Characterization of transferrin receptor in an immortalized cell line of rat brain endothelial cells, RBE4

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 +/- 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.     

Characterization of Transferrin Receptor in an Immortalized Cell Line of Rat Brain Endothelial Cells,RBE4

Abstract

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell mono-layers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (K_D) of 17.1 ± 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 ± 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.     

Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane

Insulin stimulates the accumulation of iron by isolated fat cells by increasing the uptake of diferric transferrin. Analysis of the cell-surface binding of diferric 125I-transferrin indicated that insulin caused a 3-fold increase in the cell surface number of transferrin receptors. This result was confirmed by the demonstration that insulin increases the binding of an anti-rat transferrin receptor monoclonal antibody (OX-26) to the surface of fat cells. The basis of this effect of insulin was examined by investigating the number of transferrin receptors in membrane fractions isolated from disrupted fat cells. Two methods were employed. First the binding isotherm of diferric 125I-transferrin to the isolated membranes was studied. Second, the membranes were solubilized with detergent, and the number of transferrin receptors was measured by immunoblotting using the monoclonal antibody OX-26. It was observed that insulin treatment of intact fat cells resulted in an increase in the number of transferrin receptors located in the isolated plasma membrane fraction of the disrupted fat cells. Furthermore, the increase in the number of plasma membrane transferrin receptors was associated with a concomitant decrease in the transferrin receptor number in a low density microsome fraction previously shown to consist of intracellular membranes. This redistribution of transferrin receptors between cellular membrane fractions in response to insulin is remarkably similar to the regulation by insulin of glucose transporters and type II insulin-like growth factor receptors. We conclude that insulin stimulates fat cell iron uptake by a mechanism that may involve the redistribution of transferrin receptors from an internal membrane compartment (low density microsomes) to the cell surface (plasma membrane).     

Transferrin receptors and cation-independent mannose-6-phosphate receptors deliver their ligands to two distinct subpopulations of multivesicular endosomes

The distribution of transferrin receptors (Tf-R) was determined in Clone 9 hepatocytes and compared to that of 215 kDa, cation-independent mannose-6-phosphate receptors (M6P-R) by double labeling. Cells were allowed to take up exogenous human transferrin (Tf) for 5 to 30 min, after which Tf, Tf-R, and M6P-R were localized by immunofluorescence using specific antibodies. All these proteins were found to be concentrated in the juxtanuclear or Golgi region. When Clone 9 cells were treated with NH4Cl to trap M6P-R in endosomes (Brown, W. J., J. Goodhouse, M. G. Farquhar: J. Cell Biol. 103, 1235-1247 (1986)), the distribution of the two receptors differed: Tf-R remained the same as in controls, but M6P-R were localized in large vacuolated endosomes. To carry out double labeling experiments at the electron microscope level, transferrin gold conjugates (Tf-Au) were prepared, and M6P-R were detected by immunoperoxidase labeling. Tf-Au binding to the cell surface was specific as it was reduced approximately 70 to 79% in the presence of excess native Tf. When Clone 9 cells were incubated with Tf-Au at 37 degrees C for 5 to 30 min, or binding of Tf-Au was carried out at 4 degrees C followed by warming to 37 degrees C, Tf-Au was found within a peripheral tubulovesicular network and within multivesicular endosomes that were not labeled with anti-M6P-R. Other multivesicular endosomes of similar size and morphology were heavily labeled for M6P-R but contained little or no Tf-Au. Tf-Au and M6P-R were also found in separate endosomes in cells treated with NH4Cl. Native Tf was localized in the same compartments as Tf-Au by immunoperoxidase labeling of both Clone 9 cells and mouse myeloma cells. We conclude that in Clone 9 hepatocytes, Tf/Tf-R internalized from the cell surface and M6P-R bearing newly synthesized lysosomal enzymes from the Golgi deliver their ligands to two different subpopulations of multivesicular endosomes. The endosomal subpopulation visited by Tf/Tf-R is known to correspond kinetically to early endosomes. The endosomal subpopulation heavily labeled for M6P-R presumably represent a later endosomal compartment which serves as the junction point where endocytosed ligands and newly synthesized lysosomal enzymes enroute to lysosomes meet.     

Immuno-electron-microscopic localization of enkephalin in the secretory granules of C cells in the chicken ultimobranchial glands

Interstitial cells in the pineal gland of the rat were characterized immunocytochemically using the monoclonal antibodies MRC OX-42 and ED1 for macrophages/microglia, and MRC OX-6, which recognizes major histocompatibility complex (MHC) class II antigen. A polyclonal antibody against GFAP was used to identify astrocytes. Cells immunopositive for OX-42 and/or ED1 were distributed throughout the gland; they extended processes primarily along the perivascular spaces and occasionally within the parenchyma of the gland. Ultrastructurally, these OX-42-positive cells were characterized by a nucleus with sparse heterochromatin and cytoplasmic vacuoles/lysosomes. Cells expressing MHC class II antigen had a distribution and morphology similar to OX-42-immunopositive cells, suggesting that pineal macrophages/microglia play a role as antigen-presenting cells. GFAP-positive astrocytes were concentrated at the proximal end of the pineal where the pineal stalk enters the gland. The occurrence of antigenpresenting cells in the circumventricular neuroendocrine gland has important functional implications as these cells may be mediators of neuroimmunomodulatory mechanisms, and involved in certain disease states such as autoimmune pinealitis.     

Plasma membrane and intracellular pools of transferrin receptors decline during in vitro cultivation of U937 cells

Transferrin receptor expression in the monocyte-like cell line U937 was investigated during in vitro cultivation. U937 cells expressed a single class of high affinity surface transferrin receptors (KD approximately 4 nM), with apparent subunit Mr of 90-95,000 Da as determined by SDS-reducing PAGE. [125I]-transferrin binding studies on detergent-solubilized cells revealed that half to two-thirds of the total functional binding sites were located intracellularly. Radioligand binding, immunofluorescence and flow cytometry studies were performed on intact, detergent-solubilized, or saponin-permeabilized cells, using either transferrin or the anti-transferrin receptor monoclonal antibody OKT9 IgG. These studies demonstrated that functional and antigenic transferrin receptor levels were maximal on cells 24 h after subculture at low density and declined during the culture period. Scatchard analysis of radioligand binding data suggested that the decline in functional transferrin binding sites resulted from a decline in the number of available receptors. These results demonstrate that in U937 cells there is a density-dependent regulation of transferrin receptor expression, resulting in a loss of functional and antigenic receptors from both plasma membrane and intracellular locations.     

Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies

Protein G is an immunoglobulin (IgG)-binding bacterial cell wall protein recently isolated from group G streptococci. We have investigated the avidity of protein G for various monoclonal and polyclonal Ig of the IgG class, and compared it with the binding properties of protein A, the staphylococcal Fc-binding protein. Radiolabeled Ig were mixed with Sepharose-coupled protein G or protein A, and the amounts of radioactivity bound to the matrix-coupled bacterial proteins were determined. The avidity was found to be greater for protein G than for protein A for all examined Ig. Protein G bound all tested monoclonal IgG from mouse IgG1, IgG2a, and IgG3, and rat IgG2a, IgG2b, and IgG2c. In addition, polyclonal IgG from man, cow, rabbit, goat, rat, and mouse bound to protein G, whereas chicken IgG did not. The binding property of protein G was additionally exploited in the Western blot assay, in which iodine-labeled protein G was used successfully for the detection of a rat monoclonal antibody against ovalbumin, and for the detection of rabbit and goat polyclonal whole antisera against human urinary proteins. In these experimental situations, protein G was found to be a powerful reagent for the detection of IgG, and consequently the antigen against which these antibodies are directed.     

Localisation of the MRC OX-2 Glycoprotein on the Surfaces of Neurones

The MRC OX-2 monoclonal antibody recognises membrane glycoproteins of Mr 41,000 in rat brain and 47,000 on thymocytes. It also reacts with follicular dendritic cells in lymphoid organs, endothelium, smooth muscle, and B-lymphocytes. Indirect immunoperoxidase staining of cryostat sections showed that OX-2 antigen was present throughout the cerebellum, with staining of both grey and white matter. Blood vessels were also stained. The Purkinje cell layer appeared to be unlabelled. Double-immunofluorescence staining of cerebellar interneurone cultures with MRC OX-2 antibody and tetanus toxin showed that all tetanus-positive cells (neurones) were MRC OX-2-positive. Glial fibrillary acidic protein-positive astrocytes were not labelled by MRC OX-2 antibody. Thus OX-2 antigen is one of the few biochemically characterised components of neuronal membranes and its properties are compared with those of the neuronal membrane glycoprotein Thy-1.     

Transferrin receptor activity in rat mammary epithelial cells

The binding of 125I-transferrin to rat mammary cells isolated by collagenase and hyaluronidase digestion has been investigated. Surface binding was determined at 4 degrees C and total binding also at 4 degrees C but in the presence of 0.1% w/v saponin. KD values between 20 and 25 nM were obtained. Binding assays at 37 degrees C showed the internalisation of the receptor and the bound transferrin was occurring but also provided evidence for an impaired recycling of the receptors to the cell surface in the freshly isolated cells. No differences in total binding were observed in cells prepared at different stages of lactation with a mean value of 29 fmol transferrin bound/micrograms cellular DNA, equivalent to 180,000 receptors per cell.     

Expression of leucocyte antigens by cells from the metrial gland of the pregnant rat

Summary The function of the metrial gland of the rat, and particularly of its characteristic population of granulated cells, remains unknown. However, several lines of evidence suggest that the granulated cells may derive from lymphocytes, and play a role in the immunology of pregnancy. In this study, antigen expression by granulated and other cell populations from the metrial glands of rats at Days 13 and 14 of pregnancy was studied by an indirect immunoperoxidase method. Acetone-fixed frozen sections, and cytocentrifuge preparations of collagenase-dispersed metrial gland tissue in which numbers of granulated cells had been increased by density-gradient centrifugation, were used. The primary antibodies used recognised, inter alia, B lymphocytes (MRC OX-3, MRC OX-6, MRC OX-12), T lymphocytes (MRC OX-8, W3/25, MRC OX-19), neutrophils (MRC OX-42) and cells of the monocyte/macrophage series (MRC OX-3, MRC OX-6, MRC OX-42, MRC OX43). The majority of the granulated cells, including smaller, immature forms, were unlabelled by any of these antibodies. Some lymphocytes, and varying numbers of larger, non-granulated cells, were labelled by OX-6, OX-12, W3/ 25, OX-42 and OX-43. In addition to lymphocytes, labelled cells included neutrophils (OX-42), endothelial cells (OX-43), and probably some macrophages (OX-6, OX-43). OX-12, which recognises the kappa chain of rat IgG, labelled some large cells which may have been stromal cells. These findings do not support the concept that the granulated cells are derived from lymphocytes.     

Surface phenotype of rat bone marrow-derived mononuclear phagocytes

Utilizing a panel of currently available monoclonal antibodies, the surface phenotype of a pure population of resting rat bone marrow-derived mononuclear phagocytes (BMM phi) was analyzed by means of flow cytometry. The present work provides an extensive list of surface markers expressed by BMM phi and also outlines advantages and limitations of flow cytometric analysis of this cell type. The results show that the majority of surface markers considered to be expressed selectively by T lymphocytes, such as Thy-1, CD2 and CD5 antigens, leukosialin (W3/13), or an alloantigen of peripheral T cells, are not expressed by BMM phi. On the other hand, the CD8 antigen and the leukocyte common antigen recognized by MRC OX-33, considered to represent specific markers of cytotoxic T cells and/or peripheral B cells, are expressed on a variable, often considerable proportion of BMM phi. Monoclonal antibodies W3/25, MRC OX-35, and MRC OX-38, directed against epitopes on the CD4 molecule, labeled a variable proportion of BMM phi. Among the 39 monoclonal antibodies examined, none appeared to recognize an epitope which is expressed selectively by mononuclear phagocytes.     

Binding of human C-reactive protein to mouse macrophages is mediated by distinct receptors

Human C-reactive protein (CRP) is known to activate mouse macrophages (M phi) to a tumoricidal state and to serve as an opsonin for M phi. Therefore, cell surface receptors for CRP on mouse M phi were characterized and their relationship to the IgG FcR determined. The specific binding of 125I-CRP to resident or elicited mouse M phi was saturable, reversible, and involved both a high and a low affinity receptor population. Binding of CRP to the mouse M phi cell lines PU5 1.8 and J774 was nearly identical to that observed with peritoneal M phi. The high affinity receptor population had a calculated K of 10 nM and a receptor density of approximately 10(5) sites per cell. Mouse Ig of the IgG2a, IgG2b, or IgG1 isotypes inhibited binding of 125I-CRP to PU5 1.8 cells at concentrations five-fold greater than that of the homologous ligand. In the converse experiment, unlabeled CRP failed to inhibit specific binding of 125I-labeled IgG2a, IgG2b or IgG1. Isolation of CRP binding proteins from surface iodinated PU5 1.8 cells by ligand-affinity chromatography or chemical cross-linking yielded a major protein band of 57 to 60 kDa which appeared to be distinct from the IgG1/IgG2b FcR (FcR-II) membrane proteins. Removal of radiolabeled IgG2b/IgG1 binding membrane proteins by affinity chromatography did not remove CRP-binding proteins. The rat mAb 2.4G2 which inhibits binding of radiolabeled mouse IgG2b, did not inhibit the binding of CRP. A rat polyclonal antiserum to CRP-binding membrane proteins of PU5 1.8 cells inhibited 125I-CRP binding, but not 125IgG2b binding. The rat polyclonal antibody reacted with two 57 to 60 kDa membrane proteins from PU5 1.8 cells that appear to be of a similar size on Western blots. The 125I-CRP was internalized via endosomes and intact CRP subunits could be detected intracellularly. The findings suggest that binding of CRP occurs through a receptor that is distinct from the IgG FcRs, but that CRP-R activity may be influenced by an association with an IgG FcR.     

Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation

The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, [125I]-labeled human transferrin and a [32P]-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (Ka) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 X 10(8) M-1, and the number of receptors per cell was 4.4 +/- 1.2 X 10(4). In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, greater than 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages.     

T cell-mediated cytotoxicity induced by Listeria monocytogenes. III. Phenotypic characteristics of mediator T cells

Cytotoxic thymus-derived lymphocytes (CTL) generated in vitro by restimulating rat cells with Listeria antigen- (LMA) pulsed syngeneic accessory cells were characterized in respect to their surface membrane markers. LM-dependent CTL were devoid of detectable surface immunoglobulin (Ig) and receptors for the Fc region of rabbit IgG. Experiments with monoclonal antibodies to rat T cell markers revealed that these cytotoxic cells have the phenotypic profile W3/13+, W3/25-, MRC OX 8+. LM-dependent CTL also bind the monoclonal antibody, MRC OX 3, which recognizes an Ia-antigen-like determinant on rat cells. Although LM-dependent CTL lack the W3/25 marker, their generation depends on the cooperative interplay of W3/25+ and W3/25- T cells.     

Factors affecting the level and localization of the transferrin receptor in Trypanosoma brucei

Transfer of bloodstream-form Trypanosoma brucei variant 221a from calf serum to dog serum-based medium induces acute iron starvation, as the transferrin receptor (Tf-R) of variant 221a binds dog Tf poorly. We show here that transfer to dog serum induces a 3-5-fold increase in Tf-R mRNA and protein within one doubling time (8 h). Because iron stores are still high 8 h after transfer, we infer that the signal for Tf-R overproduction is the decreased availability of cytosolic iron when cellular iron import drops. Up to 30% of the extra Tf-R spills out of the flagellar pocket onto the pellicular surface. Because the 5-fold increase in Tf-R is accompanied by a 5-fold increase in bovine Tf uptake, the up-regulation of Tf-R levels in response to Tf starvation helps the trypanosome to compete for limiting amounts of Tf. We noted that Tf-R levels also vary in calf serum medium. Cells in dense cultures contain up to 5-fold more Tf-R mRNA and protein than in dilute cultures. Only one-tenth of the extra Tf-R reaches the pellicular surface. The increase cannot be explained by a lack of Tf or to cell density sensing but is due to pericellular hypoxia. Our results show that bloodstream-form trypanosomes can regulate the expression of the two Tf-R subunit genes and the localization of their gene products in a flexible manner. This flexibility is made possible by the promoter-proximal position of the two genes in the variant surface glycoprotein expression site.     

Inhibition of cell growth by monoclonal anti-transferrin receptor antibodies.

Five anti-murine transferrin receptor monoclonal antibodies have been characterized with respect to immunoglobulin class, effects on binding of transferrin, and effects on AKR1 lymphoma cell growth in vitro. The immunoglobulin M (IgM) antibodies, but not the IgG antibodies, prevent cell growth. We suggest that the profound effects of the IgM antibodies on cell growth are probably due to extensive cross-linking of cell surface receptors. In support of this, we are able to mimic the growth-inhibiting effects of the IgM antibodies by adding antiimmunoglobulin to an IgG antibody. By flow microfluorimetry, we show that an IgG antibody by itself induces up to a 10-fold downward regulation in the cell surface transferrin receptor, which is accompanied by accelerated receptor degradation. A similar downward regulation is seen in mutant cells resistant to growth inhibition by an IgM antibody, when grown in the selecting antibody. Wild-type cells grown in the presence of IgM antibody do not show receptor downward regulation. Inhibitory effects of antibody plus antiimmuoglobulin on mutant cells are also consistent with extensive cross-linking causing inhibition of growth.     

Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor.

Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of [125I]diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 min. The effect of EGF is transient, with [125I]diferric transferrin binding returning to control values within 25 min. In contrast, PDGF and rIGF-I cause a prolonged stimulation of [125I]diferric transferrin binding that could be observed for up to 2 h. The increase in the binding of [125I]diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. Epidermal growth factor, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. This result was confirmed in human fibroblasts by the demonstration that EGF, PDGF and rIGF-I could stimulate the binding of a monoclonal antibody directed against the transferrin receptor (OKT9) to the cell surface. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of [59Fe]diferric transferrin by BALB/c 3T3 fibroblasts, while EGF transiently increased uptake. Thus the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence.     

Selective colony blotting to expand bacterial surface receptors: applications to receptors for rat immunoglobulins

Many bacterial surface receptors demonstrate a heterogeneous expression pattern among individual colonies. Methods have been developed to select bacteria expressing high levels of a stable surface receptor. This process is illustrated using a Streptococcus zooepidemicus isolate demonstrating a high level of Fc receptors for rat immunoglobulins. This strain was selected and expanded to obtain a bacterial isolate demonstrating approximately 100 fold greater reactivity with rat immunoglobulins than protein A positive Staphylococcus aureus or 30-40 fold higher reactivity for rat IgG than type III Fc receptor positive streptococcal group G strains. The optimal pH for rat IgG binding and the reactivity with rat IgG subclasses and certain rat monoclonal antibodies is described. The potential application and limitations of the selected rat Fc receptor positive bacterial strain to immunoassays based on the specificity of rat monoclonal and polyclonal antibodies is discussed.     

Antigenic specificities of human monoclonal and polyclonal IgM rheumatoid factors. The C gamma 2-C gamma 3 interface region contains the major determinants

The binding site specificity of 12 monoclonal and 11 polyclonal IgM rheumatoid factors (RF) isolated from human plasma or serum has been studied. All IgM RF bound best to sites on IgG and intact Fc. The monoclonal IgM RF did not bind at all to fragments lacking the C gamma 2 or C gamma 3 domains. In contrast, low level binding to the pFc' fragment, composed of the C gamma 3 domain, was seen with seven IgM RF, mainly from patients with rheumatoid arthritis (RA). IgG1 binding appeared to be a requisite specificity of all human IgM RF. IgM RF binding to IgG3 subclass was common among the monoclonal IgM RF. Most RA polyclonal IgM RF but only 2 of the monoclonal IgM RF possessed the IgG1, 2 and 4 binding pattern. Monoclonal IgM RF which bound best to histidine-modified IgG also bound well to IgG3. The 7-kDa fragment D of staphylococcal protein A inhibited the IgG binding of most monoclonal and to a lesser degree polyclonal IgM RF. Thus, the results indicate that the C gamma 2-C gamma 3 interface region of IgG contains the predominant determinants for monoclonal and polyclonal IgM RF. For some monoclonal IgM RF the binding site, even though at the interface of the C gamma 2 and C gamma 3 domains, is not the staphylococcal protein A site. Furthermore, polyclonal IgM RF possess specificities not encountered among the monoclonal IgM RF. These specificities may have special