Identification of secretory component as an IgA receptor on rat hepatocytes

Secretory component (SC) was found to be synthesized by isolated rat hepatocytes. SC was detected by radioimmunoassay and cultured hepatocytes were found to synthesize 0.078 microgram SC/10(6) hepatocytes in a 48-h period. SC was also present on the surface of hepatocytes as detected by the specific binding of radiolabeled anti-SC antibodies as well as by the detection of specific membrane staining in indirect immunofluorescence tests using specifically purified anti-SC antibodies. Rat SC was detected on hepatocytes and intestinal epithelial cells but not on peripheral blood lymphocytes, unfractionated spleen cells, or erythrocytes. Specific binding of radiolabeled rat dimeric IgA to rat hepatocytes was also observed and evidence was obtained to indicate that such binding was mediated by SC. Thus, prior incubation of hepatocytes with anti-SC prevented binding of radiolabeled IgA. Moreover, prior incubation of radiolabeled IgA with rat SC prevented binding of the IgA to isolated hepatocytes. Cells treated with 0.25% trypsin lost their ability to bind to radiolabeled dimeric IgA.     

Secretory IgA mediates retrotranscytosis of intact gliadin peptides via the transferrin receptor in celiac disease

Celiac disease (CD) is an enteropathy resulting from an abnormal immune response to gluten-derived peptides in genetically susceptible individuals. This immune response is initiated by intestinal transport of intact peptide 31-49 (p31-49) and 33-mer gliadin peptides through an unknown mechanism. We show that the transferrin receptor CD71 is responsible for apical to basal retrotranscytosis of gliadin peptides, a process during which p31-49 and 33-mer peptides are protected from degradation. In patients with active CD, CD71 is overexpressed in the intestinal epithelium and colocalizes with immunoglobulin (Ig) A. Intestinal transport of intact p31-49 and 33-mer peptides was blocked by polymeric and secretory IgA (SIgA) and by soluble CD71 receptors, pointing to a role of SIgA–gliadin complexes in this abnormal intestinal transport. This retrotranscytosis of SIgA–gliadin complexes may promote the entry of harmful gliadin peptides into the intestinal mucosa, thereby triggering an immune response and perpetuating intestinal inflammation. Our findings strongly implicate CD71 in the pathogenesis of CD.Celiac disease (CD) is an inflammatory enteropathy induced by gluten-derived prolamines in genetically susceptible individuals. CD affects about 1 in 100 individuals in Europe and the United States (1, 2). The associated intestinal inflammation results from synergism between innate and adaptive immune responses to gliadin peptides. The adaptive immune response is orchestrated by CD4⁺ T cells recognizing various deamidated gliadin peptides (3), including a 33-mer (peptide 56-88 [p56-88]) (4), bound to HLA-DQ2/8 molecules (5). p56-88 is a powerful immunodominant gliadin peptide extremely resistant to gastrointestinal digestion and has far higher T cell stimulatory potency than its 12-mer counterparts (6). The innate immune response in CD is triggered by a distinct set of gliadin peptides: one prototype innate peptide is p31-49, common to the N terminus of A-gliadins and shown to be toxic for CD patients both in vitro and in vivo (7–9). This peptide was recently shown to stimulate the synthesis of IL-15 (10, 11), a proinflammatory cytokine that can promote the CD4⁺ adaptive immune response (11) and activate cytotoxic activity and IFN-γ production in intraepithelial lymphocytes (12–14).This activation of the local immune system implies that undigested gliadin fragments present in the intestinal lumen somehow cross the intestinal epithelium. Indeed, apical to basal transport and processing of gliadin peptides, particularly of p31-49 and 33-mer, are severely altered in active CD, leading to the release of intact peptides on the basal side, whereas the same peptides are almost entirely degraded during their intestinal transport in control individuals and treated CD patients (15, 16). Several lines of evidence argue against simple nonspecific paracellular leakage of gliadin peptides across the celiac mucosa. In particular, a 12-mer gliadin peptide, p57-68, is completely degraded during intestinal transport in patients with active CD, suggesting no paracellular leakage of molecules of this size or larger. In addition, only 0.3% of the apical peptide crosses the epithelium during a 3-h incubation period, arguing against free diffusion across a damaged mucosa. The “protected” transport of p31-49 thus involves a transcellular pathway (15, 16), which enables the peptide to escape lysosomal degradation. As active CD is associated with high concentrations of antigliadin IgA antibodies in the intestinal lumen (17, 18), we postulated that the transport of intact gliadin peptides might result from abnormal retrotranscytosis of IgA–gliadin complexes. Indeed, although antigliadin IgA antibodies are a hallmark of CD, they have no known pathogenic role. We obtained evidence that polymeric/secretory IgA (pIgA/SIgA) can mediate protected transport of p31-49 and 33-mer gliadin peptides through their binding to CD71, the transferrin (Tf) receptor. Importantly, we found that this receptor was abnormally expressed at the apical pole of enterocytes in patients with active CD. Although initially implicated in endocytosis of iron-loaded Tf, CD71 was recently recognized as an IgA receptor mediating mesangial deposition of IgA1 complexes in IgA nephropathy (19).     

Secretory IgA enzyme immunoassay. Application of a model for computation of the standard curve

An enzyme-linked immunosorbent assay (ELISA) is applied for quantitation of secretory IgA (SIgA) in nasopharyngeal secretions (NPS) and saliva. In principle, both the alpha and secretory component determinants of SIgA are utilized in a two-site sandwich technique. Dilutions of a SIgA solution prepared from colostrum (Col-SIgA) are used for standards. A continuous SIgA standard curve, describing the relationship between the absorbance of the coloured end product and the corresponding Col-SIgA dilution, is computed by means of a fitting procedure based on a model. The variation (SD) of the absorbance was found to be proportional to the absorbance level. Knowing the SD of the absorbance, the total variation of the SIgA estimates could be calculated experimentally by means of Monte Carlo methods. Based on these results, the analytical range was chosen from 2000 micrograms/l to 50 micrograms/l of SIgA. Inhibition experiments revealed that plasma IgA could be added to the NPS in equal amounts to the SIgA content without it interfering with the SIgA measurements. Percentage recovery (median) of known amounts of SIgA added to NPS was 96%. In NPS from children suffering from recurrent upper respiratory tract infections the median SIgA level was 1.44 g/l (interquartile range 0.61-2.43 g/l); in saliva from healthy children the median SIgA content was 77 mg/l (interquartile range 56-182 mg/l).     

Sensitive solid phase enzyme immunoassay for human IgA,secretory IgA,and secretory component

Highly sensitive solid-phase immunoassay systems for human immunoglobulin A (IgA), secretory component (SC), secretory immunoglobulin A (SIgA) were developed by use of antisera against the α-chain of IgA and SC, and β-d-galactosidase from Escherichia coli as label. IgA and SC were assayed with the respective solid-phase (silicone rubber-immobilized F(ab')₂ antibody fragments and the corresponding antibody Fab'-β-d-galactosidase complex. More than 1 and 0.4 fmol (or 0.16 and 0.03 ng) of IgA, and SC, respectively, were determined, but the assay system for IgA and that for SC cross-reacted with SIgA about 90% and 2%, respectively. SIgA was specifically determined in an assay using silicone rubber with immobilized (anti-α-chain)F(ab')₂ fragments and (anti-SC) Fab'-β-d-galactosidase complex with a minimum detectable sensitivity of 2.5 fmol or 1 ng. IgA and SC values could be corrected by subtracting the amounts of cross-reacting SIgA in the same samples. Small amounts of SIgA, SC, and IgA in saliva, sweat, urine, and feces could be determined by the present method.     

pH-dependent receptor/ligand dissociation as a determining factor for intracellular sorting of ligands for epidermal growth factor receptors in rat hepatocytes.

Both epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) bind to EGF receptors and TGF alpha has been reported to be more potent than EGF as far as many biological effects are concerned. One possible reason for this is thought to be the difference in their dissociation from the receptors in intracellular acidic compartments, which may affect the final pathway (lysosomal degradation or recycling to cell-surface) of endocytosed ligands. This study was aimed at clarifying the relationship between intracellular dissociation from the receptors and the fate of the endocytosed ligands. First, (125)I-human EGF (hEGF), mouse EGF (mEGF), or human TGF alpha (hTGF alpha) was prebound to cell-surface receptors or intracellularly preloaded, followed by further incubation at 37 degrees C in primary cultured rat hepatocytes. In these experiments, the magnitude of the dissociation rate constant (k(off)) of each ligand at pH 6.0, which is similar to that inside early endosomes, was found to be in the following order: hTGF alpha>mEGF>hEGF. The recycled portion of endocytosed ligands was also in the order: hTGF alpha>mEGF>hEGF. Digitonin treatment of preloaded cells revealed that the intracellular dissociation of hTGF alpha was more rapid than that of hEGF. Moreover, several histidine-inserted or -deleted hEGF mutants were prebound to rat liver sinusoidal membrane vesicle, followed by further incubation at 37 degrees C. The dissociation rate of histidine-deleted hEGF mutants was less rapid than that of hEGF itself. These results suggested that efficient dissociation in the earlier intracellular compartment leads the endocytosed ligands to be recycled to the cell surface whereas late dissociation results in intracellular degradation in hepatocytes. Thus, one possible strategy to improve their stability in the circulation may be a change in intracellular ligand/receptor dissociation with a minimal effect on the affinity for receptors on the cell-surface and histidine residues may partly contribute to the pH-sensitive dissociation.     

Isolated rat hepatocytes as a tool for the determination of antibodies against human liver cell membrane

Antibodies against liver cell membrane are measured by counting the percentages of fluorescent rat hepatocytes ( FRH ), obtained by an indirect immunofluorescence method after incubation of isolated rat hepatocytes with sera of patients with chronic liver diseases. A close relationship exists between the percentages of FRH and the serological and histological parameters of diseases activity, there was no difference between HBsAg-positive or -negative sera.     

Fibronectin is synthesized as an acute phase reactant in rat hepatocytes

Elevated concentrations of fibronectin were found in plasma of rats under different acute phase conditions. Untreated animals showed a plasma fibronectin concentration of 150 +/- 50 mg/l, which increased to 412 +/- 59 mg/l 24 h after subcutaneous injection of turpentine. The time course of the changes in plasma fibronectin concentration showed a peak at 24 h and a decline to normal concentrations 72 h after turpentine treatment. Additional stimulation by dexamethasone resulted in plasma fibronectin concentrations of 661 +/- 49 mg/l. No or only slight elevations of fibronectin concentrations were observed after treatment with adrenaline, thyroxine and triiodothyronine as compared with saline-injected animals. The common identity of plasma fibronectin in controls, turpentine and turpentine-dexamethasone-treated animals was shown by slab gel electrophoresis under nonreducing conditions, followed by western blot and immunofluorescence staining. One dimensional immunoelectrophoresis performed with polyclonal antibodies to human fibronectin cross-reacting with rat fibronectin (shown by Ouchterlony gel diffusion) revealed identical precipitation lines for the plasma of control and acute phase animals. Hepatocytes of turpentine-pretreated rats show a threefold increase of [14C]valine incorporation into total protein and a fourfold increase of immunoreactive radioactively labeled fibronectin in the culture medium, compared with control hepatocyte cultures. These results point to the role of hepatocytes in the synthesis of plasma fibronectin, which behaves in rats as an acute phase reactant.     

Expression cloning of a human Fc receptor for IgA

IgA, the predominant isotype in secretions, mediates the neutralization and removal of environmental antigens from mucosal sites. Although cell surface receptors for the Fc region of IgA (Fc alpha R) have been implicated in a variety of immune effector mechanisms, the molecular features of Fc alpha R remain only marginally characterized. In this report, we describe the isolation of a clone from a myeloid cell line cDNA library that directs the expression of a cell surface molecule with IgA binding specificity. The cDNA encodes a peptide of Mr 30,000 including a putative transmembrane region with features atypical of conventional membrane-anchored proteins. Databank searches indicate that the human myeloid cell Fc alpha R sequence is unique, is a member of the immunoglobulin gene superfamily, and is related to Fc receptors for IgG (Fc gamma RI, II, and III) and IgE (Fc epsilon RI).     

Antigenic analysis of the IgA component of LDH-IgA immunoglobulin complexes.

LDH-IgA complexes present in some human sera were purified by using affinity chromatography on 5'-AMP-Sepharose 4-B. The IgA component of the purified complexes was analysed in haemagglutination-inhibition tests. Antigenic determinants specific for alpha1 and alpha2 heavy chains and for kappa and lambda light chains were found. Earlier studies suggested that the IgA component is of kappa light chain type only. These different results are discussed. It is suggested that the LDH association site is located in the Fab fragment of the IgA component.     

The presence of IgA on the surface of rat thoractic duct lymphocytes which contain internal IgA

The presence of lymphocytes with internal IgA among cells from rat thoracic duct lymph wdy. The number of cells detected was greater in animals kept in a convential animal house compared with those maintained under specific pathogen-free conditions. Thoracic duct lymph from B rats and adult thymectomized rats also contained cell with internal IgA. The surface Ig of the IgA-containing cells was studied using a double-labeling technique with (126I) anti-Ig to detect surface Ig, and fluorescein-conjugated anti-IgA in large amounts, but very little IgM and no surface IgG2. The surface IgA was not acquired passively.     

AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers

In liver cirrhosis, abnormal liver architecture impairs efficient transduction of hepatocytes with large viral vectors such as adenoviruses. Here we evaluated the ability of adeno-associated virus (AAV) vectors, small viral vectors, to transduce normal and cirrhotic rat livers. Using AAV serotype-1 (AAV1) encoding luciferase (AAV1Luc) we analyzed luciferase expression with a CCD camera. AAV1Luc was injected through the hepatic artery (intra-arterial (IA)), the portal vein (intra-portal (IP)), directly into the liver (intra-hepatic (IH)) or infused into the biliary tree (intra-biliar). We found that AAV1Luc allows long-term and constant luciferase expression in rat livers. Interestingly, IP administration leads to higher expression levels in healthy than in cirrhotic livers, whereas the opposite occurs when using IA injection. IH administration leads to similar transgene expression in cirrhotic and healthy rats, whereas intra-biliar infusion is the least effective route. After 70% partial hepatectomy, luciferase expression decreased in the regenerating liver, suggesting lack of efficient integration of AAV1 DNA into the host genome. AAV1Luc transduced mainly the liver but also the testes and spleen. Within the liver, transgene expression was found mainly in hepatocytes. Using a liver-specific promoter, transgene expression was detected in hepatocytes but not in other organs. Our results indicate that AAVs are convenient vectors for the treatment of liver cirrhosis.     

Identification of the transferrin receptor as a novel immunoglobulin (Ig)A1 receptor and its enhanced expression on mesangial cells in IgA nephropathy

The biological functions of immunoglobulin (Ig)A antibodies depend primarily on their interaction with cell surface receptors. Four IgA receptors are presently characterized. The FcalphaRI (CD89) expressed by myeloid cells selectively binds IgA1 and IgA2 antibodies, whereas the poly-IgR, Fcalpha/muR, and asialoglycoprotein receptors bind other ligands in addition to IgA. IgA binding by mesangial cells, epithelial cells, and proliferating lymphocytes is also well documented, but the nature of the IgA receptors on these cells remains elusive. A monoclonal antibody (A24) is described here that specifically blocks IgA binding to epithelial and B lymphocyte cell lines. Both the A24 antibody and IgA1 myelomas bind a cell surface protein that is identified as the transferrin receptor (CD71). The transferrin receptor selectively binds IgA1 antibodies, monomeric better than polymeric forms, and the IgA1 binding is inhibitable by transferrin. Transferrin receptor expression is upregulated on cultured mesangial cells as well as on glomerular mesangial cells in patients with IgA nephropathy. The characterization of transferrin receptor as a novel IgA1 receptor on renal mesangial cells suggests its potential involvement in the pathogenesis of IgA nephropathy.     

Effect of native and NH3 plasma-functionalized polymeric membranes on the gene expression profiles of primary hepatocytes

Little is known about how cells respond to different biomaterials at the molecular level. Biomaterials could stimulate specific cellular responses at the molecular level, such as activation of signalling pathways that control gene activity involved in the maintenance, growth and functional regeneration of liver tissue in vitro. This aspect is an important step in liver tissue engineering. Currently, there are no data available concerning the modulation of cellular genomic response by using synthetic membranes in a bioartificial system. For the first time we investigated gene expression profiles of primary hepatocytes cultured on different substrates: collagen sandwich, native and NH(3) plasma-grafted PEEK-WC-PU membranes. Gene expression in cell suspension prepared after cell isolation was used as a control. Generally, microarray data revealed that the expression of the majority of genes remained unchanged compared to the control. Among 31 000 genes, 52 were significantly changed: 20 were upregulated and 32 downregulated. There were similar changes in gene expression of hepatocytes cultured in the membranes and collagen sandwich. However, some genes involved in the cell proliferation and functional metabolic pathways are more expressed in cells cultured on the membranes and especially on the functionalized ones. Both membranes sustained liver functions at the molecular level, demonstrating their suitability for the reconstruction of liver and as a toxicogenomic tool to predict the liver response to novel drugs.     

A surface receptor specific for human IgA on group B streptococci possessing the Ibc protein antigen

A number of group B streptococcal strains of various serotypes, Ia, Ib, Ic, II, and III were examined for their ability to bind human IgG and IgA. No strains of group B streptococci were found to bind IgG, but many strains possessing the Ibc protein antigen(s) were found to bind a significant amount of IgA. The extent of IgA binding correlated with the amount of a 130,000 mol wt, detergent-extractable protein, and reactivity with the Ic typing sera. Using nitrocellulose blots, it was found that the 130,000 mol wt protein bound human IgA. A method was developed to purify the protein while retaining its ability to bind human IgA. Using solid phase radioimmunoassays, it was determined that the protein bound to the Fc region of monomeric or polymeric IgA and that it failed to bind IgM or any IgG isotype.     

PKH26标记原代大鼠肝细胞的实验研究

目的建立一种简单有效的肝细胞标记方法。方法原代分离的成年大鼠肝细胞,按PHK26标记程序进行标记。标记的肝细胞与胶原凝胶复合后植入体内观察植入细胞的成活情况。采用荧光显微镜对标记的细胞进行观察。结果肝细胞经PKH26染料标记后,荧光显微镜下可见红色荧光均匀的分布在整个细胞膜上。皮下植入后1周,植入的肝细胞成活,可通过标记的荧光直接探测到。连续切片显示这些细胞在成三维立体生长,形成类肝样的组织。结论用PHK26对肝细胞进行荧光标记提供了一种追踪细胞转归的方法。     

A receptor for the third component of complement in the human renal glomerulus

In the course of studying the nature of mononuclear cellular infiltrates in tissue sections of human kidney it was noted that indicator sheep erythrocytes densely coated with the third component of complement (C3) specifically adhered to all of the glomeruli in the tissue sections. The deposition of complement (C) within the glomerulus is a feature of many immunologically related renal diseases (1,2), yet the precise mechanism by which C is deposited remains unexplained. We feel that this observation, suggesting the presence of a receptor for C, is, therefore, of particular interest.     

An improved method for observing the cytoskeleton around the bile canaliculi of rat hepatocytes

In order to know more about the cytoskeletal structure around the bile canaliculi of hepatocytes, we used the following procedure. Liver specimens were passed through an 18-, a 21- and then a 23-gauge needle, by which they turned into cylindrical or oval blocks of 0.4 mm in diameter. Because of their small size, the cytosolic ground substances of every hepatocyte were washed out clearly after subsequent treatment with the solution containing 0.5% Triton X-100 and 0.5 mg/ml saponin for 15 min. This short-term extraction preserved both the cytoskeleton and the plasma membrane of the hypatocyte quite well, and we could observe the crossbridge filaments connecting between a core bundle of microfilaments and plasma membrane of the microvilli of the bile canaliculi.     

Oligomeric IgA: the major component of the in vitro primary response of mouse spleen fragments

The primary antibody response elicited from mouse spleen explants by conjugates of the 3-nitro-5-iodo-4-hydroxyphenylacetic acid (NIP) hapten consisted mostly of the IgA class. Poly-L-lysine, pneumococcal polysaccharide Type SIII, keyhole limpet hemocyanin, and sheep erythrocytes were effective carriers in this system, whereas chicken globulin was not. The anti-NIP response against all of the immunogenic conjugates was detectable in culture media 4 days after explantation and immunization, and reached peak titers by 8–10 days. IgA was identified by sucrose gradient velocity centrifugation in conjunction with the use of a class-specific antiserum. The media collected at 4 days contained low titers of IgM antibody, whereas the peak response at 8 days consisted almost entirely of IgA. The primary response IgA secreted by the spleen fragments was characterized as polymeric by its sedimentation rate through a sucrose gradient, and as polyvalent by its drastically greater avidity for NIP₁₄BSA than for free NIP-aminocaproic acid. Its haptenated phage-inactivating activity was abolished by treatment with 0.1 M 2-mercaptoethanol. These experiments indicate that precursor cells existing in the spleen before primary immunization can give rise to production of polymeric IgA.