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Biomedical subjects

W Newman

Publications and source records attributed to W Newman.

At least 73 records · Page 4Linked to original sources
PubMed1992

Altered adhesion molecule expression and endothelial cell activation accompany the recruitment of human granulocytes to the lung after segmental antigen challenge.

Mounting evidence suggests that inflammatory cells recruited to the lung can contribute to the pathogenesis of asthma. The factors governing the activation and recruitment of circulating cells to the lung remain unknown, but an early step in this process is the interaction of adhesion molecules on circulating cells with those on endothelial cells. We used a segmental antigen challenge model followed 18 h later by bronchoalveolar lavage (BAL) to study granulocyte recruitment to the lung in 14 allergic subjects. Using immunofluorescence and flow cytometry, we determined the expression of the adhesion molecules CD11b, L-selectin (LECAM-1), and VLA-4 on BAL and peripheral blood granulocytes. Total cell count and percentages of recovered eosinophils and basophils were significantly increased in BAL fluids from antigen-challenged segments. Compared with their peripheral blood counterparts, CD11b expression was increased 2- to 3-fold on BAL eosinophils, basophils, and neutrophils (n = 9, P less than 0.05). In contrast, L-selectin expression was significantly decreased on BAL cells (n = 3 to 4, P less than 0.05). Similar phenotypic changes were observed on all three cell types, and on neutrophils recovered from saline-challenged control lung segments. In two subjects, VLA-4 alpha (CD49d) expression on BAL eosinophils was 78 +/- 5% of that seen on peripheral blood eosinophils. Because ELAM-1 (endothelial leukocyte adhesion molecule-1, E-selectin) expression occurs during allergic inflammation and is shed after endothelial activation, we used a sensitive enzyme-linked immunosorbent assay to analyze BAL supernatants for a soluble form of this molecule (sELAM-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1.

Many ligands of adhesion molecules mediate costimulation of T cell activation. The generality of this emerging concept is best determined by using model systems which exploit physiologically relevant ligands. We developed such an "antigen-specific" model system for stimulation of resting CD4+ human T cells using the following purified ligands: (a) major histocompatibility complex class II plus the superantigen Staphylococcus enterotoxin A, to engage the T cell receptor (TCR); (b) adhesion proteins vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and endothelial leukocyte adhesion molecule 1 (ELAM-1), to provide potential cell surface costimulatory signals; and (c) recombinant interleukin 1 beta (rIL-1 beta)/rIL-6 as costimulatory cytokines. In this biochemically defined system, we find that resting CD4+ T cells require costimulation in order to respond to TCR engagement. This costimulation can be provided by VCAM-1 or ICAM-1; however adhesion alone is not sufficient since ELAM-1 mediates adhesion but not costimulation. The cytokines IL-1 beta and IL-6 by themselves cannot mediate costimulation, but augment the adhesion ligand-mediated costimulation. Direct comparison with the model of TCR/CD3 engagement by CD3 monoclonal antibody demonstrated comparable costimulatory requirements in both systems, thereby authenticating the commonly used CD3 model. The costimulation mediated by the activation-dependent interaction of the VLA-4 and LFA-1 integrins with their respective ligands VCAM-1 and ICAM-1 leads to increased IL-2R alpha (CD25) expression and proliferation in both CD45RA+ CD4+ and CD45RO+ CD4+ T cells. The integrins also regulate the secretion of IL-2, IL-4, and granulocyte/macrophage colony-stimulating factor. In contrast the activation-independent adhesion of CD4+ T cell to ELAM-1 molecules does not lead to T cell stimulation as measured by proliferation, IL-2R alpha expression, or cytokine release. These findings imply that adhesion per se is not sufficient for costimulation, but rather that the costimulation conferred by the VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions reflects specialized accessory functions of these integrin pathways. The new finding that VLA-4/VCAM-1 mediates costimulation adds significance to observations that VCAM-1 is expressed on a unique set of potential antigen-presenting cells in vivo.

Antibodies, Monoclonal↗
PubMed1991

Adhesion of human B cells to follicular dendritic cells involves both the lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and very late antigen 4/vascular cell adhesion molecule 1 pathways.

Presentation of antigen in the form of immune complexes to B lymphocytes by follicular dendritic cells (FDC) is considered to be a central step in the generation of memory B cells. During this process, which takes place in the microenvironment of the germinal center, B cells and FDC are in close physical contact. In the present study, we have explored the molecular basis of FDC-B cell interaction by using FDC and B cells derived from human tonsils. We found that FDC express high levels of the adhesion receptors intercellular adhesion molecule 1 (ICAM-1 [CD54]) and vascular cell adhesion molecule 1 (VCAM-1), while the B lymphocytes express lymphocyte function-associated antigen 1 (LFA-1 [CD11a/18]), very late antigen 4 (VLA-4 [CD49d], and CD44. Furthermore, we established that both the LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion pathways are involved in FDC-B lymphocyte binding, and therefore, these pathways might be essential in affinity selection of B cells and in the formation of B memory cells.

Antigens, CD↗
PubMed1991

Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules.

Cytokines such as interleukin 1 (IL-1) promote adhesiveness in human umbilical vein endothelial cells for leukocytes including basophils, eosinophils, and neutrophils, and induce expression of adherence molecules including ICAM-1 (intercellular adhesion molecule-1), ELAM-1 (endothelial-leukocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1). In the present study, blocking monoclonal antibodies (mAb) recognizing ICAM-1, ELAM-1, and VCAM-1 have been used to compare their roles in IL-1-induced adhesion of human basophils, eosinophils, and neutrophils. IL-1 treatment of endothelial cell monolayers for 4 hours induced a four- to eight-fold increase in adhesion for each cell type. Treatment of endothelial cells with either anti-ICAM-1 or anti-ELAM-1 mAb inhibited IL-1-induced adherence of each cell type. In contrast, treatment with anti-VCAM-1 mAb inhibited basophil and eosinophil (but not neutrophil) adhesion, and was especially effective in blocking eosinophil adhesion. The effects of these mAb were at least additive. Indirect immunofluorescence and flow cytometry demonstrated expression of VLA-4 alpha (very late activation antigen-4 alpha, a counter-receptor for VCAM-1) on eosinophils and basophils but not on neutrophils. These data document distinct roles for ICAM-1, ELAM-1, and VCAM-1 during basophil, eosinophil, and neutrophil adhesion in vitro, and suggest a novel mechanism for the recruitment of eosinophils and basophils to sites of inflammation in vivo.

Antibodies, Monoclonal↗
PubMed1991

Activation-independent binding of human memory T cells to adhesion molecule ELAM-1.

The induction of an ensemble of adhesion molecules on endothelial cells by inflammatory cytokines is likely to be crucial to the differential migration of T-lymphocyte subsets into inflammatory sites. Two molecular pathways involving the VLA-4 and LFA-1 integrins are known to mediate T-cell adhesion to activated endothelium. Here we show that a third pathway involving the rapidly inducible endothelial cell-surface adhesion molecule ELAM-1 contributes to the binding of resting CD4+ T cells to IL-1-induced human endothelial cells. All three pathways contribute to the greater adhesion to endothelium of memory T cells than naive T cells. There are two unique features of T-cell adhesion to purified ELAM-1: first, ELAM-1 exclusively mediates adhesion of memory T cells; second, memory T-cell binding to ELAM-1 is independent of acute activation events that regulate integrin-mediated adhesion. Thus, ELAM-1 may be of primary importance in the initial attachment of memory T cells to inflamed endothelium in vivo and to the preferential migration of memory T cells into tissue and inflammatory sites.

CD4 Antigens↗
PubMed1991

Four molecular pathways of T cell adhesion to endothelial cells: roles of LFA-1, VCAM-1, and ELAM-1 and changes in pathway hierarchy under different activation conditions.

T cell adhesion to endothelium is critical to lymphocyte recirculation and influx into sites of inflammation. We have systematically analyzed the role of four receptor/ligand interactions that mediate adhesion of peripheral human CD4+ T cells to cultured human umbilical vein endothelial cells (HUVEC): T cell LFA-1 binding to ICAM-1 and an alternative ligand ("ICAM-X"), T cell VLA-4 binding to VCAM-1, and T cell binding to ELAM-1. Contributions of these four pathways depend on the activation state of both the T cell and HUVEC, and the differentiation state of the T cell. ELAM-1 plays a significant role in mediating adhesion of resting CD4+ T cells to activated HUVEC. LFA-1 adhesion dominates with PMA-activated T cells but the strength and predominant LFA-1 ligand is determined by the activation state of the HUVEC; while ICAM-1 is the dominant ligand on IL-1-induced HUVEC, "ICAM-X" dominates binding to uninduced HUVEC. Adhesion via VLA-4 depends on induction of its ligand VCAM-1 on activated HUVEC; PMA activation of T cells augments VLA-4-mediated adhesion, both in the model of T/HUVEC binding and in a simplified model of T cell adhesion to VCAM-1-transfected L cells. Unlike LFA-1 and VLA-4, ELAM-1-mediated adhesion is not increased by T cell activation. Differential expression of adhesion molecules on CD4+ T cell subsets understood to be naive and memory cells also regulates T/HUVEC adhesion. Naive T cell adhesion to HUVEC is mediated predominantly by LFA-1 with little or no involvement of the VLA-4 and ELAM-1 pathways. In contrast, memory T cells bind better to HUVEC and utilize all four pathways. These studies demonstrate that there are at least four molecular pathways mediating T/HUVEC adhesion and that the dominance/hierarchy of these pathways varies dramatically with the activation state of the interacting cells and the differentiation state of the T cell.

Animals↗
PubMed1991

Structural and functional studies of full-length vascular cell adhesion molecule-1: internal duplication and homology to several adhesion proteins.

Full-length vascular cell adhesion molecule-1 (VCAM-1) cDNA cloned by polymerase chain reaction (PCR) of poly(A)+RNA from interleukin-1 (IL-1)-activated human umbilical vein endothelial cells (HUVEC) contained an insert of 276 nucleotides after position 1,034 of the previously published sequence. Synthetic oligomer probes, specific for each of the two possible species of VCAM-1 mRNA, detected only the longer form of VCAM-1 by Northern analysis of activated endothelial cell mRNA. This full-length VCAM-1 contains two internally repeated domains of approximately 273 amino acids with a high degree of homology. This new sequence information reveals homologies with additional members of the immunoglobulin superfamily and improves ALIGN scores for previously cited adhesion proteins. Removal of the transmembrane domain and the carboxy-terminal end of the full-length VCAM-1 molecule allows the molecule to be secreted into the culture medium from cells transfected with an expression vector containing the corresponding VCAM-1 cDNA.

Amino Acid Sequence↗
PubMed1991

Vascular cells respond differentially to transforming growth factors beta 1 and beta 2 in vitro.

Transforming growth factor beta 1 (TGF-beta 1) and beta 2 (TGF-beta 2) are equipotent in many cell systems studies thus far. Recent data, however, show different effects elicited by these two growth factors in specific biologic systems. This investigation compares the effects of TGF-beta 1 and TGF-beta 2 bovine aortic endothelial cells (BAECs), rat epididymal fat pad microvascular endothelium (RFCs), and bovine aortic smooth muscle cells (BASCs). In two-dimensional cultures, proliferation of BAECs, BASMCs, and RFCs were all inhibited by TGF-beta 1, while in response to TGF-beta 2, BASMCs were fully inhibited, RFCs were modestly inhibited, and BAECs were unaffected. Bovine aortic endothelial cell migration was significantly inhibited by TGF-beta 1, but only slightly inhibited by TGF-beta 2. In contrast, BASMC migration was enhanced by TGF-beta 1 and was not affected by TGF-beta 2. In three-dimensional cultures, RFCs were stimulated to undergo in vitro angiogenesis in response to TGF-beta 1 and TGF-beta 2 at 10-fold higher concentrations. Three distinct receptor assays demonstrated the presence of type I and type II TGF-beta 1 cell-surface-binding proteins on BAECs, BASMCs, and RFCs. Labeled TGF-beta 1 was competed off completely with 100-fold molar excess unlabeled TGF-beta 1, but only partially with equivalent excess unlabeled TGF-beta 2. Furthermore the ratios of type I to type II TGF-beta receptors in these three vascular cell types vary from 1:1 in BAECs to 1.5:1 in RFCs to 3:1 in BASMCs and can be correlated with the differences noted in cellular responses to TGF-beta 1 and TGF-beta 2 in proliferation, migration, and in vitro angiogenic assays. These findings support the hypothesis that there are different responses to the TGF-beta s, depending on the cell type and experimental conditions as well as the TGF-beta concentration and isoform used.

Animals↗
PubMed1990

T cells bind to cytokine-activated endothelial cells via a novel, inducible sialoglycoprotein and endothelial leukocyte adhesion molecule-1.

The action of human rIL-1 beta on confluent, quiescent monolayers of human umbilical vein endothelial cells (HUVEC) has been studied for the induction of new membrane proteins. Two approaches have been taken. The first is a quantitative two-dimensional gel analysis of [35S]cysteine-labeled membrane proteins of HUVEC with and without cytokine treatment. This analysis indicates that there are a restricted number of new membrane proteins synthesized in the first 6 h of IL-1 treatment, on the order of 19 out of a total of over 600 detectable proteins. Second, we have prepared two mAb (1E7 and 2G7) to different epitopes of a major inducible sialoglycoprotein with molecular mass of 114 kDa and an isoelectric point of 4.6 to 4.8. These antibodies were compared with two additional antibodies, 3B7 and 7A9, which were shown to react with the endothelial leukocyte adhesion molecule-1 (ELAM-1) protein as expressed in COS cells. The 1E7/2G7 protein is distinct from ELAM-1, based upon biochemical comparisons as well as the inability of the 1E7 and 2G7 antibodies to react with ELAM-1-transfected COS cells. The protein defined as 1E7/2G7 is neither expressed constitutively nor in an inducible manner on PBMC, granulocytes, platelets, fibroblasts, or keratinocytes. The 7A9 and 3B7 antibodies are shown to block granulocyte binding to IL-1-activated HUVEC. The 2G7 antibody is effective at inhibiting the binding of T cells but not granulocytes to IL-1-activated endothelium, suggesting this new protein is an adhesion protein that may be active in vivo in T cell-endothelial cell adhesion-related events such as inflammation or lymphocyte recirculation. In addition, T cells were shown to utilize the ELAM-1 protein in binding to cytokine-activated HUVEC. Antibodies directed to both proteins had additive effects on inhibition of T cell adhesion.

Antigens↗
PubMed1990

Immunological response of nasopharyngeal carcinoma patients to the Epstein-Barr-virus-coded thymidine kinase expressed in Escherichia coli.

A bacterial expression system which produces large amounts of the Epstein-Barr-virus-coded thymidine kinase has been developed and used to produce protein for Western blot analysis of a number of human antisera. Interestingly, only sera from nasopharyngeal carcinoma (NPC) patients had any detectable IgA antibody which reacted with the EBV TK. These findings provided the basis for ELISA tests using a crude lysate of the E. coli cells expressing the EBV TK as target antigen. Sera from NPC patients showed high levels of IgA reactive antibodies in this test while other sera did not.

Antibodies, Viral↗
PubMed1989

Biotinylated parathyroid hormone as a probe for the parathyroid hormone receptor. Structure-function analysis and detection of specific binding to cultured bone cells by flow cytometry.

The synthetic bovine parathyroid hormone (PTH) analog (Nle8, Nle18, Tyr34) bovine PTH(1-34)amide (bPTH(1-34)amide) was reacted with biotinyl-epsilon aminocaproic acid-N-hydroxysuccinimide under conditions which yielded five isoforms which were fractionated by a combination of reversed phase and ion-exchange chromatography. These reaction products were analyzed by automated Edman degradation in a manner which allowed us to specify the location and number of biotin residues on picomole quantities of hormone. The ability of each of these isoforms to induce a rise in intracellular cAMP in the ROS 17/2.8 cell line allowed us to evaluate the effect on function of biotinylation at different residues. Derivatized PTH molecules which contained a single biotin at either lysine 13, lysine 26, or lysine 27 possessed full biological activity. However, bioactivity was significantly reduced when position 13 plus either lysine 26 or 27 were biotinylated. Biological activity was lost when all 3 lysine residues were biotinylated. Biotinylation of the alpha-NH2 group of alanine at the NH2 terminus also resulted in a total loss of activity. Hence, unlike the effect of altering the alanine at position 1, modification of a single lysine residue at positions 13, 26, and 27 has a less critical effect on biological activity of the molecule. However, biotinylation of all three lysines results in a biologically inert PTH derivative and suggests that changes in isoelectric point, hydrophobicity, or tertiary structure may strongly influence hormone function. A fully bioactive-mixture of isoforms was used to detect receptors on ROS 17/2.8 cells by flow cytometry using fluorescein isothiocyanate-avidin as a fluorescent indicator. Binding to cell surface receptors was saturable and could be inhibited by native bPTH(1-34) but not by transforming growth factor beta, calcitonin or insulin. Moreover, PTH receptors could also be detected on primary cultures of human bone cells and human fibroblasts.

Animals↗
PubMed1989

Modulation of TGF-beta type 1 receptor: flow cytometric detection with biotinylated TGF-beta.

Transforming growth factor beta type 1 (TGF-beta 1) was reacted with NHS-biotin to yield a derivative of TGF-beta 1 which was biotinylated on lysine residues. The biotinylated form of TGF-beta 1 was separated from the unreacted material by reverse phase chromatography. In three separate bioassays, the derivatized peptide was as active as the starting material. The use of FITC-avidin in conjunction with flow cytometry demonstrated that the binding of biotinylated TGF-beta 1 to its receptor is saturable, competable, and specific. A 100-fold molar excess of underivatized TGF-beta 1 gave 85% inhibition of binding of the biotinylated peptide to the mink lung cell line CCL-64, while TGF-beta 2 showed no inhibition of binding, nor did insulin, calcitonin, or TGF-alpha. Both CCL-64 cells and human umbilical vein endothelial cells showed a density-dependent down-regulation of receptor expression in culture. Several factors were examined that might mediate this effect. The down-regulation was shown not to be due to the secretion of an active form of TGF-beta 1. The extracellular matrix from high-density cells did not decrease expression of the receptor. Fibronectin, collagen, and gelatin were also unable to signal changes in receptor expression, even though in other systems such matrix components can regulate the responsiveness of cells to TGF-beta 1. Lastly, staining simultaneously for DNA content and TGF-beta 1 receptor expression showed that there was no correlation between cell cycle and receptor levels.

Animals↗