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D Vestweber

Publications and source records attributed to D Vestweber.

At least 91 records · Page 5Linked to original sources
PubMed1994

Borrelia burgdorferi upregulates the adhesion molecules E-selectin, P-selectin, ICAM-1 and VCAM-1 on mouse endothelioma cells in vitro.

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd 3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (approximately 50 h), bEnd3 cells were found to bind cells of a VLA-4+ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.

Animals↗
PubMed1994

Expression of P-selectin on endothelial cells is upregulated by LPS and TNF-alpha in vivo.

P-selectin is an endothelial cell adhesion molecule which mediates the binding of neutrophils and monocytes. Its appearance at the cell surface can be induced within minutes by histamine and thrombin which rapidly stimulate the transport of P-selectin from intracellular storage granules to the plasma membrane. We have recently found a second regulation mechanism for P-selectin on mouse endothelioma cells. Like E-selectin, P-selectin is also regulated at the level of transcription. Both selectins are induced by LPS or TNF-alpha with a maximal expression level at the cell surface 3-4 h after stimulation. Here, we report that this up-regulation of the synthesis of P-selectin also occurs in vivo in endothelium of the mouse. Analysing brain tissue, which is devoid of constitutive expression of P-selectin, we found that LPS and also TNF-alpha strongly induce the expression of P-selectin on all venular endothelial cells of the leptomeninges and, at a weaker level, on some blood vessels of the brain parenchyma. Induction of P-selectin expression could also be observed in tissues, such as the tongue, where P-selectin is constitutively expressed on small venules but only rarely on larger venules. Strong staining for P-selectin on endothelium of all large venules was observed in tissues of LPS and TNF-alpha treated animals and staining for this newly synthesized P-selectin was enriched at the luminal surface of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Expression of endothelial cell adhesion molecules in joints and heart during Borrelia burgdorferi infection of mice.

The expression of adhesion molecules on endothelia was examined during chronic arthritis and carditis in SCID and immunocompetent susceptible AKR/N mice infected with Borrelia burgdorferi (B. burgdorferi). All stages of disease were associated with the upregulation or new expression of ICAM-1 and P-selectin and of VCAM-1 and E-selectin, respectively, on blood vessels of affected joint tissues of SCID and AKR/N mice as well as on heart tissue of SCID mice but not in other tissues. Moreover, ICAM-1 was also found on infiltrating mononuclear cells. The overall staining intensity for each of the four adhesion molecules on individual tissue sections of joint and heart increased with time of infection and was associated with the presence of spirochetes in the tissue. In addition it is shown that in both mouse strains inflammation of joints but not heart is accompanied by vascular proliferation. Synovial but not heart tissues of infected SCID mice were found to express both peripheral- (PNAd) and mucosal (MAdCAM-1) lymph node high endothelia venule associated vascular addressins as detected by mAb Meca-79 and Meca-367, respectively, but only at later stages of the disease and only on newly generated small venules. However, neither of the two addressins were evident in synovial lesions of AKR/N mice. Together the data suggest that the concomittant induction of ICAM-1, VCAM-1, E-selectin and P-selectin in lesions of infected mice provide a means for enhanced cellular infiltration into affected organs and that the regulation of these structures is conserved in the absence of a functional immune system. Furthermore, the differential induction of vascular proliferation in joint and heart tissues as well as the restricted expression patterns of vascular addressins indicate that the pathogenetic processes induced by B. burgdorferi are distinct for joint and heart.

Animals↗
PubMed1993

A monoclonal antibody against an activation epitope on mouse integrin chain beta 1 blocks adhesion of lymphocytes to the endothelial integrin alpha 6 beta 1.

We have generated a monoclonal antibody (mAb), 9EG7, against mouse endothelial cells that blocks adhesion of lymphocytes to endothelial cells. Sequencing of four tryptic peptides of the purified antigen revealed its identity with the integrin chain beta 1. The only beta 1 integrin that is known to mediate cell-cell adhesion is alpha 4 beta 1 (VLA-4). This is not the integrin that is functionally defined by the mAb 9EG7 on endothelial cells. First, alpha 4 is not present on the analyzed endothelial cells. Second, mAb 9EG7 does not block the cell-adhesion function of alpha 4 beta 1 on the nonactivated mouse lymphoma L1-2. Thus, the mAb 9EG7 can functionally distinguish between different beta 1 integrins and defines a beta 1 integrin other than alpha 4 beta 1 as a newly discovered cell-cell adhesion molecule. This integrin is most likely alpha 6 beta 1, since an antibody against the alpha 6 chain blocks lymphocyte adhesion to the same degree as the mAb 9EG7, the effect of both antibodies is not additive, and the alpha 6 chain is coprecipitated with beta 1 in 9EG7 immunoprecipitations. Surprisingly, activation of alpha 4 beta 1 on L1-2 cells with phorbol ester or Mn2+ allows blocking of alpha 4 beta 1-mediated adhesion of L1-2 cells to endothelial cells with mAb 9EG7. Furthermore, only the activated alpha 4 beta 1 heterodimer, but not the unactivated complex, is detectable with 9EG7 in immunoprecipitations and by flow cytometry. Thus, mAb 9EG7 defines an epitope on integrin chain beta 1, which is accessible on the alpha 4 beta 1 heterodimer only after activation of this integrin.

Animals↗
PubMed1993

Identification of a glycoprotein ligand for E-selectin on mouse myeloid cells.

E-selectin is an inducible endothelial cell adhesion molecule for neutrophils which functions as a Ca(2+)-dependent lectin. Using a recombinant, antibody-like form of mouse E-selectin, we have searched for glycoprotein ligands on mouse neutrophils and the neutrophil progenitor cell line 32D cl 3. We have identified a 150-kD glycoprotein as the only protein which could be affinity-isolated with soluble E-selectin from [35S]methionine/[35S]cysteine-labeled 32D cl 3 cells. Binding of this protein was strictly Ca(2+)-dependent, was blocked by a cell adhesion-blocking mAb against mouse E-selectin, and required the presence of sialic acid on the 150-kD ligand. This glycoprotein was also affinity-isolated from mature neutrophils, in addition to a minor component at 250 kD, but could not be isolated from several other non-myeloid cell lines. The 150-kD glycoprotein was the only protein from 32D cl 3 cells, which was detectable by silver-staining after a one-step affinity-isolation.

Animals↗
PubMed1993

Five tumor necrosis factor-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes.

We have distinguished five TNF-alpha-inducible cell adhesion mechanisms on microvasculature-derived endothelioma cells of the mouse which mediate the binding of different types of leukocytes. Three of these mechanisms could be identified as the mouse homologs of ICAM-1, VCAM-1, and E-selectin, of which the latter was defined by the novel mAb 21KC10. The fourth TNF-alpha-inducible cell adhesion mechanism was blocked by antibodies specific for mouse P-selectin. We have recently shown that TNF-alpha stimulates the synthesis of P-selectin in mouse endothelioma cells (A. Weller, S. Isenmann, D. Vestweber. 1992. J. Biol. Chem. 267:15176-15183). Here we show that this stimulation leads to maximal cell surface expression levels within 4 h after stimulation while the same endothelioma cells are also able to upregulate P-selectin at the cell surface within minutes after stimulation with PMA. Both effects are additive. The fifth TNF-induced cell adhesion mechanism is defined by mediating the binding to the mouse monocyte/macrophage cell line J774. This adhesion mechanism is not inhibited by antibodies against any of the other four CAMs; it functions well at 7 degrees C (in contrast to ICAM-1 and VCAM-1) and it is as active after 16 h of TNF induction as after 4 h (in contrast to E- and P-selectin). Furthermore, this new adhesion mechanism only functions on two of three endothelioma cell lines and is undetectable on the third, although ICAM-1, VCAM-1, E-selectin, and P-selectin could be demonstrated to function well on this cell line. Thus, in addition to the three known TNF-inducible CAMs, ICAM-1, VCAM-1, and E-selectin, also P-selectin and a fifth, as yet molecularly undefined cell adhesion mechanism, are TNF inducible at the cell surface of mouse endothelioma cells.

Animals↗
PubMed1993

VCAM-1 is not involved in LPAM-1 (alpha 4 beta p/alpha 4 beta 7) mediated binding of lymphoma cells to high endothelial venules of mucosa-associated lymph nodes.

The migration of lymphocytes from the blood into various lymphoid organs (lymphocyte homing) occurs in specialized high endothelial venules (HEV). Homing of lymphocytes into mucosa-associated lymph nodes in the mouse is mediated at least in part by the lymphocyte homing receptor LPAM-1, an integrin which has been identified as the heterodimer alpha 4 beta p (alpha 4 beta 7 in humans). The HEV-ligand for this integrin in the homing process has not yet been identified. VCAM-1, a cytokine-inducible, endothelial adhesion molecule for lymphocytes and monocytes, was recently demonstrated in vitro to be a ligand for the alpha 4 beta p integrin. We have tested whether VCAM-1 could be a candidate for an alpha 4 beta p ligand on HEV. Two findings strongly argue against this possibility: First, an anti-VCAM-1 monoclonal antibody (mAb) failed to block the binding of the mouse T-lymphoma TK-1 to HEV in cryostat sections of mesenteric lymph nodes, even though this antibody blocked alpha 4 beta p mediated binding of TK-1 cells to VCAM-1 on TNF-alpha-activated mouse endothelioma cells. Second, expression of VCAM-1 on lymph node endothelium was undetectably low as tested by immunofluorescence staining of mouse tissue sections using four different mAbs against mouse VCAM-1, three of which are described in this report. Surprisingly, expression of VCAM-1 on lymph node endothelium was not found even in mice which had been treated systemically with high doses of TNF-alpha or LPS, although VCAM-1 expression was strongly induced on endothelial of all other organs that were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Cloning of the mouse endothelial selectins. Expression of both E- and P-selectin is inducible by tumor necrosis factor alpha.

E-selectin (ELAM-1) and P-selectin (GMP-140, PADGEM, CD62) have both been described as human endothelial cell adhesion molecules for neutrophils and monocytes. Cell surface appearance of these two proteins on human umbilical vein endothelial cells can be regulated by different mechanisms: E-selectin is transcriptionally induced, within hours, by tumor necrosis factor alpha (TNF-alpha) while P-selectin is transported from storage granules to the plasma membrane within minutes upon induction by various stimulating agents. We have obtained cDNA clones covering the full-length coding sequence of the homologous mouse proteins for both endothelial selectins. We show that synthesis of mouse P-selectin, like that of E-selectin, is transiently induced by TNF-alpha in several endothelioma cell lines derived from different mouse tissues. This induction was monitored on both the RNA as well as the protein level. The TNF induced, newly synthesized P-selectin protein was detected on the cell surface. Protein expression of P-selectin increased with a slightly lower rate than expression of E-selectin. In organ cultures of lung tissue from TNF-injected mice, the synthesis of P-selectin was clearly elevated compared to control mice. The bovine P-selectin homolog recognized by cross-reaction with anti-mouse P-selectin antibodies was also TNF-inducible in primary capillary endothelial cells from adrenal cortex and in aorta-derived endothelial cells. Thus, P-selectin can be regulated on two different levels. While the transport of stored P-selectin to the cell surface is controlled by regulated secretion of storage granules, the synthesis and surface expression can be induced by TNF-alpha similarly to that of E-selectin.

Amino Acid Sequence↗
PubMed1992

Sequential translocation of an artificial precursor protein across the two mitochondrial membranes.

We have constructed a chimeric mitochondrial precursor protein consisting of a mutant bovine pancreatic trypsin inhibitor coupled to the C terminus of a purified artificial precursor protein. This construct fails to complete its import into isolated mitochondria and becomes stuck across sites of close contact between the two mitochondrial membranes. When the mitochondria are then depleted of ATP and the intramolecular disulfide bridges of the trypsin inhibitor are cleaved by dithiothreitol, the trypsin inhibitor moiety is transported across the outer membrane into the intermembrane space. This translocation intermediate can be chased across the inner membrane by restoring the ATP levels in the matrix. These results show that translocation of pancreatic trypsin inhibitor across a biological membrane is prevented by its intramolecular disulfide bridges, that import into the matrix involves two distinct translocation system operating in tandem, and that ATP is required for protein translocation across the inner but not the outer membrane.

Adenosine Triphosphate↗
PubMed1992

Selectins: cell surface lectins which mediate the binding of leukocytes to endothelial cells.

The selectins are the most recently identified family of cell adhesion molecules. The three known members of this family (L-, E- and P-selectin) mediate the binding of leukocytes to endothelial cells and are involved in the homing of lymphocytes to lymph nodes, as well as the extravasation of neutrophilic granulocytes into inflamed tissues. The lectin character of these cell adhesion molecules (CAMs) makes the selectin protein family unique among all known CAM families. The review will summarize present knowledge about the structural organization, the ligands identified (carbohydrates and glycoproteins) and the different regulation mechanisms of the cell surface activity of the three selectins.

Animals↗
PubMed1990

A yeast mitochondrial outer membrane protein essential for protein import and cell viability.

The gene encoding ISP42, an integral outermembrane protein located at the yeast mitochondrial protein import site was cloned, sequenced and modified. Yeast cells depleted of ISP42 accumulate uncleaved mitochondrial precursor proteins and then die. ISP42 is the first mitochondrial membrane protein shown to be indispensable for protein import and cell viability.

Amino Acid Sequence↗
PubMed1990

A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein.

We have probed the environment of a precursor protein stuck in mitochondrial import sites using cleavable bifunctional crosslinking reagents. The stuck precursor was crosslinked to a 70 kd protein which, by immunological techniques, was shown to be a matrix protein. The protein was purified to homogeneity by ATP-Sepharose chromatography and partially sequenced. Fourteen of its 15 N-terminal amino acids were identical to residues 24-38 of the protein encoded by the nuclear gene SSC1, which had been proposed to encode a dnaK-like 70 kd mitochondrial stress protein. Our data imply that this mitochondrial hsp70 is made with a cleavable matrix-targeting sequence composed of 23 residues. The complex containing stuck precursor, mitochondrial hsp70, and ISP42 could be solubilized from mitochondria by the non-ionic detergent Triton X-100 even without crosslinking, suggesting tight association of these three components. As the stuck precursor is arrested at an early stage of translocation, mitochondrial hsp70 may initiate the events that lead to refolding of imported precursors in the matrix space.

Biological Transport↗
PubMed1989

A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site.

An engineered precursor protein that sticks in the import site of isolated yeast mitochondria can be specifically photo-crosslinked to a mitochondrial outer-membrane protein of relative molecular mass 42,000 (42K). This protein (termed import-site protein 42 or ISP 42) is exposed on the mitochondrial surface; antibodies against it block protein import into mitochondria. ISP 42 is the first identified component of the putative transmembrane machinery that imports proteins into mitochondria.

Biological Transport↗
PubMed1989

DNA-protein conjugates can enter mitochondria via the protein import pathway.

Mitochondria import most of their proteins and small molecules from the cytoplasm. There is some tentative evidence that they import some of their RNAs, but it is not known how nucleic acids could enter mitochondria. Here, we show that isolated yeast mitochondria can import a single-stranded or double-stranded 24-base pair piece of DNA whose 5' end is covalently linked to the C-terminus of a mitochondrial precursor protein.

DNA↗
PubMed1989

Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane.

Import of precursor proteins into the yeast mitochondrial matrix can occur directly across the inner membrane. First, disruption of the outer membrane restores protein import to mitochondria whose normal import sites have been blocked by an antibody against the outer membrane or by a chimeric, incompletely translocated precursor protein. Second, a potential- and ATP-dependent import of authentic or artificial precursor proteins is observed with purified inner membrane vesicles virtually free of outer membrane components. Third, import into purified inner membrane vesicles is insensitive to antibody against the outer membrane. Thus, while outer membrane components are clearly required in vivo, the inner membrane contains a complete protein translocation system that can operate by itself if the outer membrane barrier is removed.

Antibodies↗
PubMed1989

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins.

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.

Adenosine Triphosphate↗