An autoimmune disease with multiple molecular targets abrogated by the transgenic expression of a single autoantigen in the thymus.

Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.

From coiled tubules to a secretory canaliculus: a new model for membrane transformation and acid secretion by gastric parietal cells.

The acid-secreting gastric parietal cell has a unique secretory membrane system. This membrane system exists in an inactive (non-secreting) and an active (secreting) form. The current accepted model to explain the transformation events associated with the conversion of the non-secreting membrane to the secreting membrane, and vice versa, invokes membrane recycling of elongated vesicle structures. However, recent studies employing cryopreparation have shown that the non-secreting membrane in these cells is actually a complex network of helically coiled tubules. Here, we present an alternative model to explain how the membrane in parietal cells is activated to secrete HCl.

A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins.

The mechanism by which peripheral membrane proteins are targeted to the cytoplasmic face of the Golgi apparatus is poorly understood. Previously, we have identified a carboxy-terminal domain of the trans-Golgi-network (TGN) protein p230 that is responsible for Golgi localisation [1]. Here, we report the identification of a similar Golgi-localisation domain (GLD, also termed the 'GRIP' domain - see the paper by Munro and Nichols elsewhere in this issue) in a family of putative peripheral membrane proteins from lower and higher eucaryotes. The majority of family members have a domain structure similar to that of p230, with extensive coiled-coil regions (>80%) and the potential GLD located in a non-coiled-coil domain at the carboxyl terminus. Previously reported proteins in this family include human golgin-97 and Saccharomyces cerevisiae Imh1p. By constructing chimeric cDNAs encoding carboxy-terminal regions of these family members fused to green fluorescent protein (GFP), we have directly demonstrated that the GLD of p230, golgin-97, the newly identified human protein GCC1p and yeast Imh1p functions as a Golgi-targeting domain in transfected mammalian cells. Site-directed mutagenesis of the GLDs identified two conserved aromatic residues that are critical for the function of this targeting domain. Endogenous p230 was displaced from the Golgi membranes in transfected cells expressing high levels of GFP fused to the GLD of either p230 or golgin-97, indicating that different GLDs interact with similar membrane determinants. Thus, we have identified a family of coiled-coil proteins that share a domain shown to be sufficient for the localisation of peripheral membrane proteins to the Golgi apparatus.

N-linked oligosaccharides of the murine transferrin receptor from a plasmacytoma cell line. Comparison with total cellular N-glycans.

The N-linked oligosaccharides synthesised by the murine plasmacytoma cell line NS-1 have been analysed by lectin affinity chromatography on columns of immobilised concanavalin A (Con A), Lens culinaris (lentil), Ricinus communis agglutinin (RCA) and leuko-phytohemagglutinin (L-PHA). The majority of complex N-glycans in this transformed cell line were branched structures with only a low level of biantennary complex chains detected. The analysis showed the major complex N-glycan fraction consisted of a minimum sialylated triantennary structure. [3H]Mannose-labelled transferrin receptor was isolated from NS-1 cells by immunoprecipitation followed by electroelution from SDS polyacrylamide gels. The isolated receptor was digested with Pronase and the 3H-labelled glycopeptides analysed by lectin affinity chromatography. Analysis by Con A-Sepharose indicated that approx. 50% of the labelled glycopeptides were branched complex N-glycans (unbound fraction) while the remainder were oligomannose structures (strongly bound). The presence of tri and/or tetraantennary structures in the Con A unbound fraction was further suggested by the interaction of 61% of the fraction with L-PHA. The lectin profiles obtained for the complex N-glycans of the transferrin receptor glycopeptides were similar to those for the total cellular glycopeptides of NS-1 cells. Reverse-phase HPLC analysis of tryptic glycopeptides of the isolated [3H]mannose-labelled transferrin receptor gave three 3H-labelled peaks, indicating that all three potential N-glycosylation sites on the receptor are utilised. The Con A-Sepharose profiles of the three fractions indicated the presence of branched complex N-glycans and high mannose chains at each site. The profiles of two of the tryptic glycopeptide fractions were very similar, while the third had a higher content of oligomannose oligosaccharides.

Homing and adhesion molecules in autoimmune gastritis.

The pathogenesis of autoimmune gastritis is the result of lymphocyte infiltration of the gastric mucosa, however, the events leading to the selective extravasation of autoreactive lymphocytes are unclear. Here we have examined the expression of adhesion molecules in the gastric mucosa of BALB/c mice with neonatal thymectomy-induced gastritis. The overall area of vascular endothelium was not significantly different between gastritic and non-gastritic mice. However, a significant increase in the area of mucosal endothelium expressing MAdCAM-1 in gastritic mice was observed. Treatment of neonatally thymectomized BALB/c mice with a MAdCAM-1 specific monoclonal antibody (MECA 367) reduced the incidence of autoimmune gastritis from 80 to 26%. Treatment with a monoclonal antibody (R1-2) directed to the MAdCAM-1 ligand, alpha4beta7, also resulted in a reduction in the incidence of gastritis to 40%. These findings identify the alpha4beta7/MAdCAM-I interaction as a pivotal event in the initiation of autoimmune gastritis.

Trafficking and localisation of resident Golgi glycosylation enzymes.

The localisation of glycosylation enzymes within the Golgi apparatus is fundamental to the regulation of glycoprotein and glycolipid biosynthesis. Regions responsible for specifying Golgi localisation have been identified in numerous Golgi resident enzymes. The transmembrane domain of Golgi glycosyltransferases provides a dominant localisation signal and in many cases there are also major contributions from the lumenal domain. The mechanism by which these targeting domains function in maintaining an asymmetric distribution of Golgi resident glycosylation enzymes has been intensely debated in recent years. It is now clear that the targeting of Golgi resident enzymes is intimately associated with the organisation of Golgi membranes and the control of protein and lipid traffic in both anterograde and retrograde directions. Here we discuss the recent advances into how Golgi targeting signals of glycosylation enzymes function, and propose a model for maintaining the steady-state localisation of Golgi glycosyltransferases.

Cloning and characterization of a Golgi-associated GTP-binding protein homologue from Leishmania major.

This paper describes the cloning of a Golgi-associated GTP-binding protein homologue from Leishmania major. The gene was isolated using degenerate oligonucleotides to conserved sequences amongst the small GTP-binding proteins in a polymerase chain reaction on genomic DNA of the L. major cloned line V121. The reading frame of one clone showed high similarity to the rab/YPT subfamily of small GTP-binding proteins. A full length copy of the gene was isolated from a lambda gt10 V121 genomic library and sequenced. At the amino acid level the gene showed highest similarity to the human/rat rab1 A gene and the mouse/yeast YPT gene and was named LmYPT. The LmYPT gene was present as a single copy gene in both the L. major and L. donovani genomes. Karyotype analysis localized the LmYPT gene to chromosome band 18 in V121, but it was located on a larger chromosome in the different L. major isolate L119. The LmYPT gene was transcribed as a 3.9-kb transcript in both the promastigote and amastigote forms of the parasite. Western blot analysis, using a polyclonal rabbit antiserum raised against an Escherichia coli expressed portion of the molecule, identified a doublet at 20 and 23 kDa in total promastigote protein. Immunoelectron microscopy in combination with peroxidase staining localized the LmYPT molecule to the Leishmania Golgi apparatus.

Stable expression of the human immunodeficiency virus type 1 envelope glycoprotein in transfected L cells.

An SV40-based expression vector was used to generate CD4-negative murine L cell lines which stably expressed the human immunodeficiency virus envelope glycoprotein (env). Despite the presence of abundant intracellular envelope glycoprotein, the expression of env gp120/41 was not detected on the cell surface. Pulse-chase studies showed that the majority of the gp120 detected at the end of a 20-h chase was in the culture medium. Therefore gp120 was shed and/or secreted from these cells. Transfected L cells (H-2k) served as targets for specific lysis by CTL raised against vaccinia virus-encoded env gp160. The discrepancy in relative levels of intracellular versus surface expression of env was probably due to the highly inefficient processing of newly synthesized gp160, as well as the apparent instability of the gp120/41 complex in the transfected cell lines. Digestion of immunoprecipitated gp120 and gp160 with endoglycosidase H and peptide N-glycosidase F revealed that the envelope glycoprotein in transfected L cells possessed both high mannose and complex N-glycans, analogous to the posttranslational modification of the mature envelope glycoprotein in infected T cells. These studies indicate that the relatively inefficient processing of env gp160 occurs in the absence of CD4, and that the stable surface expression of envelope gp120/41 complex may require additional factors not present in transfected cells.

Autoimmune gastritis: tolerance and autoimmunity to the gastric H+/K+ ATPase (proton pump).

The alpha and beta subunits of the gastric H+/K(+)-ATPase (proton pump) have been identified as the major molecular targets of parietal cell autoantibodies associated with pernicious anaemia and with murine experimental autoimmune gastritis (EAG) induced by neonatal thymectomy. Recent studies with EAG suggest that the mechanisms of peripheral tolerance and autoimmunity to extrathymic autoantigens are mediated by subsets of "regulator" and "effector" CD4+ T cells, respectively. The persistence of "effector" CD4+ autoreactive T cells in the periphery may be a direct consequence of the delayed developmental expression of the target autoantigen. We hypothesize that cytokines produced by the "regulator" T cells prevent the clonal expansion of the "effector" autoreactive T cells, and that neonatal thymectomy induces organ-specific autoimmunity in genetically susceptible individuals by the reduction of the "regulator" T cell population.

Diagnostic ELISA for parietal cell autoantibody using tomato lectin-purified gastric H+/K(+)-ATPase (proton pump).

Circulating parietal cell autoantibodies, a useful diagnostic marker for autoimmune gastritis and pernicious anaemia, are currently routinely tested by serum immunofluorescence reactivity with frozen sections of rodent stomach. The major molecular targets of these parietal cell autoantibodies have recently been demonstrated to be the alpha- and the beta-subunits of the gastric H+/K(+)-ATPase (proton pump). We have demonstrated that tomato lectin binds specifically to the beta-subunit of the proton pump and concomittantly co-purifies the alpha-subunit. In the present study, we have exploited the latter observation for the development of a diagnostic ELISA for the detection of parietal cell autoantibodies and compared the performance of this assay with an ELISA using crude gastric membranes. The ELISAs were tested on 72 parietal cell autoantibody-positive sera, 72 parietal cell autoantibody-negative sera and 72 disease-control sera. The ELISA using lectin-purified canine proton pump was superior to that using crude canine gastric membranes in that it was about two-fold more sensitive (82% vs. 43%). With an assay sensitivity of 82% and a specificity of 90%, we propose that the ELISA using the lectin-purified proton pump is a rapid, simple, sensitive and specific diagnostic immunoassay for parietal cell autoantibodies.

A novel method for isolating mononuclear cells from the stomachs of mice with experimental autoimmune gastritis.

Autoimmune gastritis induced in BALB/c mice by neonatal thymectomy is a CD4+ T cell-mediated disease. The disease is characterised by mononuclear cell infiltrates in the gastric mucosa, loss of gastric parietal and chief cells and autoantibodies to the gastric H/K ATPase. Here we describe a simple non-enzymatic method for isolating cellular infiltrates from stomachs of gastric mice by injection of medium directly into stomach walls, causing swelling and rupture. Using this method, large numbers of viable lymphocytes were released from stomachs for analysis by flow cytometry. An 8.3 fold increase in the total number of lymphocytes from diseased stomachs compared to normal controls was observed. Total cell numbers of CD4+ and B cells were increased 4.8 fold and 39.5 fold respectively, in diseased stomachs compared with controls. No change was observed in the CD8+ T cell population. This method will allow detailed quantitative analysis of cellular infiltrates during the development of the gastric lesion and enrichment of pathogenic T cells for analysis and cloning. This procedure may have general application for the isolation of cellular infiltrates from lesion sites of other organs.

The role of natural killer cells in the induction of autoimmune gastritis.

A number of experimental models of organ-specific autoimmunity involve a period of peripheral T cell lymphopenia prior to disease onset. In particular, experimental autoimmune gastritis, induced in susceptible mouse strains by neonatal thymectomy, is a CD4+ T cell mediated autoimmune disease. We have previously demonstrated that this disease displays the hallmarks of a Th1-mediated DTH inflammatory response with an essential role for IFN-gamma very early in the pathogenesis of disease. Given the interplay between the innate and adaptive immune responses, a potential source of early IFN-gamma production in these lymphopenic mice is the innate immune response. Here we have assessed the contribution of innate immunity to the induction of experimental autoimmune gastritis, in particular, the role of natural killer (NK) cells in production of IFN-gamma. Analysis of NK cells and macrophages revealed no difference in either the number or activation status between euthymic and neonatally thymectomised mice. Furthermore, in vivo depletion of NK cells immediately after neonatal thymectomy of (BALB/cCrSlcxC57BL/6) F1 mice demonstrated no reduction in disease incidence compared to control groups of neonatally thymectomised mice. Therefore, we conclude that NK cells are not the primary source of IFN-gamma required for the pathogenesis of autoimmune gastritis following neonatal thymectomy but rather the small cohort of T cells in the periphery of lymphopenic mice are likely to be responsible for the IFN-gamma production.

Alpha and beta subunits of the gastric H+/K(+)-ATPase are concordantly targeted by parietal cell autoantibodies associated with autoimmune gastritis.

We have previously shown that parietal cell autoantibodies predominantly react with a 60-90 kDa gastric autoantigen, subsequently identified as the beta subunit of the gastric H+/K(+)-ATPase (EC 3.6.1.3) (proton pump) whereas Karlsson et al showed that these autoantibodies primarily target the 95 kDa alpha subunit of the pump. In view of these discordant results, we have reassessed the reactivity of parietal cell autoantibodies with the two subunits of the gastric H+/K(+)-ATPase. We show here that all 26 parietal cell autoantibody-positive sera immunoblot both subunits under appropriate, but mutually exclusive, conditions. Thus, reactivity of anti-parietal cell autoantibodies with the 95 kDa alpha subunit is optimal when the SDS-PAGE is carried out with samples which are reduced but not boiled. Whereas reactivity with the 60-90 kDa beta subunit is optimal with samples which are boiled but not reduced. Autoantibody reactivity with the beta subunit is critically dependent on the presence of a full complement of N-linked glycans since partially deglycosylated protein, and recombinant beta subunit expressed in COS cells, bearing high mannose N-glycans, failed to bind to the autoantibody. These studies also suggest that B cell auto-epitopes are located on the lumenal domain of the beta subunit. Reactivity of parietal cell autoantibodies with a bacterial fusion protein incorporating the catalytic cytoplasmic domain of the alpha subunit suggests the presence of auto-epitopes in this region of the molecule.

Expression of the gastric H/K-ATPase alpha-subunit in the thymus may explain the dominant role of the beta-subunit in the pathogenesis of autoimmune gastritis.

The two subunits of the gastric H/K ATPase, namely the catalytic alpha-subunit and the glycoprotein beta-subunit, are the major targets of parietal cell autoantibodies associated with human and murine autoimmune gastritis. The murine disease induced by neonatal thymectomy is T cell-mediated. We have previously shown that transgenic expression of the H/K ATPase beta-subunit gene in the thymus prevented the development of autoimmune gastritis induced by thymectomy. However, little is known of the contribution of the H/K ATPase alpha-subunit in disease development. Here, we show that (1) in contrast to the gastric H/K ATPase beta-subunit, the alpha-subunit gene is expressed in normal BALB/c thymus. (2) transgenic expression of the gastric H/K ATPase alpha-subunit gene in the thymus failed to prevent the development of autoimmune gastritis and (3) normal BALB/c and transgenic mice expressing the alpha-subunit in the thymus develop autoimmune gastritis following immunisation with purified murine gastric H/K ATPase, whereas transgenic mice expressing the beta-subunit in the thymus do not. We propose that the expression of the H/K ATPase alpha-subunit in the normal thymus may account for the predominant role of the beta-subunit in the development of autoimmune gastritis induced either by thymectomy or by immunisation with the ATPase.

Recycling endosome-dependent and -independent mechanisms for IL-10 secretion in LPS-activated macrophages.

IL-10 is a key anti-inflammatory cytokine secreted by activated macrophages as a feedback control mechanism to prevent excessive inflammatory responses. Here, we define multiple intracellular trafficking pathways involved in the secretion of newly synthesized IL-10 from macrophages following TLR4 activation with LPS, and show how this relates to the previously defined trafficking pathways for IL-6 and TNF in macrophages simultaneously producing these proinflammatory cytokines. IL-10 exits the Golgi in multiple tubular carriers, including those dependent on p230GRIP. Some of the IL-10 is then delivered to recycling endosomes, where cytokine sorting may occur prior to its release. Another portion of the IL-10 is delivered to the cell surface in distinct vesicles colabeled for apoE. Thus, we show at least two post-Golgi pathways via which IL-10 is trafficked, ensuring its secretion from activated macrophages under different physiological conditions.

Reciprocal changes in trefoil 1 and 2 expression in stomachs of mice with gastric unit hypertrophy and inflammation.

H+/K+-ATPase beta-subunit-deficient mice (129/Sv background) display numerous pathologies in the stomach. Expression of the mutation in BALB/cCrSlc mice results in the development of an aberrant 'mucus-rich' cell population. 'Mucus-rich' cells have been described in stomachs of mice with autoimmune gastritis, a disease mediated by CD4+ T cells. Other pathological features of autoimmune gastritis are similar to those in H+/K+ beta-deficient mice and include a mononuclear cell infiltrate in the gastric mucosa, non-functional or absent parietal cells, depletion of zymogenic cells, hypergastrinaemia, and gastric unit hypertrophy caused by immature cell hyperplasia. The present study investigates further the aberrant gastric 'mucus-rich' cell lineage and analyses the mRNA expression of mucus cell products TFF1 and TFF2. 'Mucus-rich' cells stained for both acidic and neutral mucins, and with a TFF2-specific antibody. Stomachs from both models expressed decreased TFF1 mRNA and reciprocally increased TFF2 mRNA. The involvement of gastrin in regulating trefoil mRNA expression was also investigated using gastrin-deficient mice. In contrast to previous findings, gastrin did not positively regulate TFF1 mRNA expression, but there was possible augmentation of TFF2. Additionally, a clear role for inflammation was established involving both polymorphonuclear and mononuclear cells in these models, and a link was found between mucosal hypertrophy and increased interleukin-11 (IL-11) expression.

Targeting of proteins to the Golgi apparatus.

The proteins that reside in the Golgi carry out functions associated with post-translational modifications, including glycosylation and proteolytic processing, membrane transport, recycling of endoplasmic reticulum proteins and maintenance of the structural organisation of the organelle itself. The latter includes Golgi stacking, interconnections between stacks and the microtubule-dependent positioning of the organelle within the cell. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network (TGN) proteins, peripheral membrane proteins and receptors. Considerable effort has been directed at understanding the basis of the localisation of Golgi glycosyltransferases and recycling TGN proteins; in both cases there is increasing evidence that multiple signals may be involved in their specific localisation. A number of models for the Golgi retention of glycosyltransferases have been proposed including oligomerisation, lipid-mediated sorting and intra-Golgi retrograde transport. More information is required to determine the contribution of each of these potential mechanisms in the targeting of different glycosyltransferases. Future work is also likely to focus on the relationship between the localisation of resident Golgi proteins and the maintenance of Golgi structure.

Control of glycoprotein synthesis.

Hen oviduct membranes have been shown to catalyze the transfer of GlcNAc from UDP-GlcNAc to GlcNAc-beta 1-2Man alpha 1-6(GlcNAc beta 1-2 Man alpha 1-3) Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X (GnGn) to form the triantennary structure GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. The enzyme has been named UDP-GlcNAc:GnGn (GlcNAc to Man alpha 1-3) beta 4-N-acetylglucosaminyltransferase IV (GlcNAc-transferase IV) to distinguish it from three other hen oviduct GlcNAc-transferases designated I, II, and III. Since GlcNAc-transferases III and IV both act on the same substrate, concanavalin A/Sepharose was used to separate the products of the two enzymes. At pH 7.0 and at a Triton X-100 concentration of 0.125% (v/v), GlcNAc-transferase IV activity in hen oviduct membranes is 7 nmol/mg of protein/h. The product was characterized by high resolution proton NMR spectroscopy at 360 MHz and by methylation analysis. In addition to triantennary oligosaccharide, hen oviduct membranes produced about 20% of bisected triantennary material, GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3] [GlcNAc beta 1-4]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. Maximal GlcNAc-transferase IV activity requires the presence of both terminal beta 1-2-linked GlcNAc residues in the substrate. Removal of the GlcNAc residue on the Man alpha 1-6 arm or of both GlcNAc residues reduces activity by at least 80%. A Gal beta 1-4GlcNAc disaccharide on the Man alpha 1-6 arm reduces activity by 68% while the presence of this disaccharide on the Man alpha 1-3 arm reduces activity to negligible levels. A similar substrate specificity was found for GlcNAc-transferase III, the enzyme which adds a bisecting GlcNAc in beta 1-4 linkage to the beta-linked Man residue. Since a bisecting GlcNAc was found to prevent GlcNAc-transferase IV action, the bisected triantennary material found in the incubation must have been formed by the sequential action of GlcNAc-transferase IV followed by GlcNAc-transferase III. Activities similar to GlcNAc-transferase IV were also detected in rat liver Golgi-rich membranes (0.4 nmol/mg/h) and pig thyroid microsomes (0.1 nmol/mg/h).