions between leukocyte and endothelial cell adhesion molecules that are important during leukocyte extravasation. In the past few years a series of endothelial cell surface and adhesion molecules have been identified that are located at endothelial cell contacts and found to participate in leukocyte diapedesis. These junctional cell adhesion molecules are believed to have an active role in controlling the opening and closure of endothelial cell contacts to allow the passage of leukocytes between adjacent endothelial cells. Alternatively, leukocytes can cross the endothelium at nonjunctional locations, with leukocytes migrating through a single endothelial cell. Further work is clearly needed to understand, in greater detail, the molecular mechanisms that allow leukocytes to cross the endothelium via either the paracellular or the transcellular pathway.

Identification of a clonally expanding haematopoietic compartment in bone marrow.

In mammals, postnatal haematopoiesis occurs in the bone marrow (BM) and involves specialized microenvironments controlling haematopoietic stem cell (HSC) behaviour and, in particular, stem cell dormancy and self‐renewal. While these processes have been linked to a number of different stromal cell types and signalling pathways, it is currently unclear whether BM has a homogenous architecture devoid of structural and functional partitions. Here, we show with genetic labelling techniques, high‐resolution imaging and functional experiments in mice that the periphery of the adult BM cavity harbours previously unrecognized compartments with distinct properties. These units, which we have termed hemospheres, were composed of endothelial, haematopoietic and mesenchymal cells, were enriched in CD150+ CD48− putative HSCs, and enabled rapid haematopoietic cell proliferation and clonal expansion. Inducible gene targeting of the receptor tyrosine kinase VEGFR2 in endothelial cells disrupted hemospheres and, concomitantly, reduced the number of CD150+ CD48− cells. Our results identify a previously unrecognized, vessel‐associated BM compartment with a specific localization and properties distinct from the marrow cavity.

CD99 and CD99L2 act at the same site as, but independently of, PECAM-1 during leukocyte diapedesis

Leukocyte extravasation depends on various adhesion receptors at endothelial cell contacts. Here we have analyzed how mouse CD99 and CD99L2 cooperate with PECAM-1. We found that antibodies against mouse CD99 and PECAM-1 trap neutrophils between endothelial cells in in vitro transmigration assays. A sequential function, as has been suggested for human PECAM-1 and CD99, could not be demonstrated. In contrast to these in vitro results, blocking CD99 or CD99L2 or gene disruption of PECAM-1 trapped neutrophils in vivo between endothelial cells and the underlying basement membrane as revealed by electron microscopy and by 3-dimensional confocal fluorescence microscopy in the inflamed cremaster tissue. Leukocyte extravasation was inhibited in interleukin-1beta-inflamed peritoneum and in the cremaster by PECAM-1 gene disruption and was further attenuated by blocking antibodies against CD99 and CD99L2. In addition, CD99 and CD99L2 were required for leukocyte extravasation in the cremaster after stimulation with tumor necrosis factor-alpha, where the need for PECAM-1 is known to be bypassed. We conclude that CD99 and CD99L2 act independently of PECAM-1 in leukocyte extravasation and cooperate in an independent way to help neutrophils overcome the endothelial basement membrane.

Blood flow controls bone vascular function and osteogenesis

While blood vessels play important roles in bone homeostasis and repair, fundamental aspects of vascular function in the skeletal system remain poorly understood. Here we show that the long bone vasculature generates a peculiar flow pattern, which is important for proper angiogenesis. Intravital imaging reveals that vessel growth in murine long bone involves the extension and anastomotic fusion of endothelial buds. Impaired blood flow leads to defective angiogenesis and osteogenesis, and downregulation of Notch signalling in endothelial cells. In aged mice, skeletal blood flow and endothelial Notch activity are also reduced leading to decreased angiogenesis and osteogenesis, which is reverted by genetic reactivation of Notch. Blood flow and angiogenesis in aged mice are also enhanced on administration of bisphosphonate, a class of drugs frequently used for the treatment of osteoporosis. We propose that blood flow and endothelial Notch signalling are key factors controlling ageing processes in the skeletal system.

Vaccination against CD99 inhibits atherogenesis in low-density lipoprotein receptor-deficient mice

AIMS Murine CD99 was recently found to be expressed on leukocytes and endothelial cells, where it is concentrated at inter-endothelial contacts. Blockade of CD99 by specific antibodies inhibits leukocyte extravasation to inflamed sites in vivo. The aim of the present study is to show the role of CD99 in atherosclerosis using a CD99 vaccination protocol to block the function of CD99 during atherosclerosis. METHODS AND RESULTS We constructed a DNA vaccine against CD99 by cloning the extracellular domain of murine CD99 into pcDNA3. Vaccination was performed by oral administration of attenuated Salmonella typhimurium transformed with pcDNA3-CD99. This vaccination results in a CD99-specific, CD8-mediated cytotoxic response and subsequent reduction of CD99-expressing cells. We showed that CD99 is expressed on vascular endothelium overlying atherosclerotic plaques and found that CD99 expression is upregulated during western-type diet feeding. CD99 vaccination induced the formation of CD8-positive T cells that were cytotoxic against cells transfected with pcDNA3-CD99. Activation of CD8(+) T cells was demonstrated by a 30% increase in CD8(+)CD69(+) double-positive T cells in spleen and mediastinal lymph nodes. Furthermore, lymphocytes isolated from CD99-vaccinated mice specifically lysed CD99-expressing cells. More importantly, vaccination against CD99 attenuated atherosclerotic lesion formation in the aortic valve leaflets by 38% and in the carotid artery by 69% compared with mice that were vaccinated with a control vector. Furthermore, a lower number of cells were found in atherosclerotic lesions, implying that fewer leukocytes were recruited to these sites. These observations were accompanied by a decrease in CD99 expression on leukocytes. CONCLUSION We conclude that vaccination against CD99 decreases atherogenesis by the selective removal of CD99-expressing cells, which could reduce leukocyte recruitment into atherosclerotic lesions and attenuate atherogenesis.

Leukocyte trafficking in a mouse model for leukocyte adhesion deficiency II/congenital disorder of glycosylation IIc.

Leukocyte adhesion deficiency II (LAD II), also known as congenital disorder of glycosylation IIc (CDG-IIc), is a human disease in which a defective GDP-fucose transporter (SLC35C1) causes developmental defects and an immunodeficiency that is based on the lack of fucosylated selectin ligands. Since the study of in vivo leukocyte trafficking in patients with LAD II is experimentally limited, we analyzed this process in mice deficient for Slc35c1. We found that E-, L-, and P-selectin-dependent leukocyte rolling in cremaster muscle venules was virtually absent. This was accompanied by a strong but not complete decrease in firm leukocyte adhesion. Moreover, neutrophil migration to the inflamed peritoneum was strongly reduced by 89%. Previous reports showed surprisingly normal lymphocyte functions in LAD II, which indicated sufficient lymphocyte trafficking to secondary lymphoid organs. We now found that while lymphocyte homing to lymph nodes was reduced to 1% to 2% in Slc35c1(-/-) mice, trafficking to the spleen was completely normal. In accordance with this, we found a defect in the humoral response to a T cell-dependent antigen in lymph nodes but not in the spleen. Taken together, Slc35c1(-/-) mice show strongly defective leukocyte trafficking but normal lymphocyte homing to the spleen, which may explain normal lymphocyte functions in LAD II.

Migration of immature mouse DC across resting endothelium is mediated by ICAM-2 but independent of β2-integrins and murine DC-SIGN homologues

Immature dendritic cells (DC) reside in tissues where they initiate immune responses by taking up foreign antigens. Since DC have a limited tissue half‐life, the DC pool in tissues has to be replenished constantly. This implies that precursor/immature DC must be able to cross non‐activated endothelium using as yet unknown mechanisms. Here we show that immature, but not mature bone marrow‐derived murine DC migrate across resting endothelial monolayers in vitro. We find that endothelial intercellular adhesion molecule‐2 (ICAM‐2) is a major player in transendothelial migration (TEM) of immature DC, accounting for at least 41% of TEM. Surprisingly, the ICAM‐2‐mediated TEM was independent of β2‐integrins, the known ICAM‐2 ligands, since neither blocking of β2‐integrins with antibodies nor the use of CD18‐deficient DC affected the ICAM‐2‐specific TEM. In humans, the C‐type lectin DC‐specific ICAM‐3‐grabbing nonintegrin (DC‐SIGN) was shown to interact with ICAM‐2, suggesting a similar role in mice. However, we find that none of the murine DC‐SIGN homologues mDC‐SIGN, murine DC‐SIGN‐related molecule‐1 (mSIGN‐R1) and mSIGN‐R3 is expressed on the surface of bone marrow‐derived mouse DC. Taken together, this study shows that ICAM‐2 strongly supports transmigration of immature DC across resting endothelium by interacting with ligands that are distinct from β2‐integrins and DC‐SIGN homologues.

Cell-matrix signals specify bone endothelial cells during developmental osteogenesis

Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell–matrix signalling interactions. Loss of endothelial integrin β1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system.

Flow Dynamics and HSPC Homing in Bone Marrow Microvessels

Summary Measurements of flow velocities at the level of individual arterial vessels and sinusoidal capillaries are crucial for understanding the dynamics of hematopoietic stem and progenitor cell homing in the bone marrow vasculature. We have developed two complementary intravital two-photon imaging approaches to determine blood flow dynamics and velocities in multiple vessel segments by capturing the motion of red blood cells. High-resolution spatiotemporal measurements through a cranial window to determine short-time dynamics of flowing blood cells and repetitive centerline scans were used to obtain a detailed flow-profile map with hemodynamic parameters. In addition, we observed the homing of individual hematopoietic stem and progenitor cells and obtained detailed information on their homing behavior. With our imaging setup, we determined flow patterns at cellular resolution, blood flow velocities and wall shear stress in small arterial vessels and highly branched sinusoidal capillaries, and the cellular dynamics of hematopoietic stem and progenitor cell homing.

Master and commander: continued expression of Prox1 prevents the dedifferentiation of lymphatic endothelial cells

Cell differentiation occurs mostly during a specific developmental time window and is irreversible. The homeobox-containing transcription factor Prox1 is a master regulator of lymphatic endothelial cell differentiation in the embryo. A study by Johnson et al. (3282-3291) published in this issue of Genes & Development now shows that continued expression of Prox1 is required to maintain lymphatic endothelial cell identity even in adult mice. These findings indicate that Prox1 is essential for the differentiation and function of the lymphatic vasculature throughout life.