Functional ion channel formation by mouse macrophage IgG Fc receptor triggered by specific ligands

The mouse macrophage Fc gamma 2b/gamma 1R has previously been purified with the aid of the monoclonal antibody 2.4G2. That this Fc gamma R functions as a ligand-dependent ion channel is supported by the following evidence. Employing [3H]tetraphenylphosphonium ([3H]Ph4P+) as a probe for membrane potential changes in intact cells, we found a biphasic change in membrane potential following treatment with immune complexes, monoclonal antibody 2.4G2 IgG and 2.4G2 Fab-Sephadex particles. We observed an immediate depolarization followed by prolonged hyperpolarization. [3H]Ph4P+ uptake experiments with plasma membrane vesicles prepared from J774 macrophages showed that binding of ligands to the FcR led to transmembrane monovalent cation flow. Similar [3H]Ph4+ uptake experiments were done with phospholipid vesicles containing purified and reconstituted Fc gamma 2b/gamma. Following challenge with specific ligands, transmembrane monovalent cation flow was observed. Purified FcR was reconstituted into planar lipid bilayers; exposure to ligands led to transient bilayer conductance increase. THe conductance change was resolved into single channel events. Quin-2 measurements showed an increase of free cytosolic calcium levels in macrophages following exposure of cells to different ligands of the FcR. An optimal range of calcium was found to be required for phagocytosis, below and above which inhibition of ingestion occurred.     

The murine lymphocyte receptor for IgE. V. Biosynthesis, transport, and maturation of the B cell Fc epsilon receptor

The post-translational processing and maturation of the receptor for IgE (Fc epsilon R) on murine hybridoma B cells were studied to determine the carbohydrate content and the importance of processing events in cell surface expression and ligand (IgE) binding ability. Endo and exoglycosidase treatment demonstrated that the mature receptor is composed of two to three complex-type N-linked oligosaccharides and contains sialic acid. Pulse-chase experiments indicated that the receptor is synthesized as a 44,000 dalton precursor that begins to be processed by 1 hr to the mature 49,000 dalton form, and the latter is expressed at the cell surface by 2 hr. It was determined that the processing included the conversion of N-linked oligosaccharides to the complex type as well as an additional processing event, because in the presence of tunicamycin, the receptor is synthesized as a 36,000 dalton precursor that is processed to a 38,000 dalton species. Analysis of the effects of tunicamycin treatment and endo F digestion on soluble Fc epsilon R isolated from cell supernatants demonstrated the existence of several m.w. species of Fc epsilon R fragments, and indicated that only the higher m.w. fragments were N-glycosylated. The use of several inhibitors of the N-linked carbohydrate processing pathway demonstrated that the addition of core N-linked side-chains, but not their processing to the complex type, is required for cell surface expression of Fc epsilon R. Also, processing of N-linked carbohydrate is not required for ligand binding activity. Finally, IgE affinity chromatography indicated that the 49,000 and 38,000 dalton (tunicamycin) Fc epsilon R bind IgE more effectively than their precursor forms, 44,000 and 36,000 daltons, respectively, indicating that a processing event independent of N-linked glycosylation is necessary for optimal ligand binding activity.     

Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. II. Processing, subunit assembly, and intracellular transport

The biosynthesis and turnover of lipoprotein lipase (LPL) have been investigated in adipose 3T3-F442A cells labeled with [35S]methionine. Pulse-chase experiments, endo-beta-N-acetylglucosaminidase H treatment, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have indicated that LPL is synthesized in the endoplasmic reticulum as a glycoprotein of Mr = 55,500 bearing two N-oligosaccharide side chains of the high mannose-type. This precursor form of LPL is transported within 10 min to the Golgi apparatus, and this event is accompanied by the formation of a mature species of Mr = 58,000. Treatment of the Mr = 58,000 species with glycopeptidase F yielded a Mr = 51,000 protein similar to that observed after treatment of the Mr = 55,500 precursor form or after inhibition of N-glycosylation in tunicamycin-treated cells. The precursor form of LPL of Mr = 55,500 does not accumulate in the cells since, after a labeling period of 2 h, only the Mr = 58,000 species is detected. It is shown that only 20% of the newly synthesized molecules of Mr = 58,000 are constitutively secreted, whereas 80% are degraded, most likely in lysosomes, as indicated by the inhibitory effect of leupeptin upon the degradation process. Under heparin stimulation, quantitative secretion of the mature form of LPL takes place whereas the intracellular degradation is arrested. Heparin is able to mobilize intracellular LPL without changing the rate of LPL export from the endoplasmic reticulum to the cell surface. Sucrose gradient centrifugation of the material from intracellular cisternae shows that the Mr = 55,500 precursor form is present as a monomer (s = 4.1 S), whereas the Mr = 58,000 mature form is present as a homodimer (s = 6.8 S) to which LPL activity is associated. The results are interpreted as LPL being transiently stored under a dimeric form before its degradation. A sorting process of LPL in the Golgi apparatus, followed by its entry either mainly in a regulated pathway or in a constitutive pathway, is proposed.     

Analysis of the endocytic pathway upon intracellular transport of IgG molecules through Fc receptors

The uniformly distributed Fc receptors (FcRs) on the surface of many cell types are involved in a variety of immune reactions by non-specifically facilitating the entry of antigen-specific IgG molecules to the cell. Such reactions may be beneficial to the organism when foreign antigens are involved, or harmful in cases of self antigens and viruses. In order to avoid the IgG-mediated self antigen presentation or viral infection in autoimmunity and viral attack respectively, we attempt in this study to inhibit the intracellular transport of antibodies. This blockage, however, implies: efficacy of inhibition, inability of de novo exocytosis of the internalised antibody and finally maintenance of normal cell growth and morphology. We thus concentrate our interest on the endocytic pathway followed by a neutralising antibody in murine trophoblast cells where we try to inhibit antibody intracellular transport by various agents according to the criteria set above. In our model-system, IFN-gamma, upon induction of FcRs, facilitates endocytosis of the anti-p21ras antibody which blocks in turn the IFN-gamma-induced surface class II major histocompatibility complex (MHC) expression. Using various intracellular transport inhibitors, we study the required conditions by which these compounds cancel the inhibitory action of anti-p21ras and allow induction of class II MHC molecules by IFN-gamma. The effectiveness of the inhibitors in a ranking order is shown as following: monodansyl cadaverine > didansyl cadaverine > pepstatin A > leupeptin > NH4Cl > brefeldin A > ZPCK > TPCK. From these inhibitors, only brefeldin A, leupeptin, pepstatin and ZPCK do not allow exocytosis of the antibody in the culture medium and only didansyl cadaverine, pepstatin and leupeptin maintain cell viability and morphology. However, by sequential elimination based on this study's established criteria, only pepstatin A and leupeptin are shown to be effective inhibitors to specific antibody intracellular transport, protecting also the cell's viability and physiology.     

Signal transduction mechanisms through Fc gamma receptors on the mouse macrophage surface.

Mouse macrophages and macrophage cell lines such as P388D1 or J774 carry at least two distinct Fc gamma receptors (Fc gamma R): one specific for the Fc portion of IgG2a (Fc gamma aR, also classified as Fc gamma RI) and another for IgG2b (Fc gamma 2bR, also classified as Fc gamma RII beta). These Fc gamma Rs should transmit, upon binding of an appropriate ligand, a specific signal that leads to the regulation of macrophage functions, as the interaction of immune complex with cell surface receptor has been shown to lead to suppression of the humoral immune response or B cell differentiation, to the destruction of target cells by antibody-dependent cell-mediated cytotoxicity, to activation of arachidonic acid metabolic cascade, to the phagocytosis of opsonized particles, or to the generation of superoxide anion. In this review, we first describe evidence that Fc gamma 2aR and Fc gamma 2bR are associated with casein kinase II and phospholipase A2 activity, respectively. We will then discuss a potential role for these enzymatic activities in signal transduction pathways that leads to the activation of the arachidonic acid metabolic cascade and adenylate cyclase, to the regulation of phagocytosis, and to the suppression of interferon-gamma action to induce Ia antigens.     

Biosynthesis and intracellular transport of glyoxysomal malate dehydrogenase in germinating pumpkin cotyledons

Glyoxysomal malate dehydrogenase was synthesized as a larger molecular mass precursor in germinating pumpkin cotyledons. In pulse-chase experiments, the radioactive larger molecular mass precursor (38 kDa) disappeared and was converted to the mature form (33 kDa) of the enzyme. When the radiolabeled cotyledon was fractionated into cytosolic and organellar fractions, the larger molecular mass precursor was first recovered in the cytosolic fraction and then only after a 20 min chase the mature form was found in the organellar fraction. This indicates that the higher molecular mass precursor is synthesized in the cytosol and the processing of the transient precursor is coupled to the transport into glyoxysomes.     

Biosynthesis of intestinal microvillar proteins: Further characterization of the intracellular processing and transport

The effect of tunicamycin on synthesis and intracellular transport of pig small intestinal aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48–10) and maltase-glucoamylase (EC 3.2.1.20) was studied by labelling of mucosal explants with [³⁵S]methionine. The expression of the microvillar enzymes was greatly reduced by tunicamycin but could be partially restored by leupeptin, suggesting the existence of a mechanism whereby newly synthesized, malprocessed enzymes are recognized and degraded. In the presence of tunicamycin, polypeptides likely to represent non-glycosylated forms of the enzymes persisted in the Mg²⁺-precipitated membrane fraction, indicating that high mannose glycosylation is essential for transport to the microvillar membrane. Treatment of aminopeptidase N and sucrase-isomaltase with endo F reduced the size of the high mannose forms approximately to those seen in the presence of tunicamycin. The complex forms were also sensitive to endo F but did not coincide with the high mannose forms after treatment, indicating that the size difference cannot alone be ascribed to processing of N-linked carbohydrate.     

Biosynthesis of the sperm receptor during oogenesis in the mouse

During their growth phase, mouse oocytes synthesize and secrete three different glycoproteins, called ZP1, 2 and 3, that constitute the extracellular coat, or zona pellucida, of the oocyte. One of these glycoproteins, ZP3, exhibits properties expected for a sperm receptor. We have now used rabbit antisera that recognize ZP3 to immunoprecipitate [35S]methionine-labeled, intracellular precursors of this glycoprotein from growing oocytes cultured in vitro in the presence or absence of tunicamycin, a drug that prevents addition of N-linked oligosaccharides to nascent polypeptide chains. Electrophoretic analyses of these immunoprecipitates, as well as of immunoprecipitates digested with endo-beta-N-acetylglucosaminidase H (Endo H), indicate that ZP3 is synthesized as a 44,000 mol. wt. polypeptide chain to which either three or four high-mannose-type oligosaccharides are added, resulting in 53,000 and 56,000 mol. wt. ZP3 precursors, respectively. The latter species are converted to mature ZP3 (mol. wt. approximately 80,000) by processing of the high-mannose-type oligosaccharides (Endo H-sensitive) to complex-type oligosaccharides (Endo H-insensitive) prior to ZP3 secretion. The evidence presented reveals that the extreme heterogeneity of mature ZP3, with respect to both mol. wt. and isoelectric point, is partly a consequence of the N-linked oligosaccharides and not the polypeptide chain itself.     

Purification and partial characterization of rat macrophage Fc receptor and binding factor for IgA

By using a biotinylated ligand and Western blotting techniques, a receptor (RFc alpha) and a binding factor (BF) for IgA were detected, respectively, on membrane and in the cell-free culture supernatant of rat peritoneal macrophages. Extraction of the RFc alpha was obtained by solubilization of macrophages with Nonidet P-40, and purification was performed by HPLC affinity chromatography on a column derivatized with IgA. RFc alpha is formed of two subunits, with molecular masses of 56 and 70 kDa, which are both involved in the IgA binding ability of rat peritoneal macrophages. IgABF was recovered from the cell-free supernatant of a short-term culture of rat macrophages and was affinity-purified in the same manner as RFc alpha. Like RFc alpha, IgABF retained its IgA binding activity in its native, as well as denatured form. Since the molecular masses of RFc alpha and IgABF are similar, and IgABF competes with RFc alpha for IgA binding, one can assume that IgABF probably represents a shed RFc alpha.     

Biosynthesis of the complement component C1q in mouse peritoneal macrophage cultures

Biosynthesis of C1q complement component by resident peritoneal macrophages from (CBA X C57BL/6)F1 mice has been studied in in vitro experiments. Using anti-mouse C1q antibodies immobilized on CNBr Sepharose it has been demonstrated that 14C glycine incorporates both into intracellular C1q and C1q secreted into the medium. The maximum radioactivity of intracellular C1q was observed 48 h after cultivation, with it dropping drastically between hours 72-96. Kinetics of radiolabelled C1q was similar, but 24 hours delayed. Cell viability during 96 h of cultivation remained unchanged. These data can be considered as the indication of feedback regulation of C1q biosynthesis at the cellular level.     

Opposing effects of glucocorticoids on interferon-gamma-induced murine macrophage Fc receptor and Ia antigen expression

Two macrophage markers associated with differentiation are the Fc receptor (FcR) and the Ia antigen. Expression of these markers is increased with IFN-gamma treatment, although some evidence suggests that the induction pathway for Fc receptor and Ia antigen expression may be dissociable. In this study, the effect of glucocorticoids on basal and IFN-induced levels of Fc-mediated phagocytosis and Ia antigen expression was investigated. Macrophages incubated for 2 days with glucocorticoids alone showed no change in basal levels of Fc-mediated phagocytosis. However, incubation with glucocorticoids plus IFN-gamma resulted in increased Fc-mediated phagocytosis and binding to a much greater extent than IFN-gamma treatment alone. This enhancement was specific for IFN-gamma, because the IFN-beta-induced increase in Fc-mediated phagocytosis and binding was not affected by glucocorticoids. In contrast to the expression of Fc receptor capacity, both basal and IFN-gamma-induced levels of Ia antigen expression were inhibited by glucocorticoids. The glucocorticoid effect on these two markers was not observed with other steroid hormones, nor was it altered by inhibitors of the arachidonic acid pathway. The findings of this study provide additional evidence that induction of Fc receptor and Ia antigen by IFN-gamma occurs by different mechanisms.     

Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat

The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling of jejunal explants from 5-day-old suckling rats in organ culture. Explants were either continuously labeled with [35S] methionine for 15, 30, and 60 min or pulse-labeled for 30 min and chased for various periods of time up to 6 h in the presence or absence of protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor. Lactase-phlorizin hydrolase was immunoprecipitated from microvillus membrane (MVM) and ER-Golgi fractions with monoclonal antibodies. After pulse-labeling, lactase-phlorizin hydrolase from the ER-Golgi fraction appeared on SDS-PAGE as one band of approximately 220 kDa, regardless of the presence or absence of PI in the culture media. The 220-kDa protein band could also be labeled after incubation with [2-3H]mannose. In the absence of PI, the 220-kDa band appeared in the MVM by 30 min chase, simultaneously with a 180-kDa band, and by 60 min of chase an additional band of 130 kDa was seen. With increasing time of chase, the relative intensity of the 130-kDa band increased, whereas that of the 220-kDa band decreased, suggesting a precursor-product relationship. When PI were added to the medium, the formation of the 180-kDa band was not affected, but the conversion of the 180-kDa protein to the 130-kDa protein was virtually blocked. These findings suggest that lactase-phlorizin hydrolase is initially synthesized as a glycosylated precursor of 220 kDa, which is transported to the MVM. There it undergoes the following two cleavages: first, to the 180-kDa form, which is not prevented by PI used in these experiments, and second, to the 130-kDa form inhibited by PI.     

Biosynthesis and intracellular transport of alpha-2,6-sialyltransferase in rat hepatoma cells.

We investigated biosynthesis, intracellular transport and release of beta-galactoside alpha-2,6-sialyltransferase in a dexamethasone-inducible rat hepatoma cell line. Confluent cells were induced by 10 microM dexamethasone for 24 h, and metabolically labelled with [35S]methionine/cysteine, followed by immunoprecipitation of sialyltransferase and electrophoretic/fluorographic analysis. The 35S-labelled enzyme was synthesized as a 46-kDa precursor, converted to an intermediate 47-kDa form after 1 h, and gradually to a mature form of 48 kDa within the following 3 h. By means of either tunicamycin inhibition of N-glycosylation or cleavage of N-glycans from isolated sialyltransferase using N-glycosidase F, the sizes of the precursor and the mature form were reduced to 41 kDa and 43 kDa, respectively. After a 4-h chase, treatment with endoglycosidase H revealed two distinct molecular forms of sialyltransferase, bearing either two N-acetyllactosamine-type or one oligomannose-type and one N-acetyllactosamine-type N-linked sugar chain. In addition, sialyltransferase became sensitive to neuraminidase digestion after a 4-h chase. The half-life of intracellular [35S]sialyltransferase was estimated at 3 h. A soluble form was detectable in the supernatant, 2 h after the pulse. Only 12% of the initially labelled sialyltransferase was found in the medium after 12 h, while 73% of the enzyme was degraded intracellularly. To characterize a possible intracellular degradation site, we studied intracellular transport in the presence of either secretion-blocking or acidotropic agents or protease inhibitors. Degradation was significantly delayed by all treatments. Our results show that sialyltransferase follows the secretory pathway as a membrane protein and is retained at a late Golgi stage. We suggest that the bulk of sialyltransferase in rat hepatoma cells is diverted to a post-Golgi degradation pathway. This route contrasts with the post-Golgi trafficking of beta-1,4-galactosyltransferase in HeLa cells, which is constitutively secreted [Strous, G. J. A. M. & Berger, E. G. (1982) J. Biol. Chem. 257, 7623-7628].     

Role of novel rat-specific Fc receptor in macrophage activation associated with crescentic glomerulonephritis

Crescentic glomerulonephritis (Crgn) is a complex disease where the initial insult is often the glomerular deposition of antibodies against intrinsic or deposited antigens in the glomerulus. The role of Fc receptors in the induction and progression of Crgn is increasingly recognized, and our previous studies have shown that copy number variation in Fcgr3 partially explains the genetic susceptibility of the Wistar-Kyoto (WKY) rat to nephrotoxic nephritis, a rat model of Crgn. The Fcgr3-related sequence (Fcgr3-rs) is a novel rat-specific Fc receptor with a cytoplasmic domain 6 amino acids longer than its paralogue, Fcgr3. The Fcgr3-rs gene is deleted from the WKY rat genome, and this deletion is associated with enhanced macrophage activity in this strain. Here, we investigated the mechanism by which the deletion of Fcgr3-rs in the WKY strain leads to increased macrophage activation. By lentivirus-mediated gene delivery, we generated stably transduced U937 cells expressing either Fcgr3-rs or Fcgr3. In these cells, which lack endogenous Fcgr3 receptors, we show that Fcgr3-rs interacts with the common Fc-γ chain but that Fc receptor-mediated phagocytosis and signaling are defective. Furthermore, in primary macrophages, expression of Fcgr3-rs inhibits Fc receptor-mediated functions, because WKY bone marrow-derived macrophages transduced with Fcgr3-rs had significantly reduced phagocytic activity. This inhibitory effect on phagocytosis was mediated by the novel cytoplasmic domain of Fcgr3-rs. These results suggest that Fcgr3-rs may act to inhibit Fcgr3-mediated signaling and phagocytosis and could be considered as a novel mechanism in the modulation of Fc receptor-mediated cell activation in autoimmune diseases.