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Growth differentiation factor 15 (GDF-15) is the first cytokine known to counteract chemokine-induced activation of leukocyte integrins. We showed recently that this activity dampens neutrophil recruitment into inflamed tissue and is required for survival of myocardial infarction in mice. The receptor responsible for this GDF-15-triggered anti-inflammatory mechanism on myeloid cells is not known. Here, we identify this receptor as transforming growth factor β receptor I (TGF-βRI) (activin receptor-like kinase 5 [ALK-5]) and TGF-β receptor II (TGF-βRII). We show that interference with these receptors by small-molecule inhibitors, antibodies, or small interfering RNA, blocked the GDF-15 effect on leukocyte integrin activation. Likewise, gene inactivation of each of the 2 receptors in neutrophils isolated from conditional gene-deficient mice abolished the inhibitory effect of GDF-15 on CXCL1-induced β2-integrin activation and neutrophil diapedesis. Rapid neutrophil arrest induced by CXCL1 in vivo was inhibited by GDF-15 in an ALK-5 and TGF-βRII dependent way. As for GDF-15 gene-deficient mice, we found that extravasation of neutrophils deficient for ALK-5 or TGF-βRII was strongly increased in the interleukin-1β inflamed cremaster. The inhibitory effects of GDF-15 on neutrophil integrin activation and in vivo neutrophil arrest were also found for TGF-β1. Mechanistically, GDF-15 and TGF-β1 interfered with integrin activation by inhibiting the activation of Ras-related protein 1 (Rap-1), an effect that depended on CalDAG- guanine nucleotide exchange factor 1 (GEF1) and cell division control protein 42 homolog. We conclude that both GDF-15 and TGF-β1 counteract chemokine-induced integrin activation on neutrophils via the ALK-5/TGF-βRII heterodimer. This represents a novel, rapid anti-inflammatory activity of the 2 TGF-β receptors and of TGF-β1.

CD99-like 2 (CD99L2) is a membrane protein with moderate sequence homology to CD99, which initiates cell aggregation of transfected cells and that is strongly expressed on endothelial cells, neutrophils, and lymphocytes. We showed recently that Abs against CD99L2 inhibit neutrophil, but not T lymphocyte, recruitment into inflamed tissues. In this study, we have generated conditional gene–deficient mice for CD99L2 and show by analyzing them in various inflammation models several results. First, gene ablation of CD99L2 impairs neutrophil recruitment into inflamed cremaster and peritoneum. Second, despite the strong expression of CD99L2 on peripheral neutrophils, only gene ablation on endothelial cells but not on myeloid cells affects neutrophil extravasation. Third, in contrast to our previous Ab-based results, recruitment of activated T cells into inflamed skin was impaired in mice lacking CD99L2 on endothelial cells. We conclude that CD99L2 is an essential endothelial Ag for leukocyte extravasation, which does not require homophilic interactions with CD99L2 on leukocytes.

We have shown recently that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial-specific membrane protein, associates with vascular endothelial (VE)–cadherin and enhances VE-cadherin function in transfected cells (Nawroth, R., G. Poell, A. Ranft, U. Samulowitz, G. Fachinger, M. Golding, D.T. Shima, U. Deutsch, and D. Vestweber. 2002. EMBO J. 21:4885–4895). We show that VE-PTP is indeed required for endothelial cell contact integrity, because down-regulation of its expression enhanced endothelial cell permeability, augmented leukocyte transmigration, and inhibited VE-cadherin–mediated adhesion. Binding of neutrophils as well as lymphocytes to endothelial cells triggered rapid (5 min) dissociation of VE-PTP from VE-cadherin. This dissociation was only seen with tumor necrosis factor α–activated, but not resting, endothelial cells. Besides leukocytes, vascular endothelial growth factor also rapidly dissociated VE-PTP from VE-cadherin, indicative of a more general role of VE-PTP in the regulation of endothelial cell contacts. Dissociation of VE-PTP and VE-cadherin in endothelial cells was accompanied by tyrosine phoshorylation of VE-cadherin, β-catenin, and plakoglobin. Surprisingly, only plakoglobin but not β-catenin was necessary for VE-PTP to support VE-cadherin adhesion in endothelial cells. In addition, inhibiting the expression of VE-PTP preferentially increased tyrosine phosphorylation of plakoglobin but not β-catenin. In conclusion, leukocytes interacting with endothelial cells rapidly dissociate VE-PTP from VE-cadherin, weakening endothelial cell contacts via a mechanism that requires plakoglobin but not β-catenin.

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) recognize peptide epitopes of protein antigens in a hierarchical fashion. We investigated whether proteolytic cleavage, in particular by proteasomes, Is important in determining epitope hierarchy. Using highly purified 20S proteasomes, we find preferred cleavage sites directly adjacent to the N- and C-terminal ends of the immunodominant epitope of chicken ovalbumin, Ova257–264, while most of the subdominant epitope, Ova55–62, is destroyed by a major cleavage site located within this epitope. Moreover, we show that variations in amino acid sequences flanking these epitopes influence proteasomal cleavage patterns In parallel with the efficacy of their presentation. The results suggest that proteasomal cleavage within and adjacent to class I-restricted epitopes contributes to their level of presentation.

Background and objectives The LIM-and-SH3-domain-protein-1 (Lasp-1) is an actin-associated protein and is localised at focal adhesion sites where it is involved in organisation of actin polymerization and focal adhesion turnover prozesses. We investigated its role in regulating synovial fibroblast (SF) interaction with components of the extracellular matrix (ECM) and in establishing cell-cell contacts during RA. Materials and methods Lasp-1 expression was analysed in tissue from RA patients and in the hind paws of arthritic hTNFtg mice by Western blot, immunofluorescence and immunohistochemistry. Furthermore, Lasp-1-/- mice were interbred with hTNFtg mice and offsprings were analysed for the progression of joint destruction by clinical evaluation and histopathology. Migration characteristics of SF derived from wild type (wt), Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg mice were analysed in a modified scratch assay and by live cell imaging. Cell-matrix interactions and cell-cell contacts of isolated SF from all different genotypes were investigated using fibronectin coating in an electrical cell/substrate impedance sensing assay (ECIS). Additionally, we used an in vitro three-dimensional organ culture system for functional analyes. Results Lasp-1 expression levels were increased in human RA tissue and hTNFtg mice in comparison to healthy controls. Evaluation of Lasp-1-/-/hTNFtg mice revealed milder arthritis score compared to hTNFtg mice,which was confirmed by immunohistochemistry. Results of the scratch assays demonstrated a significantly reduced migration rate of Lasp-1-/- SF (-43,7% vs wt SF) and Lasp-1-/-/hTNFtg SF (-69,11% vs hTNFtg SF). Furthermore, live cell imaging studies demonstrated striking differences in the migration velocity and in migration edge formation of Lasp-1-/-/hTNFtg SF compared to hTNFtg SF. ECIS analysis demonstrated an increased cell-cell contact formation in Lasp1-/- compared to wt SF (+22% versus wt SF) and prolonged cell-cell interaction remodelling of Lasp-1-/-/hTNFtg SF in comparison to hTNFtg SF. Histological analysis of 3D matrices showed that Lasp-1 deletion in the hTNFtg background resulted in an organised cellular lining layer at the interface between the matrix and fluid phases comparable with wild type SF. In contrast, hTNFtg SF formed unorganised cellular condensations with no synovial architecture evident. Conclusion Lasp-1 regulates the migratory behaviour of synovial fibroblasts in rheumatoid arthritis by controlling the dynamics of cell-matrix and cell-cell contacts.

The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin–binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1β [IL-1β]). Endothelial domes were significantly reduced in Lsp1−/− compared with wild-type (WT) mice. Lsp1−/− animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.

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-1β-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-α, 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.

Although recent advances have enabled hematopoietic stem cells (HSCs) to be enriched to near purity, more information about their characteristics will improve our understanding of their development and stage-related functions. Here, using microarray technology, we identified endothelial cell-selective adhesion molecule (ESAM) as a novel marker for murine HSCs in fetal liver. Esam was expressed at high levels within a Rag1− c-kitHi Sca1+ HSC-enriched fraction, but sharply down-regulated with activation of the Rag1 locus, a valid marker for the most primitive lymphoid progenitors in E14.5 liver. The HSC-enriched fraction could be subdivided into 2 on the basis of ESAM levels. Among endothelial antigens on hematopoietic progenitors, ESAM expression showed intimate correlation with HSC activity. The ESAMHi population was highly enriched for multipotent myeloid-erythroid progenitors and primitive progenitors with lymphopoietic activity, and exclusively reconstituted long-term lymphohematopoiesis in lethally irradiated recipients. Tie2+ c-kit+ lymphohematopoietic cells in the E9.5–10.5 aorta-gonad-mesonephros region also expressed high levels of ESAM. Furthermore, ESAM was detected on primitive hematopoietic progenitors in adult bone marrow. Interestingly, ESAM expression in the HSC-enriched fraction was up-regulated in aged mice. We conclude that ESAM marks HSC in murine fetal liver and will facilitate studies of hematopoiesis throughout life.

The cover shows a double immunofluorescence staining of ESAM-1 and PECAM-1 in the T cell zone of the peripheral lymph node of the mouse. Red immunofluorescence of ESAM-1 colocalizes with the bright green immunofluorescence of PECAM-1, resulting in the observed yellow immunofluorescence. Lymphatic endothelial cells express PECAM-1 but lack ESAM-1 and hence are manifested as dim green immunofluorescence. The image was taken from the article by Pfeiffer et al. ( pp. 2142–2155) in which the authors demonstrate that molecularly distinct cellular junctions of blood and lymphatic endothelial cells establish different functional barriers controlling the distribution antigens and immune cells in the peripheral lymph node.

Recirculation of fluid and cells through lymphatic vessels plays a key role in normal tissue homeostasis, inflammatory diseases, and cancer. Despite recent advances in understanding lymphatic function (Alitalo, K., T. Tammela, and T.V. Petrova. 2005. Nature. 438:946–953), the cellular features responsible for entry of fluid and cells into lymphatics are incompletely understood. We report the presence of novel junctions between endothelial cells of initial lymphatics at likely sites of fluid entry. Overlapping flaps at borders of oak leaf–shaped endothelial cells of initial lymphatics lacked junctions at the tip but were anchored on the sides by discontinuous button-like junctions (buttons) that differed from conventional, continuous, zipper-like junctions (zippers) in collecting lymphatics and blood vessels. However, both buttons and zippers were composed of vascular endothelial cadherin (VE-cadherin) and tight junction–associated proteins, including occludin, claudin-5, zonula occludens–1, junctional adhesion molecule–A, and endothelial cell–selective adhesion molecule. In C57BL/6 mice, VE-cadherin was required for maintenance of junctional integrity, but platelet/endothelial cell adhesion molecule–1 was not. Growing tips of lymphatic sprouts had zippers, not buttons, suggesting that buttons are specialized junctions rather than immature ones. Our findings suggest that fluid enters throughout initial lymphatics via openings between buttons, which open and close without disrupting junctional integrity, but most leukocytes enter the proximal half of initial lymphatics.

Background and objectives Lasp-1 (LIM-and-SH3-domain-protein-1) is an actin-binding protein that modifies cytoskeleton organisation and is localised at cell-matrix interaction sites where it is involved in cell adhesion, migration and metastatic invasion. Its role in regulating synovial fibroblast (SF) interaction with components of extracellular matrix (ECM) and cell-cell contacts during RA is unknown. Furthermore, involved signalling pathways have to be elucidated. Materials and methods Lasp-1 expression in RA tissue and hind paws of arthritic hTNFtg mice was analysed via Western blot and immunohistochemistry. Furthermore, Lasp-1 ko mice were interbred with hTNFtg mice and offsprings were analysed for the progression of joint destruction by clinical evaluation and histopathology. Cell migration properties of SF derived from wild type (wt), Lasp-1 -/- , hTNFtg and Lasp1 -/- /hTNFtg mice were measured using a modified scratch assay and by live cell imaging. Extracellular matrix structure and organisation from wt, Lasp-1 -/- , hTNFtg and Lasp-1 -/- /hTNFtg SF were analysed by electron microscopy (EM). Cell-matrix interactions as well as cell-cell contacts of isolated SF from mice of all genotypes were investigated using fibronectin and self-purified collagen matrix in an electrical cell/substrate impedance sensing assay (ECIS). Via pull down assays and EM components of integrin mediated cell-matrix interaction complexes were examined. In addition, src-phosphorylation levels were evaluated by Western blot. Results Lasp-1 expression levels were increased in human RA and in hTNFtg mice compared to healthy controls. Lasp-1 -/- /hTNFtg mice displayed milder clinical symptoms confirmed by immunohistochemistry. Migration assays presented a significantly reduced migration rate of Lasp-1 -/- and Lasp-1 -/- /hTNFtg SF compared to SF from wt and hTNFtg mice. Analyses of SF ECM showed thickened collagen fibrils with less crosslinking of Lasp-1 -/- SF in comparison to wt controls. In ECIS, Lasp-1 -/- SF adhered to both fibronectin and self purified ECM faster than other genotypes (-50% vs. hTNFtg and wt SF). Additionally, Lasp-1 -/- SF formed a higher number of cell-cell contacts than wt and hTNFtg SF (-17% vs. hTNFtg and -22% wt vs. SF). Pull down assays identified cortactin, uPar and vinculin as components of cell-matrix interaction complexes with increased levels of vinculin found in complexes from Lasp-1 -/- compared to wtSF. Finally, both Lasp-1 -/- and Lasp-1 -/- /hTNFtg SF showed reduced src-phosphorylation compared to wt and hTNFtg SF. Conclusion The loss of Lasp-1 leads to altered src-phosphorylation and changes in cell-matrix interaction complexes as well as altered extracellular matrix organisation. Furthermore, Lasp-1 deficiency leads to significantly reduced migration rates of RASF and hTNFtg SF in vitro and to decreased cartilage degradation in hTNFtg mice in vivo .

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 beta2-integrins, the known ICAM-2 ligands, since neither blocking of beta2-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 beta2-integrins and DC-SIGN homologues.

Endothelial cell–selective adhesion molecule (ESAM) is specifically expressed at endothelial tight junctions and on platelets. To test whether ESAM is involved in leukocyte extravasation, we have generated mice carrying a disrupted ESAM gene and analyzed them in three different inflammation models. We found that recruitment of lymphocytes into inflamed skin was unaffected by the gene disruption. However, the migration of neutrophils into chemically inflamed peritoneum was inhibited by 70% at 2 h after stimulation, recovering at later time points. Analyzing neutrophil extravasation directly by intravital microscopy in the cremaster muscle revealed that leukocyte extravasation was reduced (50%) in ESAM−/− mice without affecting leukocyte rolling and adhesion. Depletion of >98% of circulating platelets did not abolish the ESAM deficiency–related inhibitory effect on neutrophil extravasation, indicating that it is only ESAM at endothelial tight junctions that is relevant for the extravasation process. Knocking down ESAM expression in endothelial cells resulted in reduced levels of activated Rho, a GTPase implicated in the destabilization of tight junctions. Indeed, vascular permeability stimulated by vascular endothelial growth factor was reduced in ESAM−/− mice. Collectively, ESAM at endothelial tight junctions participates in the migration of neutrophils through the vessel wall, possibly by influencing endothelial cell contacts.

The coxsackievirus-adenovirus receptor (CAR) is a cell contact protein on various cell types with unknown physiological function. It belongs to a subfamily of the immunoglobulin-superfamily of which some members are junctional adhesion molecules on epithelial and/or endothelial cells. CAR is dominantly expressed in the hearts and brains of mice until the newborne phase after which it becomes mainly restricted to various epithelial cells. To understand more about the physiological function of CAR, we have generated CAR-deficient mice by gene targeting. We found that these mice die between E11.5 and E13.5 of embryonal development. Ultrastructural analysis of cardiomyocytes revealed that the density of myofibrils was reduced and that their orientation and bundling was disorganized. In addition, mitochondria were enlarged and glycogen storage strongly enriched. In line with these defects, we observed pericardial edema formation as a clear sign of insufficient heart function. Developmental abnormalities likely to be secondary effects of gene ablation were the persistent singular cardial atrio-ventricular canal and dilatations of larger blood vessels such as the cardinal veins. The secondary nature of these defects was supported by the fact that CAR was not expressed on vascular cells or on cells of the vascular wall. No obvious signs for alterations of the histological organization of the placenta were observed. We conclude that CAR is required for embryonal heart development, most likely due to its function during the organization of myofibrils in cardiomyocytes.

Human CD99 is a small highly O-glycosylated cell-surface protein expressed on most leukocytes. It was recently found to be expressed at endothelial cell contacts and to participate in the transendothelial migration (TEM) of monocytes in vitro. In order to analyze the physiologic relevance of CD99 in vivo we searched for the mouse homolog. We cloned a mouse cDNA coding for a protein 45% identical in its sequence with human CD99. Based on the cDNA, we generated antibodies against this mouse homolog of CD99, which detected the antigen on most leukocytes, on endothelia of various tissues, and at cell contacts of cultured endothelial cells. Cell aggregation of CD99-transfected Chinese hamster ovary (CHO) cells was completely blocked by anti-CD99 antibodies. The same antibodies inhibited TEM of lymphocytes in vitro, independent of whether T cells or endothelial cells were preincubated with antibodies. In a cutaneous delayed-type hypersensitivity (DTH) reaction, anti-CD99 antibodies inhibited the recruitment of in vivo–activated T cells into inflamed skin as well as edema formation. We conclude that mouse CD99 participates in the TEM of lymphocytes and in their recruitment to inflamed skin in vivo. This establishes CD99 as a valid target for interference with cutaneous inflammatory processes.

Background and Objectives Lasp-1 is an important actin-crosslinking protein. It’s localises at focal adhesions, along stress fibres and leading edges and is involved in cellular migration and metastatic dissemination of different cancers. Rheumatoid arthritis synovial fibroblasts (RASF) are also able to enter the bloodstream from sites of cartilage destruction to new locations where they re-initiate the destructive processes. The underlying mechanisms of this process are of special interest, but currently unclear. Therefore we have investigated the role of Lasp-1 in synovial fibroblast migration during RA. Materials and Methods Lasp-1 expression and its subcellular location was analysed using Western blotting and immunofluorescence microscopy of cells seeded on various coatings, including fibronectin, laminin and a self-purified collagen matrix. Furthermore, Lasp-1 deficient mice were generated, which were interbred with hTNFtg mice, to form a model of inflammatory arthritis. Mice of all genotypes were analysed using a clinical score, measuring weight, grip strength and paw swelling over a time course of 14 weeks. Hind paws from each genotype were isolated, paraffin-embedded and toluidine blue stained in order to assess synovial inflammation, cartilage degradation and SF attachment. Cell migration properties of SFs derived from WT, Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg were measured using a modified scratch assay and by live cell imaging. Results Lasp-1 expression was up regulated in RASF and in SF from hTNFtg mice compared to healthy controls. In immunofluorescence, Lasp-1 was co-localised with cortactin, a marker for structures of cell adhesion and invasion, particularly when cultured on a purified collagen matrix. Lasp1-/-/hTNFtg mice showed milder clinical symptoms in comparison to hTNFtg mice. Notably, histopathological analyses revealed less cartilage destruction (0.4 mm vs. 1.6 mm, p Conclusion Lasp-1 deficiency leads to significantly reduced migration rates of RASF and hTNFtg SF in vitro and results in a decreased cartilage degradation and destruction and less SF attachment to articular cartilage in hTNFtg mice in vivo. Therefore, Lasp-1 is an important target for therapeutic strategies aimed to reduce the invasive and migratory behaviour of synovial fibroblast in rheumatoid arthritis.

Background Recent studies demonstrated the potential of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) to emigrate from affected joints and migrate via the bloodstream toward distant healthy cartilage in the SCID mouse model of disease. While the mechanisms of breaking endothelial barriers by RA-FLS are largely unclear, the junctional adhesion molecule C (JAM-C) has become of interest in this context due to its involvement in trans-endothelial migration of leukocytes. Using the human TNF transgenic (hTNFtg) mouse as a model for human RA, we studied the role of JAM-C in the transmigration of FLS derived from these mice. Methods The expression pattern of JAM-C on wild-type and hTNFtg FLS was analyzed by Western-blot and immunocytochemistry. We investigated the transmigratory capacity of these cells in a transmigration assay using murine endothelioma cells (bEnd.5) as an endothelial barrier, IL-1alpha treated murine cartilage tissue served as chemoattractant stimulus within this setting. Functional analyses included the knock down of JAM-C expression by siRNA against murine JAM-C as well as its neutralization by blocking antibodies. Results The expression of JAM-C could be detected on the surface of both wild-type and hTNFtg FLS and it was primarly located on sites of cell-cell interactions. Additionally, Western blot data showed an elevated expression of JAM-C in FLS from hTNFtg mice. Our transmigration experiments demonstrated a markedly higher potential of hTNFtg FLS to migrate through the endothelial monolayer than FLS from wild-type mice (+40%, p≤0.05), and cartilage explants pre-treated with IL-1alpha as chemoattractant strengthened the migratory capacity of hTNFtg FLS. Interestingly, knock down of JAM-C expression on hTNFtg FLS mediated by siRNA decreased the number of transmigrated cells of about 40% comparing with mock control (p≤0.01). Equally, the neutralization of JAM-C by blocking antibodies against murine JAM-C resulted in a reduction of transmigration on a similar level. Conclusions The inflammatory environment characteristic for joints of hTNFtg mice induces an up-regulation of JAM-C on FLS similar to that of human FLS during RA. Moreover, the differentiation of a trans-migrating phenotype of FLS, capable to break through endothelial barriers is supported by this environment. In this context, JAM-C seems to be contributed to this process of transmigration and, thus, to the extravasation of FLS and, therefore targeting JAM-C may be a promising therapeutic strategy for RA. Disclosure of Interest None Declared

Background and Objectives RA synovial fibroblasts (SF) have been suggested to contribute to the spreading of disease through their ability to leave cartilage destruction sites, migrate via the bloodstream and re-initiate the destructive process at distant articular cartilage surfaces. In this context, the actin-crosslinking protein Lasp-1 is of interest, because it is localised at leading edges of migrating cells and regulates metastatic dissemination of different tumours. Therefore, it is particularly important to investigate the role of Lasp-1 in SF migration and its effects on RA. Materials and Methods To identify different Lasp-1 expression levels in the hind paws of wt and hTNFtg mice, an established model for human RA, Western- blot analyses were performed. In parallel, Lasp-1 expression and its sub-cellular distribution was investigated in SF from wt and hTNFtg mice by Western-blot analyses and immunofluorescence. The migratory capacity of SFs derived from wild-type, Lasp-1 -/- , hTNFtg and Lasp1 -/- /hTNFtg mice was studied in a modified scratch assay as well as in live cell imaging studies. Furthermore, a transmigration assay using SF from all four genotypes and murine endothelioma cells (bEnd.5) as an endothelial barrier was carried out. For more detailed information, SF transmigration was evaluated when endothelial cells were also pre-treated with TNF-alpha, mimicking inflammatory conditions. Results Lasp-1 expression is upregulated in SF from hTNFtg mice and localises to structures of cell adhesion and invasion. In the scratch assay, a significantly reduced migration rate was detected in Lasp-1 -/- SFs after 24 hrs (–43.7% versus wt, p -/- /hTNFtg, respectively (–69.11% versus hTNFtg, p -/- /hTNFtg compared to hTNFtg SF. Furthermore, analyses showed a significant reduction of transmigration of Lasp1 -/- /hTNFtg compared to hTNFtg SF that was even enhanced by TNF-alpha stimulation of the endothelial cells. Interestingly, interbred Lasp1 -/- /hTNFtg mice presented milder clinical symptoms and analyses of histopathology revealed less cartilage degradation and less attachment of synovial tissue to the cartilage than hTNFtg mice at an age of 14 weeks. Conclusions Our data provide that the migratory capacity of SF is regulated by Lasp-1 and influences the severity of arthritis in hTNFtg mice. SF – when activated – migrate through the formation of invasive and adhesive membrane structures such as invadopodia, where Lasp-1 is prominently localised. Thus, targeting Lasp-1 may be a promising strategy to reduce the invasive and migratory behaviour of synovial fibroblasts in RA.

Background and objectives Recent studies demonstrated the potential of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) to migrate long distances via the bloodstream and invade distant cartilage in the SCID mouse model of disease.While the mechanisms of trans-endothelial migration of RA-FLS are largely unclear the junctional adhesion molecule C (JAM-C) has become of interest due to its involvement in trans-endothelial migration of leucocytes. Here, the authors used the hTNFtg mouse as a model for human RA and studied the role of JAM-C in the transmigration of FLS derived from these mice. Materials and methods The expression of JAM-C on wild-type and hTNFtg FLS was investigated by western-blot analysis and immunocytochemistry. The transmigratory capacity of these cells was studied in a transmigration assay using murine endothelioma cells (bEnd.5) as an endothelial barrier and IL-1alpha treated murine cartilage tissue as chemoattractant stimulus. Functional analyses included the knock down of JAM-C expression by siRNA against murine JAM-C as well as its neutralisation by blocking antibodies. Results The authors found the expression of JAM-C on the surface of both wild-type and hTNFtg FLS which is prominently located on sites of cell-cell interactions. Moreover, Western blot data revealed an elevated expression of JAM-C in FLS from hTNFtg mice. Transmigration experiments demonstrated a significantly higher potential of hTNFtg FLS to migrate through the endothelial monolayer than FLS from wild-type mice (+40%, p≤0,05), and cartilage explants pretreated with IL-1α enhanced the migratory capacity of hTNFtg FLS. Interestingly, siRNA-mediated knock down of JAM-C expression on hTNFtg FLS resulted in a reduction of transmigration of about 40% compared to mock control (p≤0,01). Likewise, the neutralisation of JAM-C by blocking antibodies against murine JAM-C reduced the number of transmigrated hTNFtg FLS on a similar level. Conclusion Our data demonstrate that the inflammatory environment within the joints of hTNFtg mice induces an up-regulation of JAM-C on FLS, which is characteristic for human RA-FLS. Moreover, they indicate that this environment supports the development of a trans-migrating phenotype of FLS able to get through endothelial barriers. JAM-C seems to be involved functionally in the transmigration and, thus, in extravasation of FLS and, therefore targeting JAM-C may be a promising therapeutic strategy for RA.

In the course of an analysis of cell-cell adhesion in the Xenopus embryo, antibodies directed against α and β catenin were applied to investigate their relation to the cadherins occurring early in this system. The results demonstrate that α and β-catenin are provided maternally and increase in amount throughout embryogenesis. Immunoprecipitations indicate that both of the catenins are complexed to U-cadherin in the early phase of embryogenesis and to β-cadherin, when it appears during gastrulation. An excess of α-catenin occurs in free form in the early embryo, whereas all of the catenin seems to be complexed to cadherin. Synthesis of the two components throughout early embryogenesis and their binding to newly synthesized cadherins were demonstrated by metabolic labelling. The spatial distribution of α-catenin was analysed by immunohistology. During cleavage β-catenin is deposited evenly along the plasma membranes within the embryo, while the cell peripheries at the surface of the embryo remain devoid of α-catenin. At later stages, the pattern of α-catenin distribution becomes more complex. Quantitative differences in the intensity of staining along the plasma membranes in the different regions of the embryo can be distinguished. Particularly the appearance of β-cadherin in the gastrula ectoderm is accompanied by conspicous depositions of α-catenin along the respective plasma membranes in this layer. All cells in the later embryo, apart from the neural crest cells, carry α-catenin on their plasma membranes indicating the universal character of cadherin-mediated cell-cell adhesion in the Xenopus embryo.

Hematopoietic stem cells (HSC) are an important cell type with the capacity for self-renewal as well as differentiation into multi-lineage blood cells, maintaining the immune system throughout life. Many studies have attempted to identify unique markers associated with these extremely rare cells. In bone marrow of adult mice, the Lin-c-kitHi Sca1+ CD34−/Lo Thy1.1Lo subset is known to include HSC with long-term repopulating capacity. However, several of these parameters differ between strains of mice, change dramatically during developmental age and/or are expressed on many non-HSC during inflammation. Efficient HSC-based therapies and the emerging field of regenerative medicine will benefit from learning more about what defines stem cells. We previously determined that the most primitive cells with lymphopoietic potential first develop in the paraaortic splanchnopleura/aorta-gonad-mesonephros (AGM) region of embryos using Rag1/GFP knock-in mice. We also reported that Rag1/GFP-c-kitHi Sca1+ cells derived from E14.5 fetal liver (FL) reconstituted lympho-hematopoiesis in lethally irradiated adults, while Rag1/GFPLo c-kitHi Sca1+ cells transiently contributed to T and B lymphopoiesis. To extend those findings, microarray analyses were conducted to search for genes that characterize the initial transition of fetal HSC to primitive lymphopoietic cells. The comparisons involved mRNA from Rag1Lo ckitHi Sca1+, early lymphoid progenitors (ELP) and the HSC-enriched Rag1-ckitHi Sca1+ fraction isolated from E14.5 FL. While genes potentially related to early lymphopoiesis were discovered, our screen also identified genes whose expression seemed to correlate with HSC. Among those, endothelial cell-selective adhesion molecule (ESAM) attracted attention because of its conspicuous expression in the HSC fraction and sharp down-regulation on differentiation to ELP. ESAM was originally identified as an endothelial cell-specific protein, but expression on megakaryocytes and platelets was also reported (J. Biol. Chem., 2001, 2002). Flow cytometry analyses with anti-ESAM antibodies showed that the HSC-enriched Rag1-c-kitHi Sca1+ fraction could be subdivided into two on the basis of ESAM levels. The subpopulation with the high density of ESAM was enriched for c-kitHi Sca1Hi cells, while ones with negative or low levels of ESAM were found in the c-kitHi Sca1Lo subset. Among endothelial-related antigens on HSC, CD34 and CD31/PECAM1 were uniformly present on Rag1-c-kitHi Sca1+ cells in E14.5 FL and neither resolved into ESAMHi and ESAM−/Lo fractions. Expression profiles of Endoglin and Tie2 partially correlate with ESAM. The primitive ESAMHi fraction uniformly expressed high levels of Endoglin and Tie2, but many of the more differentiated ESAM−/Lo cells still retained the two markers. ESAM expression correlated well with HSC activity. Cells in the ESAMHi Rag1-ckitHi Sca1+ fraction formed more and larger colonies than those in the ESAM-/Lo Rag1-ckitHi Sca1+ fraction. Particularly, most CFU-Mix, primitive progenitors with both myeloid and erythroid potential, were found in the ESAMHi fraction. In limiting dilution stromal cell co-cultures, we found that 1 in 2.1 ESAMHi Rag1-ckitHi Sca1+ cells and 1 in 3.5 ESAM−/Lo Rag1-ckitHi Sca1+ cells gave rise to blood cells. However, while only 1 in 125 ESAM−/Lo Rag1-ckitHi Sca1+ cells were lymphopoietic under these conditions, 1 in 8 ESAMHi Rag1-ckitHi Sca1+ cells produced CD19+ B lineage cells. In long-term reconstituting assays, ESAMHi Rag1-ckitHi Sca1+ cells contributed highly to the multi-lineage recovery of lympho-hematopoiesis in recipients, but no chimerism was detected in mice transplanted with ESAM−/Lo Rag1-ckitHi Sca1+ cells. These results suggested that HSC in E14.5 FL are exclusively present in the ESAMHi fraction. Tie2+ c-kit+ lympho-hematopoietic cells of E10.5 AGM also expressed high levels of ESAM. Furthermore, ESAM expression in adult bone marrow was detected on primitive progenitors and cells in the side population within the Lin-ckitHi Sca1+ fraction. Interestingly, the expression was up-regulated in aged mice. Based on these observations, we conclude that ESAM marks HSC throughout life in mice. We also observed that many of human cord blood CD34+ CD38− cells express ESAM, suggesting potential application for the purification of human HSC.