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W A Muller

Publications and source records attributed to W A Muller.

At least 55 records · Page 3Linked to original sources
PubMed1993

PECAM-1 is required for transendothelial migration of leukocytes.

Platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) is crucial to the process of leukocyte transmigration through intercellular junctions of vascular endothelial cells. A monoclonal antibody to PECAM, or recombinant soluble PECAM, blocks transendothelial migration of monocytes by 70-90%. Pretreating either the monocytes or the endothelial junctions with antibody blocks transmigration. If the endothelium is first activated by cytokines, anti-PECAM antibody or soluble recombinant PECAM again block transmigration of both monocytes and neutrophils. Anti-PECAM does not block chemotaxis of either cell type. Light and electron microscopy reveal that leukocytes blocked in transmigration remain tightly bound to the apical surface of the endothelial cell, precisely over the intercellular junction. Thus, the process of leukocyte emigration can be dissected into three successive stages: rolling, mediated by the selectin class of adhesion molecules; tight adhesion, mediated by the leukocyte integrins and their endothelial cell counter-receptors; and now transmigration, which, based on these studies, requires PECAM-1.

Antibodies, Monoclonal↗
PubMed1993

Platelet/endothelial cell adhesion molecule-1 (CD31)-mediated cellular aggregation involves cell surface glycosaminoglycans.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa integral membrane glycoprotein expressed on endothelial cells, platelets, and leukocytes. Experiments analyzing the aggregation of mouse L-cells stably transfected with full-length PECAM-1 cDNA have demonstrated that PECAM-1 is capable of mediating calcium-dependent heterophilic aggregation. In this report the ligand interactions involved in the aggregation process were studied. This aggregation was inhibited by heparin and chondroitin sulfate, but not by other glycosaminoglycans. Enzymatic removal of cell surface glycosaminoglycans confirmed a PECAM-1-glycosaminoglycan interaction and suggested that this interaction involved glycosaminoglycans on adjacent cells. PECAM-1 contains a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like domain of the molecule's extracellular domain. A comparable region in the related adhesion protein N-CAM has been shown to mediate the adhesive properties of N-CAM. Cells expressing mutant PECAM-1 protein missing the second domain failed to aggregate. Synthetic peptides mimicking the consensus glycosaminoglycan binding sequence, L-K-R-E-K-N, inhibited aggregation. These results demonstrate that PECAM-1-mediated aggregation is dependent on the binding of PECAM-1 to specific glycosaminoglycans on adjacent cells via a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like homology domain.

3T3 Cells↗
PubMed1993

Molecular cloning and adhesive properties of murine platelet/endothelial cell adhesion molecule 1.

We describe the isolation and characterization of a functional murine platelet/endothelial cell adhesion molecule (PECAM) 1 cDNA clone from a mouse lung library. At the nucleotide level, the coding sequence of murine PECAM-1 is 73% identical to human PECAM-1, and at the amino acid level, the sequence is 79% homologous to its human counterpart. Southern hybridization reveals that one copy of the gene exists in the mouse genome; Northern hybridization reveals a single mRNA species in mouse lung tissue. COS-7 and mouse L cells transfected with murine PECAM-1 expressed a 130-kDa glycoprotein on their surfaces that reacted with anti-murine PECAM-1 monoclonal antibody and comigrated on SDS/PAGE with human PECAM-1. Stable L-cell transfectants aggregate with each other in a PECAM-dependent, homophilic manner.

Amino Acid Sequence↗
PubMed1992

Monocyte-selective transendothelial migration: dissection of the binding and transmigration phases by an in vitro assay.

We describe a quantitative assay of transendothelial migration (TEM) that allows us to selectively study the interaction of monocytes with confluent human endothelial cell (HEC) monolayers. The HEC are grown on hydrated collagen gels; the monocytes need not be purified. 100% of monocytes transmigrated the monolayer within 1 h at 37 degrees C and accumulated in the subendothelial collagen; TEM of lymphocytes was not detected within this time. Migration of neutrophils from the same donor was much slower and incomplete, with only 14% of PMN transmigrating in 2 h. This rapid TEM occurs in the absence of exogenous chemoattractants, and HEC in this system do not express cytokine-inducible leukocyte adhesion molecules. A slight modification of the TEM assay allowed us to separate binding to the apical HEC surface from TEM. We found that tight apical surface binding was the rate-limiting step for TEM. Two-thirds of this binding and TEM could be blocked by a monoclonal antibody against the leukocyte beta 2 integrin chain CD18. This assay will allow us to dissect the mechanisms of both the binding and transmigration stages of diapedesis.

Cell Cycle↗
PubMed1992

A heterophilic adhesion mechanism for platelet/endothelial cell adhesion molecule 1 (CD31).

The molecular nature of cell adhesion mediated by platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) was examined using stably transfected L cells in a PECAM-dependent aggregation assay. This adhesion was temperature sensitive and divalent cation dependent, with Mg2+ supporting aggregation to a greater degree than Ca2+. PECAM-dependent aggregation was heterophilic: PECAM-1 transfectants bound as readily to control-transfected L cells as to other PECAM-1 transfectants, demonstrating that a molecule endogenously expressed on the L cells serves as the ligand for PECAM in this system and presumably substitutes for the natural human ligand.

Animals↗
PubMed1991

Cloning of a human alpha(1,3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on Chinese hamster ovary cell transfectants.

In previous studies, Chinese hamster ovary (CHO) cell genomic DNA transfectants that expressed a human alpha(1,3)-fucosyltransferase (alpha(1,3)Fuc-T) gene were isolated and shown to possess a common approximately 7.5-kilobase (kb) EcoRI fragment that hybridized to an Alu probe (Potvin, B., Kumar, R., Howard, D. R., and Stanley, P. (1990) J. Biol. Chem. 265, 1615-1622). One of these transfectants was used to make a genomic DNA library in lambda ZAP-II from EcoRI-digested, size-selected (6-8 kb) DNA, and plaques that hybridized to an Alu probe were purified. After in vivo excision, two plasmids with DNA inserts greater than or equal to 6 kb were obtained and one of these (D2.1) conferred human alpha(1,3)-Fuc-T activity on CHO transfectants. A partial restriction map of this clone revealed an approximately 3.6-kb PstI fragment that contained an Alu sequence. This fragment was subcloned into pGEM-3Zf(+) and compared by restriction analyses with a previously described approximately 3.6-kb PstI DNA fragment isolated from a human peripheral blood lymphocyte library and shown to encode an alpha(1,3)-Fuc-T gene (Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berhend, T. L., and Marks, R. M. (1990) Cell 63, 475-484). Both approximately 3.6-kb fragments gave identical restriction patterns. In addition, they both caused CHO transfectants to synthesize the Lex determinant Gal beta(1,4)[Fuc alpha(1,3)]GlcNAc beta 1 but not the alpha(2,3)-sialyl-Lex determinant. As expected, these transfectants did not bind to ELAM-1 on activated endothelial cells, since sialyl-Lex is a carbohydrate ligand recognized by ELAM-1. Surprisingly, however, an open reading frame encoded within the approximately 3.6-kb PstI fragment had a sequence identical to that of ELFT, an alpha(1,3)-Fuc-T previously reported to confer ELAM-1 binding on a previously reported to confer ELAM-1 binding on a CHO transfectant (Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R., (1990) Cell 63, 1349-1356). Possible explanations for these apparently disparate results are discussed.

Amino Acid Sequence↗
PubMed1991

Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule.

PECAM-1 is a 130-120-kD integral membrane glycoprotein found on the surface of platelets, at endothelial intercellular junctions in culture, and on cells of myeloid lineage. Previous studies have shown that it is a member of the immunoglobulin gene superfamily and that antibodies against the bovine form of this protein (endoCAM) can inhibit endothelial cell-cell interactions. These data suggest that PECAM-1 may function as a vascular cell adhesion molecule. The function of this molecule has been further evaluated by transfecting cells with a full-length PECAM-1 cDNA. Transfected COS-7, mouse 3T3 and L cells expressed a 130-120-kD glycoprotein on their cell surface that reacted with anti-PECAM-1 polyclonal and monoclonal antibodies. COS-7 and 3T3 cell transfectants formed cell-cell junctions that were highly enriched in PECAM-1, reminiscent of its distribution at endothelial cell-cell borders. In contrast, this protein remained diffusely distributed within the plasma membrane of PECAM-1 transfected cells that were in contact with mock transfectants. Mouse L cells stably transfected with PECAM-1 demonstrated calcium-dependent aggregation that was inhibited by anti-PECAM antibodies. These results demonstrate that PECAM-1 mediates cell-cell adhesion and support the idea that it may be involved in some of the interactive events taking place during thrombosis, wound healing, and angiogenesis.

Amino Acid Sequence↗
PubMed1990

PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily.

An antibody to a platelet integral membrane glycoprotein was found to cross-react with the previously identified CD31 myelomonocytic differentiation antigen and with hec7, an endothelial cell protein that is enriched at intercellular junctions. This antibody identified a complementary DNA clone from an endothelial cell library. The 130-kilodalton translated sequence contained six extracellular immunoglobulin (Ig)-like domains and was most similar to the cell adhesion molecule (CAM) subgroup of the Ig superfamily. This is the only known member of the CAM family on platelets. Its cell surface distribution suggests participation in cellular recognition events.

Amino Acid Sequence↗
PubMed1989

A human endothelial cell-restricted, externally disposed plasmalemmal protein enriched in intercellular junctions.

We have raised an mAb to a previously undescribed 135-kD externally disposed integral membrane protein that is enriched in the intercellular junctional domain of cultured human umbilical vein endothelial cells. This protein localizes at the appositional surfaces of cells as they become confluent and is stably expressed in the junctional zones of confluent monolayers. This protein is expressed in situ on continuous endothelia of all blood vessels in all human tissues examined. Moreover, this protein, as determined by mAb immunocytochemistry, is not expressed by any other cell type. This protein may mediate endothelial-specific functions restricted to the intercellular domain. It may also serve as a unique cell surface marker for the identification and purification of human endothelial cells.

Antibodies, Monoclonal↗
PubMed1987

Interleukin 2 receptors are expressed by alveolar macrophages during pulmonary sarcoidosis and are inducible by lymphokine treatment of normal human lung macrophages, blood monocytes, and monocyte cell lines.

Expression of receptors for IL 2 was believed initially to be restricted to T cells after their activation by IL 1 and antigen. However, recently IL 2 receptors (IL 2R) were demonstrated on activated B cells by using an anti-IL 2R monoclonal antibody (anti-Tac). In this study, we examined the capacity of cultured human alveolar macrophages, blood monocytes, and myelomonocytic (HL-60) or monoblast (U937) cell lines to bind three different anti-IL 2R monoclonal antibodies before or after stimulation with the monocyte-activating agents IFN-gamma, LPS, phorbol ester, or lymphokine-containing conditioned medium. For each of the four cell populations examined, resting unstimulated cells bound little or no anti-IL 2R antibody, as shown independently by quantitative cell binding assay and by immunoperoxidase labeling. By contrast, incubation with recombinant IFN-gamma, conditioned medium, or to a lesser extent, native or recombinant IL 2 itself, resulted in a significant enhancement of anti-IL 2 receptor monoclonal antibody binding by all four populations, whereas LPS, PMA, or IL 1 had no effect. In addition, membrane binding of anti-Tac antibody, similar to that seen after stimulation of normal lung macrophages with IFN-gamma, was detected by using macrophages obtained by bronchoalveolar lavage of five patients with active pulmonary sarcoidosis. These findings are consistent with the expression of a functional IL 2R on activated cells of the monocyte lineage, since anti-Tac binding to IFN-gamma-treated HL-60 cells was inhibited by addition of excess IL-2; specific binding of anti-IL 2 monoclonal antibodies was detected in the presence of exogenous IL 2; and a 50 to 55 kD molecule was immunoprecipitated from both activated lung macrophages and T lymphoblasts by using anti-Tac antibody. We conclude that human mononuclear phagocytes can be induced by lymphokines to express IL 2R, and that such IL 2R+ macrophages can be detected in vivo during inflammation.

Cell Line↗
PubMed1986

Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination.

Vascular endothelium in vivo appears to function as a polarized epithelium. To determine whether cellular polarity exists at the level of the plasma membrane, we have examined cultured endothelial monolayers for evidence of differential distribution of externally disposed plasmalemmal proteins at apical and basal cell surfaces. Lactoperoxidase beads were used to selectively label the apical surfaces of confluent endothelial monolayers, the total surfaces of nonenzymatically resuspended cells, and the basal surfaces of monolayers inverted on poly-L-lysine-coated coverslips, while maintaining greater than 98% viability in all samples. Comparison of the SDS PAGE radioiodination patterns obtained for each surface revealed a number of specific bands markedly enriched on either apical or basal surface. This polarized distribution involved membrane-associated as well as integral membrane proteins and was observed in several strains of bovine aortic endothelial cells, as well as in both primary and passaged human umbilical vein endothelial cells. In contrast, two morphologically nonpolarized cell types, bovine aortic smooth muscle and mouse peritoneal macrophages, did not display differential localization of integral membrane proteins. Polarized distribution of integral membrane proteins was established before the formation of a confluent monolayer. When inverted (basal-side-up) monolayers were returned to culture, the apical-side-up pattern was reexpressed within a few days. These results demonstrate that cell surface-selective expression of plasmalemmal proteins is an intrinsic property of viable endothelial cells in vitro. This apical/basal asymmetry of membrane structure may provide a basis for polarized endothelial functions in vivo.

Animals↗
PubMed1983

Membrane proteins of the vacuolar system. III. Further studies on the composition and recycling of endocytic vacuole membrane in cultured macrophages.

In previous publications (Muller, W.A., R.M. Steinman, Z.A. Cohn. 1980, J.Cell Biol. 86:292-314), we found that the membrane of macrophage phagolysosomes could be selectively radioiodinated in living cells, The technique required phagocytosis of lactoperoxidase covalently coupled to latex spheres (LPO-latex), followed by iodination on ice with Na(125)I and hydrogen peroxide. In this paper, we use the LPO-latex system to further analyze the composition and recycling of phagocytic vacuole membrane. Three approaches were employed to examine the polypeptide composition of the phagolysosome (PL) and plasma membranes (PM). (a) The efficiency of intracellular iodination was increased by increasing lysosomal pH with chloroquine. By one-dimensional SDS PAGE, the heavily labeled chloroquine-treated PL exhibited the same labeled polypeptides as PM iodinated extracellularly with LPO-latex. (b) Iodinated PL and PM were compared by two-dimensional gel electrophoresis. No differences in the isoelectric point and molecular weight of the major iodinated species were detected. (c) Quantitative immune precipitation was performed with five specific antibodies directed against cell surface antigens. Four antibodies precipitated similar relative amounts of labeled antigen on the cell surface and endocytic vacuole. One antibody, secreted by hybridoma 2.6, detected a 21-kdalton polypeptide that was enriched sevenfold in PL membrane. This enrichment was cell surface-derived, since the amount of labeled 2.6 was increased sevenfold when iodinated PM was driven into the cell during latex uptake. Therefore, intracellular iodination primarily detects PL proteins that are identical to their PM counterparts. Additional studies employed electron microscope autoradiography to monitor the centrifugal flow of radiolabeled polypeptides from PL to PM. Cells were iodinated intralysosomally and returned to culture for only 5-10 min at 37 degrees C. Most of the cell-associated label then redistributed to the cell surface or its adjacent area. Significant movement out of the lysosome compartment occurred even at 2 degrees C and 22 degrees C. Extensive and rapid membrane flow through the secondary lysosome presumably contributes to the great similarity between PM and PL membrane polypeptides.

Animals↗
PubMed1980

In vitro and in vivo studies on glucagonoma tissue.

A 66 year old patient with diabetes had a necrolytic migratory erythema, weight loss and anaemia. Plasma immunoreactive glucagon (IRG) of 2465 pmoles/l (normal 35 +/- 5 SEM pmoles/l) suggested the existence of a glucagonoma which was confirmed by arteriography and subsequently removed by surgery. Although plasma IRG returned to normal, glucose tolerance and insulin secretion remained pathological. Plasma amino acid levels had been reduced but were corrected by surgery. Pancreatic polypeptide, however, 298 pmoles/l before was still 206 pmoles/l after the operation (normal 12-48 pmoles per litre). Column chromatography of plasma and tumor extracts showed quantitatively important IRG fractions with molecular weights above 9000 daltons, possibly precursors of glucagon. Beside a 50-fold IRG excess, the tumour concentrations of insulin and somatostatin were 4 to 150 times increased. By contrast, pancreatic polypeptide was present in normal amounts. Electron microscopic examination showed atypical A-cell granula and unusual abundance of mitochondria.

Aged↗
PubMed1980

The membrane proteins of the vacuolar system I. Analysis of a novel method of intralysosomal iodination.

A method has been developed to deliver an idoinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane. Lactoperoxidase (LPO) is covalently coupled to carboxylated latex spheres (LPO-latex) in a stable, enzymatically active form. The addition of LPO-latex to cultured macrophages leads to their rapid attachment, ingestion, and enclosure in a plasma membrane-derived phagocytic vacuole. These organelles rapidly fuse with preexisting lysosomes and are converted to phagolysosomes (PL) that demonstrates both acid phosphatase and lactoperoxidase activities. The exposure of LPO-latex containing cells to 125I- and an extracellular peroxide-generating system, glucose oxidase-glucose, at 4 degrees C leads to incorporation of label into TCA-precipitable material. The incorporated cel-associated label was present as monoiodotyrosine, and negligible amounts were found in lipids. Cell viability remained > 99%. Autoradiography at both the light and EM level revealed that > 97% of the cells were labeled, and quantitative analysis demonstrated the localization of grains to LPO-latex containing PL. PL were separated on sucrose gradients, and their radiolabel was confined almost exclusively to the membrane rather than soluble contents. SDS-polyacrylamide gel electrophoretic analysis of the peptides iodinated from within PL demonstrated at least 24 species with molecular weights ranging from 12,000 to 250,000. A very similar group of proteins was identified on the plasma membrane (PM) after surface iodination, and on latex phagosomes derived from iodinated PM. No novel proteins were detected in PL, either immediately after phagosome-lysosome fusion or after 1 h of intracytoplasmic residence. We conclude that the membrane proteins accessible to LPO-catalyzed iodination on the luminal surface of the PL and on the external face of the PM are similar, if not identical.

Animals↗
PubMed1980

The membrane proteins of the vacuolar system. II. Bidirectional flow between secondary lysosomes and plasma membrane.

Lactoperoxidase covalently coupled to latex spheres (LPO-latex) has been used to selectively iodinate the phagolysome (PL) membrane within living macrophages, as discussed in the accompanying article. This procedure labeled approximately 24 polypeptides in the PL membrane; these were similar to those iodinatable on the external surface of the plasma membrane (PM). We now report on the translocation and fate of these proteins when the cells are returned to culture. TCA-precipitable radioactivity was lost from cells with biphasic kinetics. 20-50% of the cell-associated radiolabel was rapidly digested (t 1/2 approximately equal to 1 h) and recovered in the culture medium as monoiodotyrosine. 50-80% of the label was lost slowly from cells ( 1/2 approximately equal to 24-30 h). Quantitative analysis of gel autoradiograms showed that all radiolabeled proteins were lost at the same rate in both the rapid and slow phases of digestion. Within 15-30 min aftr labeling of the PL membrane, EM autoradiography revealed that the majority of the cell-associated grains, which at time 0 were associated with PL, were now randomly dispersed over the plasmalemma. At this time, analysis of PM captured by a second phagocytic load revealed the presence of all labeled species originally present in the PL membrane. This demonstrated the rapid, synchronous centrifugal flow of PL polypeptides to the cell surface. Evidence was also obtained for the continuous influx of representative samples of the PM into the PL compartment by way of pinocytic vesicles. This was based on the constant flow of fluid phase markers into latex-containing PL and on the internalization of all iodinatable PM polypeptides into this locus. These observations provide evidence for the continuous, bidirectional flow of membrane polypeptides between the PM and the secondary lysosome and represent an example of a membrane flow and recycling mechanism.

Animals↗