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Biomedical subjects

M Gröger

Publications and source records attributed to M Gröger.

14 recordsLinked to original sources
PubMed2001

Overexpression of transcription factor Ets-1 in rheumatoid arthritis synovial membrane: regulation of expression and activation by interleukin-1 and tumor necrosis factor alpha.

OBJECTIVE: To investigate the expression of the transcription factor Ets-1 in synovial tissue and cultured synovial fibroblasts from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to study the regulation of Ets-1 expression and activation in synovial fibroblasts by proinflammatory cytokines. METHODS: In situ expression of Ets-1 in synovial tissue from RA and OA patients was examined by double immunohistochemistry. The effects of interleukin-1 (IL-1) or tumor necrosis factor alpha (TNFalpha) on Ets-1 expression and activation (DNA binding) in cultured synovial fibroblasts were analyzed by Western blotting and DNA gel shift assay, respectively. In addition, the intracellular location of Ets-1 in synovial fibroblasts was determined by immunofluorescence. RESULTS: Pronounced expression of Ets-1 was detected in synovial tissues from all RA patients evaluated, particularly in the synovial lining layer and the sublining areas. Ets-1 was expressed by both fibroblasts and macrophages as well as by endothelial cells, while only a few T cells stained positive for Ets-1. In synovial specimens from OA patients, Ets-1 expression was much less frequently observed and was largely restricted to vascular cells. Ets-1 was expressed to a similar degree in cultured synovial fibroblasts from RA and OA patients, as demonstrated by reverse transcriptase-polymerase chain reaction and Western blotting. Both IL-1 and TNFalpha induced pronounced up-regulation of Ets-1 in synovial fibroblasts. Moreover, binding of Ets-1 to its specific DNA binding site was induced by both cytokines, although with different time courses. Immunofluorescence staining revealed a dominant nuclear localization of Ets-1 in IL-1- or TNFalpha-stimulated synovial fibroblasts. CONCLUSION: The overexpression of Ets-1 observed in RA synovial tissue appears to be caused by TNFalpha and IL-1, suggesting that Ets-1 may be an important factor in the cytokine-mediated inflammatory and destructive cascade characteristic of RA.

Arthritis, Rheumatoid↗
PubMed2001

Dimethylfumarate inhibits tumor-necrosis-factor-induced CD62E expression in an NF-kappa B-dependent manner.

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 < or = 70 microM, 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-kappa B, or AP-1-responsive promoter constructs, dimethylfumarate inhibited tumor-necrosis-factor-induced activation of the CD62E and the NF-kappa B but not the AP-1 promoter construct. In summary, at a dose range < or = 70 microM, dimethylfumarate appeared to be a specific inhibitor of CD62E expression in an NF-kappa B-dependent manner.

Capillaries↗
PubMed2001

Platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin cooperatively regulate fibroblast growth factor-induced modulations of adherens junction functions.

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.

Adherens Junctions↗
PubMed2000

Dermal microvascular endothelial cells express the 180-kDa macrophage mannose receptor in situ and in vitro.

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.

Cell Membrane↗
PubMed2000

Endothelial adherens junctions.

The principle of the molecular organization of adherens junctions follows a uniform pattern, which is found in epithelial, muscular, neuroneal as well as in endothelial cells and is highly conserved among species. Transmembrane molecules of the cadherin family link to catenins, which anchor the adhesion plaque to the cytoskeleton. The kind of cadherin used in adherens junctions is cell-type specific, vascular endothelial (VE)-cadherin is specific for endothelial cells. The assembly and disassembly of the cadherin/catenin complex is dynamic and regulated by growth factors. The functional status of adherens junctions controls endothelial cell-to-cell adhesion, cell scattering, vessel morphogenesis and has intracellular signaling properties, thereby playing an important role in vasculogenesis and angiogenesis.

Adherens Junctions↗
PubMed1999

Survival, maturation, and function of CD11c- and CD11c+ peripheral blood dendritic cells are differentially regulated by cytokines.

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.

Animals↗
PubMed1999

A standardized, a computer-assisted in vitro assay for the assessment of neutrophil transmigration across endothelial monolayers.

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.

Animals↗
PubMed1999

Immune complexes from vasculitis patients bind to endothelial Fc receptors independent of the allelic polymorphism of FcgammaRIIa.

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.

Alleles↗
PubMed1999

Activation of Fas inhibits heat-induced activation of HSF1 and up-regulation of hsp70.

Activation of heat shock factor (HSF) 1-DNA binding and inducible heat shock protein (hsp) 70 (also called hsp72) expression enables cells to resist various forms of stress and survive. Fas, a membrane-bound protein, is a central proapoptotic factor; its activation leads to a cascade of events, resulting in programmed cell death. These two mechanisms with contradictory functions, promoting either cell survival or death, were examined for their potential to inhibit each other's activation. Induction of FAS-mediated signaling was followed by a rapid decrease in HSF1-DNA binding and inducible hsp70 expression. Inhibition of HSF1-DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS signaling. These effects of FAS activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1) inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase in apoptosis rates from 20% to 50% in response to heat stress. When analyzing the effects of HSF1/hsp70 activation on Fas-mediated apoptosis, protection from apoptosis was seen in cells with induced hsp70 protein levels, but not in cells that were just induced for HSF1-DNA binding. Thus, we conclude that inhibition of HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis, without the influence of inhibitory signals.

Apoptosis↗
PubMed1998

Enhanced expression of heat shock protein 70 (hsp70) and heat shock factor 1 (HSF1) activation in rheumatoid arthritis synovial tissue. Differential regulation of hsp70 expression and hsf1 activation in synovial fibroblasts by proinflammatory cytokines, shear stress, and antiinflammatory drugs.

Heat shock proteins (hsp) have been repeatedly implicated to participate in the pathogenesis of rheumatoid arthritis (RA). Herein, we investigated the regulation of synovial hsp70 expression by analyzing the DNA-binding activity of heat shock transcription factor 1 (HSF1) as well as inducible hsp70 expression. Experiments were performed both on synovial tissue and on synovial fibroblast-like cells (SFC). Gel mobility shift analysis revealed increased HSF1 activation, and Western blotting and immunohistochemistry revealed increased hsp70 expression in RA synovial tissue, but not in synovial tissue derived from patients with osteoarthritis. Proinflammatory cytokines (TNF-alpha, IL-1alpha, IL-6), but not IFN-gamma or TGF-beta, induced activation of HSF1-DNA binding and hsp70 expression in cultivated SFC. Activation of HSF1 in SFC was accompanied by hyperphosphorylation and nuclear translocation of HSF1. Furthermore, shear stress also induced a complete heat shock response in cultivated synovial cells. In contrast, nonsteroidal antiinflammatory drugs triggered only an incomplete heat shock response, with HSF1 activation but not hsp70 induction, whereas steroids and immunosuppressive drugs did not affect the heat shock response at all. In summary, these data suggest that induction of hsp70 expression in rheumatoid synovial tissue is based on transcriptional activation of HSF1 due to the presence of proinflammatory cytokines (and possibly also shear stress).

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1997

HECA-452+ T cells migrate through superficial vascular plexus but not through deep vascular plexus endothelium.

The skin is nourished by two interconnected vascular systems, the superficial vascular plexus coursing just beneath the epidermis and the deep vascular plexus located above the subcutaneous tissue. Skin inflammatory T cells in diseases, such as psoriasis or dermatitis, strikingly aim for the superficial vascular plexus without involving the deep vascular plexus, and the infiltrating T cells bear a distinct phenotype expressing the cutaneous lymphocyte-associated antigen, which is recognized by mAb HECA-452. We wanted to know whether HECA-452+ lymphocytes indeed are able to distinguish between superficial and deep vascular plexus homing sites. Employing the hu-SCID mouse model grafted with human skin and human T cells, as described previously, we developed a new skin-grafting strategy providing superficial and deep vascular plexus skin specimens placed separately onto the same mouse. Fourteen days after allogeneic human T cell grafting, both human skin sites were densely infiltrated by human T cells, but only T cells within the superficial vascular plexus, but not within the deep vascular plexus, expressed the cutaneous lymphocyte-associated antigen. IL-2 and IFN-gamma expression and allogeneic vessel destruction were present within both superficial and deep vascular plexus skin. This model provides direct evidence that expression of a specific homing receptor is indeed able to direct lymphocyte traffic, not only to a distinct organ but also to a distinct vascular bed within one organ.

Animals↗
PubMed1996

Dermal microvascular endothelial cells express CD32 receptors in vivo and in vitro.

Immune complexes are thought to be the major cause of cutaneous necrotizing vasculitis, but the mechanism of immune complex targeting to specific vessels is largely unknown. In myelomonocytic cells, immune complex binding and receptor-mediated endocytosis are mediated by Fc gamma R. We asked whether dermal microvascular endothelial cells (DMEC) express Fc gamma Rs. In cryostat sections of normal human skin, mAb IV.3 or AT10, both recognizing CD32 (Fc gamma RII), localizes to the luminal surface of DMEC of the superficial but not of the deep vascular plexus. All DMEC do not express CD16 (Fc gamma RIII) or CD64 (Fc gamma RI) molecules. Adult skin-derived DMEC in culture express CD32 (Fc gamma RII) molecules, as measured by FACS, but are negative for CD16 or CD64. HUVEC, tested for comparison, do not express CD16, 32, or 64 proteins. By reverse-transcriptase PCR and subsequent Southern blot analysis, the isoform of the CD32 molecule expressed on DMEC is determined as Fc gamma RIIa. HUVEC do not contain Fc gamma RIIa or Fc gamma RIIb mRNA. In DMEC, Fc gamma RIIa cross-linking results in immediate intracellular free Ca2+ ([Ca2+]i) concentration fluxes and in rapid internalization of the occupied receptors. We conclude that DMEC are equipped with fully functional Fc gamma RIIa molecules.

Adult↗
PubMed1996

Human delayed-type hypersensitivity reaction in a SCID mouse engrafted with human T cells and autologous skin.

We have developed and animal model to study human delayed-type hypersensitivity reactions occurring in a human environment within a mouse host. Human skin was grafted onto the backs and autologous human immune cells were injected into the peritoneal cavity of mice with severe combined immunodeficiency. Seven and 14 d after grafting, 2-50% of total white blood and spleen cells were of human origin. Mouse spleen-derived human T cells from tetanus toxoid-sensitized donors proliferated in response to tetanus toxoid as measured by [3H]thymidine uptake, and the strength of this proliferative response equaled that with pre-graft T cells from the same donor. Proliferation was blocked with monoclonal antibodies to human but not to mouse major histocompatibility complex antigens and with anti-human CD4 monoclonal antibodies. In vivo vaccination of mice with tetanus toxoid did not enhance proliferation of mouse spleen-derived human T cells in response to antigen. Injection of tetanus toxoid into the human skin graft caused a perivascular human CD4+/CD25+ T-cell infiltrate, which was not present when tetanus toxoid was injected into adjacent mouse skin. We conclude that human T cells grafted into mice with severe combined immunodeficiency retain their function, that human T cells specifically recognize human but not mouse skin as homing sites, and that human T-cell responses depend on the human micro-environment. This model lends itself to studies of endothelium-T-cell interactions, T-cell activation within skin, and chronic inflammatory skin diseases.

Animals↗