Biomedical subjects

J R Bender

Publications and source records attributed to J R Bender.

At least 19 recordsLinked to original sources

PubMed2001

Inhibition of interferon-gamma-mediated microvascular endothelial cell major histocompatibility complex class II gene activation by HMG-CoA reductase inhibitors.

BACKGROUND: Graft vascular disease, a major cause of late graft failure in cardiac transplant patients, is associated with the presence of class II major histocompatibility complex molecules on the endothelium. 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, e.g., simvastatin, have been shown to reduce the incidence of graft vascular disease. We studied the effect of simvastatin on interferon (IFN)-gamma-induced human leukocyte antigen (HLA)-DR expression in human microvascular endothelial cells (MVECs). METHODS AND RESULTS: Simvastatin pretreatment inhibited MVEC HILA-DR induction by IFN-gamma, as detected by flow cytometry. Simvastatin's inhibitory effect was reversed by the cholesterol synthesis pathway intermediates mevalonate and geranylgeranyl pyrophosphate but not squalene, indicating the involvement of protein prenylation in this process. Reverse transcription-polymerase chain reaction analysis demonstrated that induction of class II transactivator (CIITA), and consequently, HLA-DRalpha mRNA, is abrogated by simvastatin. Although signal transducer and activator of transcription (STAT)-1 is a critical CIITA gene transactivator, immunofluorescence studies, Western blotting, and electrophoretic mobility shift assays demonstrated that IFN-gamma-induced STAT-1 phosphorylation, nuclear translocation, and DNA binding are not affected by simvastatin. However, simvastatin inhibited IFN-gamma-induced transactivation of a CIITA promoter IV reporter construct, indicating the involvement of this promoter in the inhibitory effect of simvastatin. CONCLUSIONS: Simvastatin pretreatment inhibits CIITA and consequent HLA-DR induction by IFN-gamma in MVECs through interference with protein prenylation. This inhibitory effect occurs at the level of transcription and is directed, at least in part, at the CIITA promoter IV. These results explain some of the beneficial effects of HMG-CoA reductase inhibitors in cardiac transplantation.

Cholesterol↗

PubMed2001

Ex vivo evaluation of PBMNCs collected with a new cell separator.

BACKGROUND: This study reports on an evaluation of the ability of a cell separator (Amicus, Baxter Healthcare) and the integral MNC computer software program to collect a variety of MNC subsets. The collection efficiency (CE) of the Amicus for these MNC subsets was compared to that of another cell separator (CS-3000 Plus, Baxter). The collected MNCs were also assayed ex vivo to determine if these cells remained functional. STUDY DESIGN AND METHODS: Healthy volunteer blood donors were recruited to provide PBMNCs for the isolation of CD3+, CD4+, CD8+, CD19+, NK, and gammadelta+ cells and monocytes. Cells were collected with an Amicus (test arm; n = 16) or a CS-3000 Plus (control arm; n = 11) cell separator. Cells were counted on a flow cytometer and CEs were calculated. For functional studies, the Amicus-collected MNC data were compared to CS-3000 Plus historical data. Functional studies performed included surface antigen expression assays (CD8+), proliferation assays (CD4+ and CD8+ cells), NK cytotoxicity assays for K562 and HUVE cells, and E-selectin induction on endothelial cells through NK+ contact dependency. Dendritic cells (DCs) were generated from CD34+ cells collected on the Amicus, positively selected by the use of antibody-bound, magnetic bead technology, and then cultured ex vivo with a combination of growth factors to generate the DCs. RESULTS: CEs were higher on the Amicus than on the CS-3000 Plus for CD3+ (68 vs. 54%), CD4+ (70 vs. 56%), CD8+ (68 vs. 52%), and CD19+ (60 vs. 48%) cells (p<0.05). For the two separators, CEs were equivalent for monocytes, NK+, and gammadelta+ cells. The Amicus separator collected significantly fewer platelets than did the CS-3000 Plus (p<0.00001). CD4+, CD8+, and NK cells proliferated normally. NK cells appropriately stimulated E-selectin expression on endothelial cells. Culture-generated DCs obtained by using Amicus-collected CD34+ cells expressed appropriate cell surface markers. CONCLUSION: The Amicus separator is acceptable for the collection of PBMNC subsets. The device collects CD3+, CD4+, CD8+, and CD19+ T- and B-cell subsets with greater efficiency and collects MNCs with significantly fewer contaminating platelets than does the CS-3000 Plus. Cells collected on the Amicus are suitable for use in a variety of research and clinical immunobiologic studies.

Blood Donors↗

PubMed2000

Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells.

17beta-Estradiol (E(2)) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E(2)-stimulated endothelial NO release can occur without an increase in cytosolic Ca(2+). Here we demonstrate for the first time, to our knowledge, that E(2) rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase-Akt pathway. E(2) treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182, 780. E(2) stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E(2)-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca(2+) levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E(2)-stimulated NO release. The membrane-impermeant E(2)BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase-Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca(2+) fluxes and describes an important pathway relevant to cardiovascular pathophysiology.

Adenoviridae↗

PubMed2000

Simvastatin modulates cytokine-mediated endothelial cell adhesion molecule induction: involvement of an inhibitory G protein.

Endothelial cell adhesion molecules (CAMs) E-selectin, ICAM-1, and VCAM-1 play variably important roles in immune-mediated processes. They are induced by the proinflammatory cytokines IL-1 and TNF-alpha, and NF-kappaB is required for the regulated expression of all three genes. Regulators of this pathway could potentially be potent immune modulators. We studied the effect of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin, on cytokine-induced expression of CAMs in HUVEC. Unexpectedly, pretreatment with simvastatin potentiated the induction of all three endothelial CAMs by IL-1 and TNF, but not LPS or PMA, as detected by flow cytometry. Northern blot analysis demonstrated an increase in steady state IL-1-induced E-selectin mRNA levels in cells pretreated with simvastatin. This was associated with an increase in nuclear translocation of NF-kappaB, as detected by EMSA. The effect of simvastatin was reversed by mevalonate and geranylgeranyl pyrophosphate but not squalene, indicating that an inhibitory prenylated protein is involved in endothelial responses to proinflammatory cytokines. Pertussis toxin mimicked the effect of simvastatin, and the G protein activator NaF inhibited the cytokine-induced expression of endothelial CAMs, indicating that a Gialpha protein is involved. These results demonstrate that cytokine-mediated activation of the endothelium, and specifically CAM induction, can be modulated by a heterotrimeric G protein-coupled pathway. This may represent a "basal tone" of endothelial inactivation, which can either be disinhibited or amplified, depending on the stimulus.

Adjuvants, Immunologic↗

PubMed2000

Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling.

Estrogen induces both rapid and delayed effects on the cardiovascular system. The early effects take place within minutes (e.g., changes in vasomotor tone) and are mediated through rapid intracellular signaling pathways; whereas the delayed effects (e.g., remodeling or lipid alterations) require hours to days to occur and require transcriptional effects with subsequent modulation of protein expression. To study the acute effects of 17beta-estradiol (E2) treatment on vascular function, we have investigated the rapid (on the order of minutes) effects of E2 treatment on intracellular signaling in human endothelial cells (EC). Our previous data have shown that E2 induces rapid release of NO from and activation of guanylate cyclase in human EC. In this study, we demonstrate that E2 also activates mitogen-activated protein kinase (extracellular signal-related kinase) signaling within minutes in EC. We hypothesized that this effect might be mediated by estrogen receptors (ER) localized to the cell surface. Our data show that membrane-impermeant forms of E2 also activate EC mitogen-activated protein kinase as well as stimulate cGMP production and NO release. The ER antagonist ICI 182,780 blocks this effect. Using confocal microscopy and flow cytometric analysis, we demonstrate that EC contain surface binding sites for E2, detectable by cell-impermeant ligand binding and equally with an anti-ERalpha antibody. Immunoreactive bands of 66 and 45 kDa are detectable with an anti-ERalpha mAb in human EC, and their individual presence correlates functionally with E2-stimulated genomic and rapid nongenomic responses, respectively. Membrane ERs may provide key molecular switches in these novel, rapid signaling pathways induced by E2 in EC.

Antibodies, Monoclonal↗

PubMed2000

Estrogen stimulates heat shock protein 90 binding to endothelial nitric oxide synthase in human vascular endothelial cells. Effects on calcium sensitivity and NO release.

Estradiol (E(2)) causes endothelium-dependent vasodilation, mediated, in part, by enhanced nitric oxide (NO) release. We have previously shown that E(2)-induced activation of endothelial nitric oxide synthase (eNOS) reduces its calcium dependence. This pathway of eNOS activation is unique to a limited number of stimuli, including shear stress, the response to which is herbimycin-inhibitable. Consistent with this, herbimycin and geldanamycin pretreatment of human umbilical vein endothelial cells (HUVEC) abrogated E(2)-stimulated NO release and cGMP production, respectively. These benzoquinone ansamycins are potent inhibitors of Hsp90 function, which has recently been shown to play a role in stimulus-dependent eNOS activation. As in response to shear, E(2) induced an Hsp90-eNOS association, peaking at 30 min and completely inhibited by the conventional estrogen receptor antagonist ICI 182,780. These findings suggest that Hsp90 plays an important role in the rapid, estrogen receptor-mediated modulation of eNOS activation by estrogen.

Benzoquinones↗

PubMed2000

CD28 and LFA-1 contribute to cyclosporin A-resistant T cell growth by stabilizing the IL-2 mRNA through distinct signaling pathways.

In clinical transplantation, the occurrence of cyclosporin A (CsA)-resistant production of IL-2 in vitro correlates with graft rejection in vivo. In this study we investigated the role of the costimulatory molecules CD28 and LFA-1 in this process in the setting of TCR-induced proliferation of primary T lymphocytes in vitro. Co-stimulation with ICAM-1 and B7.2 led to strong and CsA-resistant proliferation, which was found to be largely IL-2 dependent. All of the known calcineurin-dependent events, such as induction of NF-AT and NF-kappaB or stress-activated protein kinase activation, were markedly modulated by CsA independently of costimulation. In contrast, both ICAM-1 and B7.2 enhanced the half-life of the inducible IL-2 transcript in a CsA-resistant manner. LFA-1- but not CD28-induced IL-2 mRNA stabilization required the integrity of the actin-based cytoskeleton, suggesting that the two costimulatory molecules impact on qualitatively different signaling pathways. This is further suggested by the demonstration that LFA-1 and CD28 acted synergistically to confer CsA resistance in a model of co-stimulation using superantigen-pulsed dendritic cells. We propose that IL-2 transcript accumulation and subsequent T cell proliferation at the low transcriptional rate imposed by CsA are the result of co-stimulation-dependent stabilization of IL-2 mRNA.

Antigens, CD↗

PubMed1999

A tyrosine-based sorting signal in the beta2 integrin cytoplasmic domain mediates its recycling to the plasma membrane and is required for ligand-supported migration.

Integrins play pivotal roles in supporting shear- and mechanical-stress-resistant cell adhesion and migration. These functions require the integrity of the short beta subunit cytoplasmic domains, which contain multiple, highly conserved tyrosine-based endocytic signals, typically found in receptors undergoing regulated, clathrin-dependent endocytosis. We hypothesized that these sequences may control surface integrin dynamics in statically adherent and/or locomoting cells via regulated internalization and polarized recycling of the receptors. By using site-directed mutagenesis and ectopic expression of the alphaL/beta2 integrin in Chinese hamster ovary cells, we found that Y735 in the membrane-proximal YRRF sequence is selectively required for recycling of spontaneously internalized receptors to the cell surface and to growth factor-induced membrane ruffles. Disruption of this motif by non-conservative substitutions has no effect on the receptor's adhesive function, but diverts internalized integrins from a recycling compartment into a degradative pathway. Conversely, the non-conservative F754A substitution in the membrane-proximal NPLF sequence abrogates ligand-dependent adhesion and spreading without affecting receptor recycling. Both of these mutants display a severe impairment in ligand-supported migration, suggesting the existence in integrin cytoplasmic domains of independent signals regulating apparently unrelated functions that are required to sustain cell migration over specific ligands.

Amino Acid Sequence↗

PubMed1999

Anchorage dependence of mitogen-induced G1 to S transition in primary T lymphocytes.

Anchorage dependence defines the cellular requirement for integrin-mediated adhesion to substrate to initiate DNA replication in response to growth factors. In this study we investigated whether normal T cells, which spend extended periods in a nonadherent state, show similar requirements for cell cycle progression in response to TCR stimulation. Resting primary T lymphocytes were induced to enter the cell cycle by TCR triggering, and leukocyte integrins were either engaged using purified ICAM-1 or inhibited with function-blocking mAbs. Our data indicate that leukocyte integrins complement TCR-driven mitogenic signals not as a result of their direct clustering but, rather, via integrin-dependent organization of the actin cytoskeleton. Leukocyte integrin-dependent reorganization of the actin cytoskeleton cooperates with the TCR to effect mitogen-activated protein kinase activation, but also represents a required late (4-8 h poststimulation) component in the mitogenic response of normal T cells. Prolonged leukocyte integrin-dependent spreading, in the context of intercellular contact, is a requisite for the production of the mitogenic cytokine IL-2, which, in turn, is involved in the induction of D3 cyclin and is primarily responsible for the decrease in the cyclin-dependent kinase inhibitor p27kip, resulting in retinoblastoma protein inactivation and S phase entry. Thus, T lymphocytes represent a peculiar case of anchorage dependence, in which signals conveyed by integrins act sequentially with the activating stimulus to effect a sustained production of the essential mitogenic cytokine.

Antibodies, Monoclonal↗

PubMed1999

A MHC-encoded ubiquitin-like protein (FAT10) binds noncovalently to the spindle assembly checkpoint protein MAD2.

Recently a number of nonclass I genes were discovered in the human MHC class I region. One of these, FAT10, encodes a protein consisting of two domains with homology to ubiquitin. FAT10 mRNA is expressed constitutively in some lymphoblastoid lines and dendritic cells and in certain other cells after gamma-interferon induction. FAT10 protein expression is controlled at several levels including transcription, translation, and protein stability. Yeast two-hybrid screening of a human lymphocyte library and immunoprecipitation studies revealed that FAT10 noncovalently associated with MAD2, a protein implicated in a cell-cycle checkpoint for spindle assembly during anaphase. Thus, FAT10 may modulate cell growth during B cell or dendritic cell development and activation.

Amino Acid Sequence↗

PubMed1999

Mortality from nephritis and nephrosis in the fibreglass manufacturing industry.

OBJECTIVES: To investigate the question of whether there is an association between exposure to silica or respirable glass fibre and mortality from nephritis or nephrosis among workers in fibrous glass wool manufacturing facilities. METHODS: A case-control study with cases and controls derived from the Owens Corning mortality surveillance system. Two case-control analyses were carried out, one where the cases are defined with nephritis or nephrosis as the underlying cause of death and one where cases are defined as those where nephritis or nephrosis is either the underlying or a contributing cause of death. RESULTS: There is no consistent relation between respirable fibres or respirable silica and nephritis or nephrosis when the analysis is based either on underlying cause only or on underlying plus contributing cause of death. None of the sociodemographic variables considered suggests an increased risk when considering both underlying and contributing cause of death. CONCLUSIONS: These data would seem to support the contention that the most accurate picture of renal disease will be gained from the use of all information on the death certificate and not only the underlying cause. For these data, all odds ratios (ORs) for respirable fibres and silica based on both underlying and contributing cause of death are < 1 with the exception of the highest exposure to silica which is slightly > 1 (OR = 1.04). Although these results do not prove that there is no association between nephritis and nephrosis and exposure to fibreglass or silica in the fibreglass manufacturing environment, they do not support the assertion that such an association exists.

Case-Control Studies↗

PubMed1999

Neuregulin activation of ErbB receptors in vascular endothelium leads to angiogenesis.

The ErbB, or epidermal growth factor receptor (EGF-r), family of transmembrane tyrosine kinase receptors has been demonstrated to play an important role in growth regulation and intracellular signaling in a wide variety of cell types. Targeted deletion of neuregulin (an ErbB ligand) in mice results in endocardial cushion abnormalities, suggesting that these receptor-ligand interactions have important effects on vascular endothelial growth and development. To study the role of ErbB receptor signaling in vascular endothelium, we investigated the expression pattern of the various receptor family members and the effect of ErbB receptor stimulation in human umbilical vein endothelial cells (HUVEC). We demonstrate that ErbB2 (neu), ErbB3, and ErbB4 are highly expressed, whereas ErbB1 (EGF-r) is undetectable. Stimulation of HUVEC with recombinant neuregulin-beta (an ErbB3/4 ligand) induces rapid calcium fluxes, receptor tyrosine phosphorylation, and cell proliferation. We demonstrate marked in vitro and in vivo angiogenic responses to neuregulin-beta, which are independent of vascular endothelial cell growth factor. These findings support an important role for the ErbB family of receptors in endothelial cell signaling and function, including neuregulin-induced angiogenesis.

Animals↗

PubMed1998

Lymphotoxin alpha3 induces chemokines and adhesion molecules: insight into the role of LT alpha in inflammation and lymphoid organ development.

Lymphotoxin (LT) plays an important role in inflammation and lymphoid organ development, though the mechanisms by which it promotes these processes are poorly understood. Toward this end, the biologic activities of a recently generated recombinant murine (m) LT alpha preparation were evaluated. This cytokine preparation was effective at inducing cytotoxicity of WEHI target cells with 50% maximal killing observed with 1.2 ng/ml. mLT alpha also induced the expression of inflammatory mediators in the murine endothelial cell line bEnd.3. rmLT alpha induced expression of the adhesion molecules VCAM, ICAM, E-selectin, and the mucosal addressin cellular adhesion molecule, MAdCAM-1. When mLT alpha, human (h) LT alpha, and mTNF-alpha were compared, mLT alpha was the most potent inducer of MAdCAM-1. None of these cytokines induced the peripheral node addressin, PNAd. mLT alpha also induced expression of the chemokines RANTES, IFN-inducible protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1). mRNA levels peaked 4 h following treatment with mLT alpha and declined through the 24-h treatment period. LT alpha also induced chemokine protein within 8 h of treatment, which increased through the 24-h treatment period. These data demonstrate that the proinflammatory effects of LT alpha3 may be mediated in part through the induction of adhesion molecule and chemokine expression. Further, LT alpha3 may promote development of lymphoid tissue through induction of chemokines and the mucosal addressin MAdCAM-1. These data confirm previous observations in transgenic and knockout mice that LT alpha3 in the absence of LT beta carries out unique biologic activities.

Animals↗

PubMed1998

Class II transactivator-independent endothelial cell MHC class II gene activation induced by lymphocyte adhesion.

NK cells induce MHC class II molecules on the surface of allogeneic endothelial cells in an adhesion-dependent, IFN-gamma-independent manner. Here, we demonstrate that NK cells induce HLA-DR on the surface of a mutant cell line that is defective in IFN-gamma-induced MHC class II expression. RNA analysis in these cells and in a cell line that is defective in class II transactivator (CIITA) demonstrates that NK cell-induced HLA-DR alpha mRNA expression is also CIITA-independent. The Janus kinase-1-deficient cell line U4A expresses HLA-DR alpha mRNA in response to NK cell activation, and HLA-DR alpha promoter constructs transfected into these cells are induced by NK cells but not IFN-gamma. These data indicate that the IFN-gamma-independent component of the target cell HLA-DR expression induced by lymphocyte adhesion uses a signaling pathway that is distinct from the IFN-gamma-dependent mechanism and also suggest that CIITA is not required.

Adult↗

PubMed1998

Retroviral gene transfer: effects on endothelial cell phenotype.

BACKGROUND: Endothelial cells (EC) are an attractive target for somatic cell gene therapy, both for the treatment of cardiovascular disease and for the systemic delivery of recombinant gene products directly into the circulation. Recent evidence, however, suggests that viral transduction may induce unfavorable changes in EC phenotype. We examined the proliferative capacity and cell adhesion molecule (CAM) profile of EC after retroviral gene transfer (GT), employing a clinically relevant ex vivo GT protocol. METHODS: Human umbilical vein EC (HUVEC, N = 14 isolates) were exposed to supernatants containing the MFG.nlsLACZ vector, which codes for a nuclear localized beta-galactosidase. Control HUVEC were exposed to empty virus (CRIP) or no virus (NT). Efficiency of GT was quantitated by direct counting of beta-galactosidase-stained cells on a grid. Proliferation was quantitated by a 1-week assay of viable cell counts. Expression of EC activation molecules (Class II major histocompatibility antigen [MHC II], E-selectin, intercellular adhesion molecule-1 [ICAM-1], and vascular cell adhesion molecule-1 [VCAM-1]) was examined using fluorescent cytometry (FACS) at rest and after cytokine stimulation. RESULTS: GT was reproducibly efficient (mean 57%, range 40-77%) using sequential viral exposures without selection. NT, CRIP, and LACZ-transduced HUVEC exhibited identical FACS profiles for E-selectin, ICAM-1, VCAM-1, and MHC II at rest, consistent with a nonactivated state. Upregulation of expression by cytokine was quantitatively similar for all groups. Growth rates were likewise not different between groups. CONCLUSIONS: Retroviral vectors may be employed to achieve high percentages of transduced EC for ex vivo GT without the use of selection. Transduced EC generated in this fashion are not activated, demonstrate an unaltered pattern of inducible CAM expression, and exhibit normal cell growth. The effects of GT on target cell phenotype are likely to be both vector and protocol specific and should be carefully assessed in each case prior to in vivo applications.

Cell Division↗

PubMed1998

Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement.

As an adhesion receptor, the beta2 integrin lymphocyte function-associated antigen-1 (LFA-1) contributes a strong adhesive force to promote T lymphocyte recirculation and interaction with antigen-presenting cells. As a signaling molecule, LFA-1-mediates transmembrane signaling, which leads to the generation of second messengers and costimulation resulting in T cell activation. We recently have demonstrated that, in costimulatory fashion, LFA-1 activation promotes the induction of T cell membrane urokinase plasminogen activator receptor (uPAR) and that this induced uPAR is functional. To investigate the mechanism(s) of this induction, we used the RNA polymerase II inhibitor 5, 6-dichloro-1-beta-D-ribobenzimidazole and determined that uPAR mRNA degradation is delayed by LFA-1 activation. Cloning of the wild-type, deleted and mutated 3'-untranslated region of the uPAR cDNA into a serum-inducible rabbit beta-globin cDNA reporter construct revealed that the AU-rich elements and, in particular the nonameric UUAUUUAUU sequence, are crucial cis-acting elements in uPAR mRNA degradation. Experiments in which Jurkat T cells were transfected with reporter constructs demonstrated that LFA-1 engagement was able to stabilize the unstable reporter mRNA containing the uPAR 3'-untranslated region. Our study reveals a consequence of adhesion receptor-mediated signaling in T cells, which is potentially important in the regulation of T cell activation, including production of cytokines and expression of proto-oncogenes, many of which are controlled through 3' AU-rich elements.

Amino Acid Sequence↗

PubMed1998

Induction of transporter associated with antigen processing by interferon gamma confers endothelial cell cytoprotection against natural killer-mediated lysis.

T lymphocytes react minimally with nonactivated endothelial cells (ECs). However, natural killer (NK) lymphocyte interactions with resting ECs are rapid, avid, and result in endothelial activation and/or cytotoxicity. The molecular basis for these interactions and EC sensitivity to NK-mediated lysis is unclear. To address the EC-specific nature of NK sensitivity, we used syngeneic human umbilical vein ECs, dermal microvascular ECs, dermal fibroblasts, and B lymphoblastoid cell lines in calcein-AM retention NK assays with allogeneic NK effector cells and found the EC lines consistently more NK-sensitive. Because NK inhibitory receptors are engaged by membrane major histocompatibility complex (MHC) I molecules and MHC I-deficient targets are NK-sensitive, we investigated the quantitative levels of membrane MHC I on the panel of syngeneic lines. Highly sensitive ECs expressed similar (or higher) levels of membrane MHC I than their syngeneic NK-resistant counterparts. Pretreatment of ECs with gamma interferon (IFN-gamma) conferred protection against NK-mediated lysis, with much more rapid kinetics (2-6 hr) than those required for membrane MHC I hyperinduction (>8 hr). These kinetics are consistent with induction of transporter associated with antigen processing (TAP) expression and function. As opposed to NK-resistant cell lines, TAP-1 was undetectable in resting ECs. Recombinant expression of the TAP inactivator ICP47 by adenoviral-mediated transduction was used to selectively inhibit IFN-gamma-mediated EC TAP function. ICP47 expression abrogated EC cytoprotection conferred by IFN-gamma. We demonstrate a relationship between both basal and induced TAP-1 expression/function and EC sensitivity to NK-mediated cytotoxicity. We discuss the influence of an induced MHC I-associated peptide repertoire on vascular vulnerability to cytotoxic lymphocytes.

B-Lymphocytes↗

PubMed1998

Modulation of circulating cellular adhesion molecules in postmenopausal women with coronary artery disease.

OBJECTIVES: The present study examined the association of estrogen (E2) and the inflammatory response of endothelium in coronary artery disease (CAD) by measuring circulating cellular adhesion molecules (cCAMs) in subjects with atherosclerosis. BACKGROUND: Atherosclerotic plaque demonstrates features similar to inflammation. Endothelial cell activation by inflammatory cytokines induces expression of cellular adhesion molecules (CAMs), thereby perhaps augmenting leukocyte adhesion and recruitment and subsequent development of atherosclerosis. The incidence of CAD is lower in women; this may be due to the cardioprotective effects of E2. METHODS: Consecutive eligible subjects with CAD admitted for cardiac catheterization were studied. The groups evaluated were men, postmenopausal women receiving E2 replacement therapy (ERT), postmenopausal women not receiving ERT and premenopausal women. Control groups included men and women without CAD. Preprocedural blood samples were drawn from all groups. Measurements of cCAMs, E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 were performed by enzyme-linked immunoabsorbant assay. E2 levels were assessed by radioimmunoassay. RESULTS: We observed a statistically significant increase in all cCAMs in men with CAD and postmenopausal women with CAD not receiving ERT compared with postmenopausal women with CAD receiving ERT. Premenopausal women with CAD and postmenopausal women with CAD receiving ERT had a significant increase in VCAM-1 alone compared with the female control group. CONCLUSIONS: A possible mechanism by which E2 exerts one of its cardioprotective effects is by limiting the inflammatory response to injury by modulating the expression of CAMs from the endothelium.

Adult↗