The endothelium is a complex organ influenced by circulating mediators, adjacent cells, physico-chemical factors, and shear stress. During systemic inflammation and sepsis, excessive and sustained activation of the endothelium result in the loss of its anticoagulant and anti-adhesive characteristics as well as in a loss of endothelial barrier function. We set up a cell-culture model to study endothelial activation induced by lipopolysaccharide (LPS) or by plasma from septic patients and studied the effect of adsorbent-based mediator modulation on endothelial activation.

Matrilysin (MMP7) is a secreted matrix metalloproteinase, which contributes to angiogenesis by breaking down basement membranes. We show that the angiogenic factor FGF-2 induces MMP7 expression in human endothelial cells. The promoter contains a Lef/Tcf consensus sequence, but using wildtype or Lef/Tcf-mutated promoter constructs, FGF-2-induced MMP7 reporter activity is independent from Lef/Tcf sites. Instead, we show that overexpression of a dominant negative Stat3 mutant reduces FGF-2-mediated MMP7 promoter activity. However, Stat3 does not bind to the MMP7 promoter, but activates MMP7 gene expression indirectly via AP-1. This is confirmed by MMP7 promoter constructs with mutated AP-1 sites which did not respond to FGF-2 and by siRNAs against Stat1 and Stat3, which repressed FGF-2-induced MMP7 protein expression. In conclusion, we show that FGF-2-induced MMP7 expression in endothelium depends on AP-1 and FGF-2 signaling to AP-1 involves a Stat1/3-dependent pathway.

Factors determining lymphatic differentiation in the adult organism are not yet well characterized. We have made the observation that mixed primary cultures of dermal blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) grown under standard conditions change expression of markers during subculture: After passage 6, they uniformly express LEC-specific markers Prox-1 and podoplanin. Using sorted cells, we show that LEC but not BEC constitutively express IL-3, which regulates Prox-1 and podoplanin expression in LEC. The addition of IL-3 to the medium of BEC cultures induces Prox-1 and podoplanin. Blocking IL-3 activity in LEC cultures results in a loss of Prox-1 and podoplanin expression. In conclusion, endogenous IL-3 is required to maintain the LEC phenotype in culture, and the addition of IL-3 to BEC appears to induce transdifferentiation of BEC into LEC.

Factors determining lymphatic differentiation in the adult organism are not yet well characterized. We have made the observation that mixed primary cultures of dermal blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) grown under standard conditions change expression of markers during subculture: After passage 6, they uniformly express LEC-specific markers Prox-1 and podoplanin. Using sorted cells, we show that LEC but not BEC constitutively express IL-3, which regulates Prox-1 and podoplanin expression in LEC. The addition of IL-3 to the medium of BEC cultures induces Prox-1 and podoplanin. Blocking IL-3 activity in LEC cultures results in a loss of Prox-1 and podoplanin expression. In conclusion, endogenous IL-3 is required to maintain the LEC phenotype in culture, and the addition of IL-3 to BEC appears to induce transdifferentiation of BEC into LEC.

Plasmacytoid dendritic cells (pDCs) recognize microbes, viruses in particular, and provide unique means of innate defense against them. The mechanism of pDC tissue recruitment remained enigmatic because the ligands of CXCR3, the cardinal chemokine receptor on pDCs, have failed to induce in vitro chemotaxis of pDCs in the absence of additional chemokines. In this study, we demonstrate that CXCR3 is sufficient to induce pDC migration, however, by a migratory mechanism that amalgamates the features of haptotaxis and chemorepulsion. To mediate "haptorepulsion" of pDCs, CXCR3 requires the encounter of its cognate ligands immobilized, optimally by heparan sulfate, in a form of a negative gradient. This is the first report of the absolute requirement of chemokine immobilization and presentation for its in vitro promigratory activity. The paradigmatic example of pDC haptorepulsion described here may represent a new pathophysiologically relevant migratory mechanism potentially used by other cells in response to other chemokines.

Plasmacytoid dendritic cells (pDCs) recognize microbes, viruses in particular, and provide unique means of innate defense against them. The mechanism of pDC tissue recruitment remained enigmatic because the ligands of CXCR3, the cardinal chemokine receptor on pDCs, have failed to induce in vitro chemotaxis of pDCs in the absence of additional chemokines. In this study, we demonstrate that CXCR3 is sufficient to induce pDC migration, however, by a migratory mechanism that amalgamates the features of haptotaxis and chemorepulsion. To mediate “haptorepulsion” of pDCs, CXCR3 requires the encounter of its cognate ligands immobilized, optimally by heparan sulfate, in a form of a negative gradient. This is the first report of the absolute requirement of chemokine immobilization and presentation for its in vitro promigratory activity. The paradigmatic example of pDC haptorepulsion described here may represent a new pathophysiologically relevant migratory mechanism potentially used by other cells in response to other chemokines.

Fumaric acid esters, mainly dimethylfumarate (DMF), have been successfully used to treat psoriasis. Based on previous observations that DMF inhibited expression of several TNF-induced genes in endothelial cells, we wished to explore the molecular basis of DMF function in greater detail. In first experiments we analyzed DMF effects on tissue factor expression in human endothelial cells in culture, because tissue factor is expressed by two independent sets of transcription factors, by NF-kappa B via TNF and by early gene response-1 transcription factor via vascular endothelial growth factor (VEGF). We show that DMF inhibits TNF-induced tissue factor mRNA and protein expression as well as TNF-induced DNA binding of NF-kappa B proteins, but not VEGF-induced tissue factor protein, mRNA expression, or VEGF-induced early gene response-1 transcription factor/DNA binding. To determine where DMF interferes with the TNF/NF-kappa B signaling cascade, we next analyzed DMF effects on I kappa B and on the subcellular distribution of NF-kappa B. DMF does not inhibit TNF-induced I kappa B alpha phosphorylation and I kappa B degradation; thus, NF-kappa B is properly released from I kappa B complexes even in the presence of DMF. Importantly, DMF inhibits the TNF-induced nuclear entry of NF-kappa B proteins, and this effect appears selective for NF-kappa B after the release from I kappa B, because the constitutive shuttling of inactive NF-kappa B/I kappa B complexes into and out from the nucleus is not blocked by DMF. Moreover, DMF does not block NF-kappa B/DNA binding. In conclusion, DMF appears to selectively prevent the nuclear entry of activated NF-kappa B, and this may be the basis of its beneficial effect in psoriasis.

Lef/Tcf proteins belong to a family of architectural transcription factors that control developmental processes and play an important role in oncogenesis. Classical activators of Lef/Tcf-dependent transcription comprise the Wnt family of proteins, which translocate beta-catenin into the nucleus and allow the formation of transactivation-competent Lef/Tcf-beta-catenin complexes. Here we show that in human endothelial cells fibroblast growth factor-2 (FGF-2) reduces GSK-3 activity and augments nuclear levels of beta-catenin. FGF-2 induced Lef/Tcf-dependent transcription of a cyclin D1-luciferase construct. Gel shift assays revealed binding of Tcf-4 as the only Lef/Tcf family member and of P-catenin to the Lef/Tcf site in the cyclin D1 promoter. Cotransfection with a dominant negative Tcf-4 construct inhibited the FGF-2-induced cyclin D1 promoter activity. Overexpression of an uninhibitable GSK-3beta mutant resulted in partial inhibition of FGF-2-mediated cyclin D1 induction. The importance for cyclin D1 in FGF-2-induced angiogenesis in vivo is shown in cyclin D1(-/-) mice, where FGF-2-induced new vessel formation was significantly reduced compared with FGF-2-induced angiogenesis in cyclin D1(+/+) mice. In conclusion, FGF-2 is a novel modulator of Lef/Tcf-beta-catenin signaling in endothelial cells, suggesting that angiogenic properties of FGF-2 are at least in part mediated by Lef/Tcf-beta-catenin activation.

Cellular adherens junctions are formed by cadherins linked to proteins of the catenin family. In endothelial cells, not only vascular endothelial cadherin but also platelet endothelial cell adhesion molecule-1 localizes into junctions and associates with beta-catenin. To explore a putative cooperation of platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin, we analyzed transfectants expressing either platelet endothelial cell adhesion (CD31 cells) or vascular endothelial cadherin (CD144 cells) or both molecules (CD31/CD144 cells), and, for comparison, human umbilical vein endothelial cells. Basic fibroblast growth factor completely dissociated vascular endothelial cadherin/beta-catenin complexes and robustly moved beta-catenin into the nucleus in CD144 cells, whereas in CD31/CD144 cells as well as in human umbilical vein endothelial cells, fibroblast growth factor only partially dissociated the junctional complex followed by a significantly reduced nuclear translocation of beta-catenin. In contrast, in CD31 cells, the subcellular distribution of beta-catenin remained unaffected by fibroblast growth factor. As a functional consequence, fibroblast growth factor induced a complete collapse of the F-actin network in CD144 cells, a limited rearrangement of F-actin fibers in CD31/CD144 cells and no F-actin rearrangement in CD31 cells. We also analyzed the effect of fibroblast growth factor-induced rearrangement of junctions on junction permeability for leukocytes: in line with our observation that vascular endothelial cadherin was required for cells to respond to fibroblast growth factor, only in CD31/CD144 cells, but not in CD31 cells, leukocyte transmigration was significantly enhanced by fibroblast growth factor. In conclusion platelet endothelial cell adhesion molecule-1 cooperates with vascular endothelial cadherin in a mutual fashion; platelet endothelial cell adhesion molecule-1 reduces and temporarily limits fibroblast growth factor-induced dissociation of vascular endothelial cadherin/beta-catenin complexes, but requires vascular endothelial cadherin to control leukocyte transmigration in dependence of fibroblast growth factor.

Fumaric acid esters are thought to improve psoriasis by altering leukocyte, keratinocyte, and/or endothelial functions. To determine specificity, kinetics, and molecular mechanisms of different fumaric acid esters in their ability to inhibit endothelial cell activation, we analyzed CD62E and CD54 expression in endothelial cells in vivo and in vitro. In lesional skin of psoriatic patients, oral fumaric acid ester treatment resulted in a marked reduction of CD62E but not CD54 expression on dermal microvessels. Using human umbilical vein endothelial cells, dimethylfumarate almost completely inhibited tumor-necrosis-factor-induced CD62E, but not CD54 expression at concentrations less than or equal to 70 muM, mimicking the situation in vivo. A 60 min dimethylfumarate preincubation was sufficient to block tumor-necrosis-factor-induced CD62E expression for up to 24 h. In contrast, equimolar concentrations of methylhydrogenfumarate, the hydrolysis product of dimethylfumarate, did not suppress tumor-necrosis-factor-induced CD62E expression. Likewise, all fumaric acid esters other than dimethylfumarate were ineffective. Using CD62E, NF-kappaB, or AP-1-responsive promoter constructs, dimethylfumarate inhibited tumor-necrosis-factor-induced activation of the CD62E and the NF-kappaB but not the AP-1 promoter construct. In summary, at a dose range less than or equal to 70 muM, dimethylfumarate appeared to be a specific inhibitor of CD62E expression in an NF-kappaB-dependent manner.

Expression of the 180-kDa mannose receptor (MR) is mainly found on cells of the macrophage lineage. MR mediates the uptake of micro-organisms and host-derived glycoproteins. We demonstrate that endothelium of the human skin in situ and dermal microvascular endothelial cells (DMEC) in vitro expressed MR at both the protein and mRNA levels. In contrast, HUVEC were consistently negative for MR expression. DMEC internalized dextran as well as Escherichia coli by the way of MR into acidic phagosomes, only a few of which fused with CD63- and lysosomal-associated membrane glycoprotein-2-positive lysosomes. This contrasts with the situation in monocyte-derived dendritic cells, where almost all of the MR-Ag complexes reached CD63- and lysosomal-associated membrane glycoprotein-2-positive compartments, indicating differences in the phagolysosomal fusion rate between DMEC and dendritic cells. In conclusion, DMEC express functional MR, a finding that corroborates a role of skin endothelium in Ag capture/clearing.

We describe an automated, observer-independent and highly reproducible assay for the quantification of transmigrated neutrophils across endothelial monolayers. Endothelial cells grown on collagen gels were loaded with a dye emitting red fluorescence. Neutrophils loaded with dye emitting green fluorescence were allowed to adhere to and transmigrate across endothelial monolayers. For quantification of adherent and migrated cells, randomly selected fields were scanned by confocal laser scan microscopy at defined depths within and below the endothelial monolayers. The images obtained were transferred into the public domain NIH image program and numbers and distribution of cells within scanned sectors were automatically calculated. We demonstrate that adherent neutrophils are easily discriminated from transmigrated cells; absolute numbers of migrated cells can be reproducibly calculated by counting cells at a depth of -20 microm, thus permitting evaluation of large-scale experiments: the efficacy of neutrophil transmigration depends on the level of endothelial activation after TNF stimulation and mAbs to cell surface adhesion molecules interfere with migration in a manner similar to that previously shown in in vivo experiments. This assay lends itself to the identification of molecules influencing in cell migration in each phase of EC activation and to the screening of pro- and anti-migratory properties of biological or pharmacological reagents.

Two types of dendritic cells (DC) are circulating in human blood and can be identified by their differential expression of the myeloid Ag CD11c. In this study, we show that CD11c- peripheral blood (PB)-DC correspond to plasmacytoid DC of lymphoid tissue not only by their surface Ag expression profile but, more impressively, by their peculiar ultramorphology. We also demonstrate that CD11c- and CD11c+ DC differ in the quality of their response to and in their requirement for certain cytokines. Freshly isolated CD11c- cells depend on IL-3 for survival and use autocrine or exogenous TNF-alpha as maturation signal, leading to the appearance of a highly dendritic phenotype, the up-regulation and redistribution of MHC class II from lysosomal compartments to the plasma membrane, the increased expression of costimulatory molecules, and the switch from a high Ag-processing to a low Ag-processing/potent accessory cell mode. Surprisingly, IL-4 efficiently killed freshly isolated CD11c- PB-DC, but did not impair the viability of CD11c+ PB-DC and, together with GM-CSF, induced maturation of these cells. A direct functional comparison revealed that neo-Ag-modified and subsequently matured CD11c- but to a lesser extent CD11c+ DC were able to prime naive Ag-specific CD4+ T cells. Our findings show that two diverse DC types respond to certain T cell-derived cytokines in a differential manner and, thus, suggest that suppression or activation of functionally diverse DC types may be a novel mechanism for the regulation of the quantity and quality of immune responses.

UNLABELLED Cutaneous leukocytoclastic vasculitis is characterized by the deposition of circulating immune complexes, neutrophil extravasation, and vessel destruction, but mechanisms of circulating immune complexes capture within postcapillary venules are unknown. We demonstrate that circulating immune complexes from sera of vasculitis patients bind to cultured endothelium in an Fc gamma receptor IIa-dependent fashion. In lesional skin, endothelial cells bind immunoglobulin G2 > immunoglobulin G3 and immunoglobulin G4, but not immunoglobulin G1, even before obvious neutrophil transmigration and vessel damage. As the human Fc gamma receptor IIa proteins exist in two allotypes (one with a histidine at position 131, which binds immunoglobulin G1, 2, 3 and the other with an arginine at position 131, which binds immunoglobulin G1, and 3, but is unable to bind immunoglobulin G2), we expected an altered prevalence of histidine 131 forms in vasculitis patients. Sequence analysis, however, revealed an equal distribution of allotypes in patients and controls. In conclusion, circulating immune complex binding to endothelial Fc gamma receptor IIa is among the initial steps in the development of vasculitis. Although immunoglobulin G2 is the predominant subtype precipitated at endothelial surfaces, it is not required for fixing circulating immune complexes to endothelium, because patients homozygote for Fc gamma receptor IIa-arginine 131 equally develop leukocytoclastic vasculitis as those bearing the Fc gamma receptor IIa-histidine 131 allele. As immunoglobulin G1 is virtually absent in leukocytoclastic vasculitis lesions and immunoglobulin G4 does not bind to both Fc gamma receptor IIa alleles, these complexes, in addition to immunoglobulin G2, should contain immunoglobulin G3 in order to fix to vascular Fc gamma receptor IIa, at least in persons homozygous for Fc gamma receptor IIa-arginine 131. KEYWORDS CD32/immunoglobulin G subtypes/leukocytoclastic vasculitis/microvessels.