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At least 757 records · Page 42Linked to original sources
PubMed2000

A sensitive and versatile bioassay for ligands that signal through receptor clustering.

The induced expression of xanthine-guanine phosphoribosyl transferase (XGPRT) by low concentrations (-2 pg/ml) of interferon-alpha (IFN-alpha) or IFN-beta, in the 2fTPGH cell line caused a 50% cytotoxicity when these cells were grown in medium containing 6-thioguanine. We extended the application of this sensitive, reliable, and easy bioassay to other members of the cytokine family. To activate the IFN signaling pathway, we made receptor chimeras, consisting of the IFN type I receptor intracellular and transmembrane domains, fused to either the interleukin-5 (IL-5) receptors or erythropoietin (Epo) receptor extracellular domains as model systems. 2fTGH cells, stably transfected with these receptor chimeras, responded to very low concentrations of IL-5 or Epo (IC50 values of approximately 15 pg and 3 pg/ml, respectively) and thus can be used as a very sensitive bioassay for both ligands. Background activity of IL-5, Epo, tumor necrosis factor (TNF), IL-6, or leptin on cells that did not carry the receptor chimeras was very low. This methodology can in principle be extended to any ligand that acts via clustering of its receptors.

Biological Assay↗
PubMed2001

Receptor-mediated endocytosis of CD34 on hematopoietic cells after stimulation with the monoclonal antibody anti-HPCA-1.

CD34 most probably acts as a receptor molecule on hematopoietic progenitor cells; however, its precise function remains to be elucidated. To track the intracellular pathway of CD34 after binding of a stimulatory antibody (anti-HPCA-1), immuno-electron microscopical analysis was performed on cells of normal bone marrow (NBMPC), acute leukemias, and the KG1a cell line. Before stimulation, CD34 was evenly distributed over the cell surface. After binding of the anti-HPCA-1, but not the anti-HPCA-2 antibody to CD34 and labeling with 30-nm immunogold, a rapid capping of CD34 and a subsequent internalization from the cell surface via clathrin-coated pits and coated vesicles was observed. The percentage of internalized CD34/immunogold complexes ranged from 8 to 80% in the NBMPC and the leukemic blasts, whereas KG1a cells showed an internalization rate of only 0.42%. Moreover, in the KG1a cells, the CD34/immunogold complexes were not associated with the coated pits. These differences in CD34 internalization did not correlate to the mRNA expression for the full-length or truncated CD34 assessed by isotype-specific real-time PCR. Taken together, evidence was found that CD34 is a surface receptor molecule that is modulated by receptor-mediated endocytosis. CD34 on KG1a cells appears to have a functional and/or structural defect, preventing modulation of this epitope.

Acute Disease↗
PubMed2000

Effects of I domain deletion on the function of the beta2 integrin lymphocyte function-associated antigen-1.

A subset of integrin alpha subunits contain an I domain, which is important for ligand binding. We have deleted the I domain from the beta2 integrin lymphocyte function-asssociated antigen-1 (LFA-1) and expressed the resulting non-I domain-containing integrin (DeltaI-LFA-1) in an LFA-1-deficient T cell line. DeltaI-LFA-1 showed no recognition of LFA-1 ligands, confirming the essential role of the I domain in ligand binding. Except for I domain monoclonal antibodies (mAbs), DeltaI-LFA-1 was recognized by a panel of anti-LFA-1 mAbs similarly to wild-type LFA-1. However, DeltaI-LFA-1 had enhanced expression of seven mAb epitopes that are associated with beta2 integrin activation, suggesting that it exhibited an "active" conformation. In keeping with this characteristic, DeltaI-LFA-1 induced constitutive activation of alpha4beta1 and alpha5beta1, suggesting intracellular signaling to these integrins. This "cross-talk" was not due to an effect on beta1 integrin affinity. However, the enhanced activity was susceptible to inhibition by cytochalasin D, indicating a role for the cytoskeleton, and also correlated with clustering of beta1 integrins. Thus, removal of the I domain from LFA-1 created an integrin with the hallmarks of a constitutively active receptor mediating signals into the cell. These findings suggest a key role for the I domain in controlling integrin activity.

Antibodies, Monoclonal↗
PubMed1995

Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking.

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.

Actins↗
PubMed1990

Increased beta 2-adrenoceptor number in peripheral sympathetic ganglia of spontaneously hypertensive rats.

We determined beta-adrenoceptor concentrations in sympathetic ganglia from adult Spontaneously Hypertensive Rats (SHR) and Wistar-Kyoto (WKY) control rats by quantitative autoradiography. Adjacent tissue sections were incubated with [125I]iodocyanopindolol, with or without excess of unlabeled (-)-propranolol, the beta 1-antagonist CGP 20712A or the beta 2-antagonist ICI 118,551. Most beta-adrenoceptors were of the beta 2-type. Their concentration was higher in the superior cervical and stellate ganglia of SHR when compared to normotensive WKY. Our results indicate that beta 2-adrenoceptor stimulation may be enhanced in sympathetic ganglia of SHR, and could play a role in the maintenance of their increased sympathetic activity.

Animals↗
PubMed2000

MEKK2 gene disruption causes loss of cytokine production in response to IgE and c-Kit ligand stimulation of ES cell-derived mast cells.

Ligation of the high-affinity IgE receptor (FcepsilonRI) or of c-Kit stimulates cytokine production in mast cells. We show that MEK kinase 2 (MEKK2), a MAPK kinase kinase (MAP3K) that regulates the JNK and ERK5 pathways, is required for cytokine production in embryonic stem (ES) cell-derived mast cells (ESMC). Targeted disruption of the MEKK2 or MEKK1 gene was used to abolish expression of the respective kinases in ESMC. Transcription of specific cytokines in response to IgE or c-Kit ligand was markedly reduced in MEKK2(-/-) ESMC relative to wild-type ESMC. Cytokine production in MEKK1(-/-) ESMC was similar to that of wild-type ESMC, demonstrating the specificity of MEKK2 in signaling cytokine gene regulation. MEKK2(-/-) ESMC also lost receptor-mediated stimulation of JNK. In contrast, JNK activation in response to UV irradiation was normal, showing that MEKK2 is required for receptor signaling but not for cellular stress responses. MEKK2 is the first MAP3K shown to be required for mast cell tyrosine kinase receptor signaling controlling cytokine gene expression.

Animals↗
PubMed1995

Cross-linking of the CAMPATH-1 antigen (CD52) triggers activation of normal human T lymphocytes.

The CAMPATH-1 (CD52) antigen is a 21-28 kDa glycopeptide which is highly expressed on lymphocytes and macrophages and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The function of this molecule is unknown. However, it is an extremely good target for complement-mediated attack and antibody-mediated cellular cytotoxicity. The humanized CAMPATH-1H antibody, which is directed against CD52, is very efficient at mediating lymphocyte depletion in vivo, and is currently being used in clinical trials for lymphoid malignancy and rheumatoid arthritis. It is therefore important to examine the functional effects of this antibody on different lymphocyte sub-populations. Because several other GPI-linked molecules expressed on the surface of T lymphocytes are capable of signal transduction resulting in cell proliferation, we have investigated whether the CAMPATH-1 antigen can also mediate these effects. In the presence of phorbol esters and cross-linking anti-Ig antibodies, mAbs specific for CD52 induced proliferation and lymphokine production in highly purified resting CD4+ and CD8+ T lymphocytes. The rat IgG2c YTH 361.10 anti-CD52 antibody, however, was able to activate resting CD4+ and CD8+ T cells directly without cross-linking or phorbol myristate acetate in the absence of Fc-bearing cells. Anti-CD52 antibodies also augmented the anti-CD3 mediated proliferative response of CD4+ and CD8+ T cells when the two antibodies were co-immobilized onto the same surface or cross-linked in solution by the same second antibody. Both CD4+ CD45RA and CD4+ CD45RO T cells were stimulated to proliferate by anti-CD52 antibodies in the presence of appropriate co-stimulatory factors. Anti-CD52 mAbs did not, however, synergize with anti-CD2 or CD28 mAb to induce CD4+ T cell proliferation. The activation of CD4+ T cells by anti-CD52 antibodies was inhibited by cyclosporin A, suggesting a role for the calcineurin-dependent signal transduction pathways. Although CD52 could transduce a signal in T cells, anti-CD52 antibodies did not inhibit antigen-specific or polyclonal T cell responses, suggesting this molecule does not play an essential co-stimulatory role in normal T cell activation.

Antibodies, Monoclonal↗
PubMed2003

Impaired signaling via the high-affinity IgE receptor in Wiskott-Aldrich syndrome protein-deficient mast cells.

Wiskott-Aldrich syndrome protein (WASP) is the product of the gene deficient in boys with X-linked Wiskott-Aldrich syndrome. We assessed the role of WASP in signaling through the high-affinity IgE receptor (FcepsilonRI) using WASP-deficient mice. IgE-dependent degranulation and cytokine secretion were markedly diminished in bone marrow-derived mast cells from WASP-deficient mice. Upstream signaling events that include FcepsilonRI-triggered total protein tyrosine phosphorylation, and protein tyrosine phosphorylation of FcepsilonRIbeta and Syk were not affected by WASP deficiency. However, tyrosine phosphorylation of phospholipase Cgamma and Ca(2+) mobilization were diminished. IgE-dependent activation of c-Jun N-terminal kinase, cell spreading and redistribution of cellular F-actin in mast cells were reduced in the absence of WASP. We conclude that WASP regulates FcepsilonRI-mediated granule exocytosis, cytokine production and cytoskeletal changes in mast cells.

Actins↗
PubMed2006

CMRF-35-like molecule-5 constitutes novel paired receptors, with CMRF-35-like molecule-1, to transduce activation signal upon association with FcRgamma.

The murine CMRF-35-like molecule (CLM) family belongs to the Ig superfamily, and consists of nine mapped genes. Here we report that CLM-5 and CLM-1 constitute novel paired Ig-like receptors (PIRs). CLM-1 and CLM-5 genes are encoded in close vicinity on chromosome 11. CLM-1 has been reported to possess immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic region and to inhibit the differentiation of osteoclast. CLM-5 has an Ig domain that is highly homologous with that of CLM-1 in its extracellular domain, and possesses a negatively charged residue in its transmembrane domain. CLM-5, like CLM-1, is expressed in myeloid cells, such as dendritic cells, macrophages and granulocytes. We also show that CLM-5 interacts with FcRgamma to be expressed on the plasma membrane and that the cross-linking of CLM-5 leads to tyrosine phosphorylation of proteins, including FcRgamma, in the monocyte/macrophage cell line. Taken together, these characteristics suggest that CLM-1 and CLM-5 constitute novel PIRs and that CLM-5 may transduce activation signals as the activating receptor in myeloid cells.

Amino Acid Motifs↗
PubMed1997

Induction of the human sperm acrosome reaction with mannose-containing neoglycoprotein ligands.

In the interest of classifying cases of male factor infertility, we have paid particular attention to the sugar ligand binding properties of the human sperm surface and the functional capacity of the acrosome for exocytosis--key parameters for assessing sperm fertilizing ability. Zona recognition and binding involve the interactions of sperm surface mannose receptors (lectins) with mannose ligands on the zona pellucida. Sperm surface mannose lectins can be visualized by their ability to bind a synthetic model zona ligand, fluorescein isothiocyanate (FITC)-conjugated mannosylated bovine serum albumin (BSA) (Man-FITC-BSA). We now report that Man-FITC-BSA biologically also mimics the effects of solubilized authentic human zonae, in that binding of Man-FITC-BSA results in a time-dependent receptor aggregation and the induction of acrosome exocytosis in capacitated sperm populations from fertile donors. In our assay, the addition of mM amounts of mannose monosaccharide to Man-FITC-BSA increases the number of polyvalent mannose ligands bound by individual spermatozoa and increases the rate of the acrosome reactions induced by Man-FITC-BSA, thereby increasing specimen processing efficiency. We conclude that exposure of human spermatozoa to polyvalent mannose ligands + D-mannose monosaccharide offers a new, convenient and readily available system to study sperm capacity for induced acrosome loss.

Acrosome↗
PubMed1995

Inhibition of the growth of a human nasopharyngeal carcinoma cell line by bFGF is mediated via FGFR-1.

The growth of CG-1 human nasopharyngeal carcinoma cell line and five of its randomly selected, single cell-derived subline cells is inhibited by bFGF in an autocrine and paracrine manner. In contrast, aFGF, which has a 55% homology in amino acid sequence with bFGF, stimulates cell growth. Basic FGF binds to specific cell surface high-affinity receptor sites with an apparent Kd of 105 pM. Of the two lines examined, the high-affinity binding sites for bFGF are calculated to be 1200 and 2600 per cell. The biological effect of bFGF is conveyed through its binding to the high-affinity receptor sites and the binding is dependent on the presence of cell surface heparin-like molecules, as treatment of cells with heparitinase or sodium chlorate abolishes high-affinity binding and growth inhibition. In contrast, similar treatment has no obvious effect on the growth-stimulatory effect of aFGF. Experimental results are also presented showing that the growth inhibition by bFGF is mediated through type I FGF receptors. These results suggest that bFGF and aFGF act via distinct receptor types to oppositely regulate the growth of CG-1 and subline cells.

Base Sequence↗
PubMed2000

Activity-dependent and use-dependent regulation of dopamine-receptor clustering.

In this study, fluorescence-conjugated ligands were employed to label dopaminergic D1-like and D2-like receptors, respectively, in neurons derived from the frontal cortex of embryonic rats. The receptor binding sites were visualized and analyzed using confocal microscopy. Our results showed that fluorescently labeled receptors tended to form clusters with a diameter of about one micrometer and were distributed on both somata and dendrites. Chronic treatment with tetrodotoxin reduced the number of fluorescent clusters of both D1-like and D2-like receptors, while chronic treatment with a high concentration of potassium increased the number of fluorescent clusters of both D1-like and D2-like receptors. Further, chronic treatment with SCH23390 up-regulated the number of D1-like receptor clusters, whereas chronic treatment with bromocriptine down-regulated the number of D2-like receptor clusters. In addition, chronic treatment with spiperone down-regulated the number of D1-like receptor clusters. These results suggest that both neuronal activity and dopaminergic receptor occupancy are important factors that determine dopaminergic receptor clustering which is an essential step toward synaptogenesis during neuronal maturation process.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed2004

Neuregulin potentiates agrin-induced acetylcholine receptor clustering in myotubes.

Agrin and neuregulin (HRG-beta1) play complementary roles in synapse formation. While HRG-beta1 induces transcriptional up-regulation of postsynaptic proteins, here we present evidence that it can potentiate agrin-induced acetylcholine receptor (AChR) clustering in C2 myotubes. Agrin induced maximal AChR clustering in 4 h. HRG-beta1 treatment for 4 h produced no increase over basal AChR cluster numbers. When myotubes were treated for 4 h with 100 pM agrin, HRG-beta1 augmented AChR cluster numbers by 2-fold compared to myotubes treated with 100 pM agrin alone. Thus, HRG-beta1 can potentiate agrin-induced AChR clustering.

Agrin↗
PubMed1991

The molecular basis for the synergism between the CD3/alpha beta T cell receptor and the CD2 antigen-derived signals in promoting T-cell proliferation.

Northern blot analysis and a highly sensitive methodology for mRNA phenotyping, polymerase chain reaction (PCR), were used to explore the basis for the synergism between CD3/alpha beta T cell receptor (TCR) and the CD2 antigen-derived signals in promoting proliferation of T cells. Northern blotting of RNA isolated from highly purified normal human T cells revealed that crosslinking of anti-TCR-1 (a mAb directed at a framework determinant of the TCR) and OKT11 (a mAb directed at the SRBC-binding epitope of the CD2 antigen) induced the expression of the interleukin-2 gene and the gene for IL-2 receptor alpha, mRNA phenotyping by PCR revealed that crosslinkage of TCR with the CD2 antigen, and not independent crosslinking of TCR or the CD2 antigen, results in the induction of IL-2, IL-2 receptors alpha and beta, and IL-4-specific transcripts. Highly purified CD4+ T cells, as well as CD8+ T cells proliferated by crosslinking TCR with CD2 antigen. Moreover, crosslinkage of TCR with the CD2 antigen and not of either antigen with the CD4 antigen (on the surface of CD4+ T cells) or the CD8 antigen (on the surface of CD8+ T cells) resulted in marked proliferation. Our demonstration that the CD2 antigen-derived signal(s) contribute to the expression of growth promoting genes elicited via the TCR, and that the CD2 antigen is more efficient compared with the CD4 or CD8 antigen in evoking T cell proliferation, suggests that autoimmunity as well as alloimmunity might be regulated by targeting the CD2 antigen.

Antigens, CD↗
PubMed1992

Inflammatory mediator release from human monocytes via immobilized Fc receptors. Its potential role in adverse reactions to systemic monoclonal antibody therapy.

Human monocytes released superoxide anion, IL-1, and TNF subsequent to binding of their Fc receptor I to murine IgG2a or rabbit IgG. Fc receptor II binding to murine IgG2b or IgG1 had similar consequences. Immobilized murine monoclonal antibodies, IgG2a anti-CD3 (OKT3) or IgG1 anti-CD44 also induced superoxide anion and monokine production. Monocytes bound OKT3 via FcRI and responded to immobilized OKT3 by inflammatory mediator release in the absence of T cells. These results suggest that direct interaction of immunoglobulins with monocytes via FcR may represent an important phase of the pathophysiology of adverse reactions to systemic monoclonal antibodies.

Humans↗
PubMed2001

Role of Epstein-Barr virus-encoded latent membrane protein 2A on virus-induced immortalization and virus activation.

To quantitatively evaluate the role of Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in immortalization of peripheral B-lymphocytes, we used the Akata cell system to generate an EBV recombinant in which the first exon of the LMP2A gene was disrupted. The results indicated that deletion of the LMP2A gene did not affect the immortalization efficiency of EBV in B-lymphocytes. Deletion of the LMP2A gene made EBV-transformed lymphocytes more permissive for virus replication in response to surface immunoglobulin cross-linking. On the other hand Akata cells, in which LMP2A expression was much lower than in EBV-transformed lymphocytes, were equally permissive for virus replication whether they were infected with wild EBV or LMP2A-knockout EBV. The results raise a question as to the role of LMP2A in inhibition of disruption of virus latency in vivo, where LMP2A expression has been expected to be low as in Akata cells.

Antigens, Viral↗
PubMed1993

Isotype-specific cross-linking of select human Fc gamma R isoforms triggers release of IL-6.

Anti-CD3 MoAbs are widely used in T cell activation studies, and are effective in immunosuppressive therapy. We used a panel of mouse (m) anti-CD3 switch variant MoAbs of five different isotypes to study IL-6 release from accessory cells. Incubation of human (h) mononuclear cells with anti-CD3 MoAbs resulted in increased IL-6 levels with MoAbs of mIgG1 and mIgG2a isotypes, with no effect of mIgG2b or mIgA. This suggested involvement of IgG Fc receptors (Fc gamma R) in triggering IL-6 production. To evaluate the role of different Fc gamma R molecules individually we used a panel of hFc gamma R-transfected mouse fibroblasts, and Jurkat T cells as a model. IL-6 secretion by CD32 transfectants expressing the hFc gamma RIIa high-responder (HR) allelic form was triggered by mIgG1 anti-CD3 MoAb, with no effect of four other isotypes. None of the anti-CD3 MoAbs induced IL-6 secretion by CD32 transfectants expressing either a variant of this receptor, containing only a single intracellular amino acid (CT-), the hFc gamma RIIa low-responder (LR) allelic form, or hFc gamma RIIb1. hFc gamma RI (CD64) transfectants exhibited IL-6 production after incubation with mIgG2a anti-CD3 MoAb, and to a lesser extent with mIgG2b, and mIgG1 MoAb. Indirect involvement of T cells in triggering IL-6 secretion could be excluded by experiments in which transfectants were cultured with immobilized anti-CD3 MoAb. These data indicate that cross-linking of either hFc gamma RI, or hFc gamma RIIaHR by appropriate anti-CD3 MoAbs triggers IL-6 production of accessory cells, and not T cells. This may also take place in vivo during immunosuppressive therapy with anti-CD3 MoAbs, and related antibody-mediated immune responses.

3T3 Cells↗