Biomedical subjects
Publications and source records attributed to C S Kaetzel.
The polymeric immunoglobulin receptor (pIgR) is important in host defense, transporting antibodies across mucosal epithelial cells. Recent work has shown that, using a protein that binds directly to the pIgR, Streptococcus pneumoniae can co-opt the transcytosis machinery and gain entry into airway epithelial cells.
The fourth exon of the mouse polymeric immuno-globulin receptor (pIgR) is 654 nt long and, despite being surrounded by large introns, is constitutively spliced into the mRNA. Deletion of an 84 nt sequence from this exon strongly activated both cryptic 5' and 3' splice sites surrounding a 78 nt cryptic intron. The 84 nt deletion is just upstream of the cryptic 3' splice site; the cryptic 3' splice site was likely activated because the deletion created a better 3' splice site. However, the cryptic 5' splice site was also required to activate the cryptic splice reaction; point mutations in either of the cryptic splice sites that decreased their match to the consensus splice site sequence inactivated the cryptic splice reaction. The activation and inactivation of these cryptic splice sites as a pair suggests that they are being co-recognized by the splicing machinery. Interestingly, the large fourth exon of the pIgR gene encodes two immunoglobulin-like extracellular protein domains; the cryptic 3' splice site coincides with the junction between these protein domains. The cryptic 5' splice site is located between protein subdomains where an intron is found in another gene of the immunoglobulin superfamily.
The polymeric IgR (pIgR) mediates transcytosis of IgA across epithelial barriers of mucous membranes and exocrine glands. Synthesis of pIgR is up-regulated by the proinflammatory cytokines TNF-alpha, IFN-gamma, and IL-1 in HT-29 human colon carcinoma cells. We previously reported that IFN-gamma and TNF-alpha induce production of the transcription factor IFN regulatory factor-1 (IRF-1) in HT-29 cells and that IRF-1 binds to an element in exon 1 of the PIGR gene. We now report that levels of IRF-1 and pIgR mRNA are coordinately regulated in HT-29 cells by TNF-alpha, IFN-gamma, and IL-1beta. Furthermore, we demonstrate that in vivo expression of pIgR mRNA is greatly depressed in the intestine and liver of IRF-1-deficient mice. Our findings indicate a major role for the IRF-1 transcription factor in regulation of the PIGR gene and suggest a model for regulation of important genes in the mucosal immune system by proinflammatory cytokines.
To emphasize the requirement for a J chain in native polymeric immunoglobulins for their selective transport into exocrine secretions, IgG, purified from two different antisera specific for the human J chain, was shown to: (i) bind in vitro to human polymeric IgA (pIgA) by density gradient ultracentrifugation; (ii) inhibit binding in vitro of rat secretory component to human pIgA; (iii) inhibit hepatic transport of human pIgA into rat bile in vivo; and (iv) inhibit apical transcytosis of pIgA in vitro by polarized human polymeric immunoglobulin receptor (pIgR)-expressing Madin-Darby canine kidney cells. Inhibition of biliary transport increased with the molar ratio of anti-J chain antibodies against pIgA and their incubation time. Anti-J chain F(ab')2 and Fab fragments also inhibited biliary transport, excluding a role for phagocytic clearance or excessive size of the immune complexes. Anti-human-Fc alpha Fab, bound to human pIgA in complexes of larger size than those with anti-J chain Fab, did not inhibit biliary transport of human pIgA. Propionic acid-denatured human pIgA, although containing J chains, was very poorly transported into rat bile. Altogether, our data strongly support, now also by in vivo experiments, the crucial role of the J chain of native pIgA in its selective pIgR-mediated transport into secretions, as suggested long ago by in vitro data only. Recent data on J chain-knockout mice, with low IgA levels in bile and feces, cannot explain the role of the J chain in contributing to the secretory component/pIgR-binding site of normal pIgA, but otherwise agree with our study.
IgA is transported into external secretions by the polymeric Ig receptor (pIgR). Interferon-gamma (IFN-gamma), a major regulator of pIgR expression, has been shown to increase pIgR mRNA levels in HT-29 human colon carcinoma cells. To determine the molecular mechanisms of pIgR regulation, genomic DNA containing the 5'-flanking region of the human pIgR gene was isolated and a single start site of transcription in human intestinal epithelial cells was identified. Using chimeric reporter plasmids containing flanking regions of the pIgR gene, a segment of the pIgR promoter which is necessary and sufficient for induction of transcription by IFN-gamma in HT-29 cells was identified. Significantly, the pIgR promoter contains three motifs homologous to the interferon-stimulated response element (ISRE), two in the 5'-flanking region and one in exon 1 of the pIgR gene. The upstream ISREs bind nuclear protein(s) which are constitutively expressed by HT-29 cells, while the exon 1 ISRE binds interferon regulatory factor-1 (IRF-1), following stimulation with IFN-gamma. Furthermore, induction of the IRF-1 promoter by IFN-gamma correlates with induction of the pIgR promoter by IFN-gamma. It has previously been demonstrated that induction of pIgR mRNA by IFN-gamma, requires de novo protein synthesis. It is now shown that IRF-1 is not detected in nuclear extracts from HT-29 cells stimulated with IFN-gamma in the presence of cycloheximide, suggesting that de novo synthesis of IRF-1 is required for induction of pIgR transcription by IFN-gamma.
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Epithelial transport of polymeric IgA (pIgA) from its site of synthesis to the mucosal lumen is mediated by the polymeric Ig receptor (pIgR). During transcytosis, a disulfide bond forms between pIgR and pIgA, resulting in secretion of a covalently linked complex. To dissect further the intracellular processing and functions of pIgR, we have expressed the entire coding sequence of human pIgR cDNA in Madin-Darby canine kidney (MDCK) cells. Cloned transfected cells express human pIgR, as detected by immunofluorescence and by quantification of the cleaved extracellular domain of pIgR in culture supernatants. The function of transfected pIgR was confirmed by measuring vectorial transcytosis of 125I-labeled pIgA and its disulfide bonding to pIgR. Species specificity of transcytosis was determined by comparing transport of human, rat, and mouse pIgA in MDCK cells expressing either human or rabbit pIgR. pIgA from all three species was transported by both human and rabbit pIgR, with rat pIgA being transported to the greatest extent in each case. However, disulfide bonding was observed only with human pIgR, and was found to occur mainly inside the cell. Our results suggest that conformational differences between human and rabbit pIgR may account for differences in disulfide bonding to pIgA, and show that efficient transcytosis of pIgA is correlated better with noncovalent than covalent binding to pIgR.
Transcytosis of polymeric Ig (pIg) by mucosal epithelial cells is mediated by the polymeric Ig receptor (pIgR). Here we describe the characterization of a 3095-bp mouse pIgR cDNA, which encodes a protein of 771 amino acids. Northern blot analysis detected a single mouse pIgR transcript of 3.9 kb, expressed at high levels in small intestine and liver, and at low levels in lung. Alignment of the amino acid sequences of mouse, rat, human, bovine, and rabbit pIgR revealed that functional regions of the molecule are conserved across species. In the extracellular region, conserved motifs include: a 23-amino acid pIg-binding site, 11 intradomain disulfide bonds, consensus sites for N-glycosylation, and a putative cleavage site at which the extracellular region of pIgR (secretory component) is released from the plasma membrane. A 10-amino acid sequence within the transmembrane region is highly conserved, possibly reflecting a mechanism for transmitting signals from the extracellular region to the cytoplasmic tail. Conservation within the cytoplasmic tail of pIgR is clustered in motifs that mediate polarized sorting, endocytosis, and transcytosis.
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The traditional role of IgA antibodies in mucosal defense has been considered as providing an immune barrier to keep exogenous substances, including microbial pathogens, from penetrating the mucosa. In this way infections can be prevented. More recently, studies in vitro and in vivo are providing evidence to suggest that IgA may have additional roles in mucosal defense. For example, during their passage through the lining epithelial cells of mucous membranes en route to the secretions, IgA antibodies may have an opportunity to neutralize intracellular pathogens like viruses. Also, IgA antibodies in the mucosal lamina propria have opportunities to complex with antigens and excrete them through the adjacent mucosal epithelium, again by the same route to the secretions that is taken by free IgA. These latter functions could aid in recovery from infection.
Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.
The polymeric IgR (pIgR) mediates transcytosis of polymeric IgA across mucosal epithelia. Expression of this receptor in HT-29.74 human colon carcinoma cells is up-regulated by the recombinant cytokines IFN-gamma, TNF-alpha, and IL-4. Here, we demonstrate that activation of freshly isolated human intestinal lamina propria mononuclear cells (LPMC) induces production of natural cytokines, and these act synergistically as potent stimulators of pIgR expression in HT-29.74 cells. LPMC from normal colonic mucosa were stimulated with PMA and calcium ionophore A-23187. The resulting supernatants consistently induced dose-dependent increases in pIgR expression by HT-29.74 cells, up to 65-fold. Analysis of four separate LPMC supernatants revealed mean concentrations of 8260 pg/ml for IFN-gamma, 420 pg/ml for TNF-alpha, and 15 pg/ml for IL-4. Ab-mediated neutralization of these cytokines suggested that the central regulator of pIgR expression in these supernatants was IFN-gamma. IL-4 neutralization had no effect on induction and TNF-alpha neutralization slightly reduced induction. In contrast, IFN-gamma neutralization abolished up to 93% of pIgR induction and had essentially the same effect as simultaneous neutralization of all three cytokines. In conclusion, our data demonstrate that natural cytokines, predominantly IFN-gamma, produced by stimulated human intestinal lymphocytes and macrophages have the capacity to up-regulate dramatically pIgR expression in an intestinal epithelial cell line, strongly suggesting that their action in vivo leads to enhancement of local defense functions mediated by IgA.
We recently demonstrated that the polymeric IgR (pIgR) mediates epithelial transcytosis of immune complexes (IC) containing dimeric IgA (dIgA). In vivo, this "excretory" pathway could allow direct elimination of IgA IC at the mucosal sites where they are likely to form. In contrast, IC containing only monomeric IgA (mIgA) were not transported, consistent with the specificity of pIgR for polymeric IgA. However, the potential exists in vivo that monomeric Ig like mIgA or IgG could become associated through binding to multivalent Ag with IC containing dIgA, and that such mixed IC could act as ligands for pIgR. In the present work, using Madin-Darby canine kidney epithelial cells that express pIgR, we showed that 125I-labeled anti-DNP mIgA or IgG in the same IC with unlabeled dIgA antibody and DNP-BSA Ag was vectorially transported from the basolateral to the apical surface and then released. However, 125I-mIgA IC or 125I-IgG IC (without dIgA) and 125I-mIgA or 125I-IgG in the presence of dIgA antibody (but without Ag) were not transported, demonstrating the necessity for monomeric Ig to be in an IC with dIgA to be transported. Transcytosis of mixed mIgA/dIgA or IgG/dIgA IC was mediated by pIgR because no transport was observed in untransfected, wild-type Madin-Darby canine kidney cells lacking pIgR. The data demonstrate that mIgA and IgG can participate along with dIgA in the "excretory" pathway for local elimination of IgA IC, thus providing a means by which monomeric antibodies have the potential to participate in the mucosal immune system.
The polymeric immunoglobulin receptor on rat hepatocytes binds dimeric IgA on the sinusoidal surface and mediates its transport to the canaliculus, where the complex of dimeric IgA and secretory component, the cleaved extracellular domain of polymeric immunoglobulin receptor, is secreted into bile. This process is unique in that disulfide bonds are formed between dimeric IgA and polymeric immunoglobulin receptor during transcytosis, permanently preventing their dissociation. Here we present three lines of evidence that disulfide bonding between dimeric IgA and polymeric immunoglobulin receptor occurs predominantly in a late transcytotic compartment and that hepatic transcytosis can proceed in the absence of disulfide bond formation. First, throughout the course of transcytosis the percentage of intracellular dimeric IgA disulfide bonded to polymeric immunoglobulin receptor is less than half that in bile, suggesting that disulfide bond formation is a late event in transcytosis. Second, dimeric IgA that recycles from early endocytotic compartments into the circulation is mostly noncovalently bound to secretory component. Finally, the rate of transcytosis of dimeric IgA and its appearance in bile are not affected when disulfide bond formation with polymeric immunoglobulin receptor is inhibited by blocking of free thiol groups on dimeric IgA with iodoacetamide. These results are consistent with other findings in the literature and indicate that the main physiological role of disulfide bond formation between dimeric IgA and polymeric immunoglobulin receptor is not to facilitate transcytosis but, rather, to stabilize the dimeric IgA-secretory component complex after its release into external secretions such as bile and intestinal secretions.
Platelet-derived growth factor-AA and -BB homodimers and -AB heterodimers bind with high affinity to the platelet-derived growth factor (PDGF) alpha-receptor. Basic polypeptides such as polylysine and protamine sulfate compete with PDGF for receptor binding, suggesting a role for ligand positive charge in the binding interaction. A pentapeptide amino acid sequence with a cationic tripeptide core is perfectly conserved between the A- and B-chains (Val158-Arg159-Lys160-Lys161-Pro162) and was therefore considered as a possible alpha-receptor-binding domain. We have investigated the functional importance of positive charge within this region of the PDGF A-chain by using site-directed mutagenesis to convert the cationic core amino acids to the acidic sequence triglutamic acid. cDNAs encoding wild-type (PDGF-AAwt) and charge mutant (PDGF-AAcm) proteins were expressed following stable transfection of Chinese hamster ovary cells. Proper assembly and secretion of PDGF-AAcm was verified by metabolic labeling with [35S]cysteine, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis analysis under nonreducing and reducing conditions. PDGF-AAcm was secreted as two major species of disulfide-linked A-chain homodimers identical in molecular mass to those observed for PDGF-AAwt (32 and 35 kDa). Secreted PDGF-AAwt and PDGF-AAcm proteins were purified to homogeneity and subjected to structural and functional analyses. Compared to purified PDGF-AAwt, PDGF-AAcm displayed a marked reduction in both binding affinity for PDGF alpha-receptors and mitogenic activity in Swiss 3T3 cells. Large reductions were also observed in the ability of semipurified PDGF-AAcm to stimulate calcium influx and the production of inositol phosphates. Measurement of circular dichroism spectra of highly purified PDGF-AAcm and PDGF-AAwt revealed no significant difference in secondary structure. Collectively, these results indicate that the cationic Arg159-Lys160-Lys161 region plays a critical role in the biological activity of PDGF-AA by direct participation in ligand binding to the PDGF alpha-receptor.
Transport of secretory IgA into external fluids is mediated by the polymeric immunoglobulin receptor (pIgR) on the surface of mucosal epithelial cells. We studied the mechanism by which interferon-gamma (IFN-gamma) induces pIgR expression in HT-29.74 cells, a subclone of the HT-29 cell line selected for high concns of pIgR. Here we report the isolation of genomic DNA and cDNA clones encoding human pIgR and development of a sensitive ribonuclease protection assay for pIgR mRNA. This assay was used to determine if induction of pIgR by IFN-gamma is mediated by accumulation of pIgR mRNA. After an initial lag of 12 hr, pIgR mRNA increased seven-fold in response to IFN-gamma, reaching a plateau at 24 hr. Concentrations of pIgR protein also increased seven-fold, but the increase was delayed until 48 hr following stimulation with IFN-gamma. Cycloheximide treatment abolished the IFN-gamma induced increase in pIgR mRNA, indicating that induction of pIgR mRNA by IFN-gamma requires de novo protein synthesis. These results suggest that induction of pIgR expression by IFN-gamma involves an increase in steady-state concns of pIgR mRNA via a protein synthesis dependent mechanism.
Disulfide bonds are a major force in stabilizing the three-dimensional structure of immunoglobulins. To determine the pattern of interchain disulfide bonding between the four H chains, four L chains and single J chain of rat dimeric IgA (dIgA), we analyzed dIgA from the LO DNP-64 hybridoma by diagonal SDS-PAGE. Bands corresponding to one, two, three and four H chains, one and two L chains and the free J chain were observed under non-reducing conditions, suggesting that the interchain disulfide bonds in rat dIgA are unstable under denaturing conditions. Similar patterns of disulfide bonding were observed in three other hybridoma or myeloma dIgAs from LOU/CN rats. In contrast, when dIgA pretreated with iodoacetamide (IA) was analyzed by the same technique, only bands corresponding to four H chains, one and two L chains and the free J chain were observed, suggesting that blocking free sulfhydryl groups stabilizes the inter-H chain disulfide bonds. Reaction of dimeric LO DNP-64 dIgA with 5,5'-dithiobis-(2-nitrobenzoic acid) or with 14C-IA demonstrated that this dIgA contains an average of 4 moles of free sulfhydryl groups per mole of protein under non-denaturing conditions and 9 moles of free sulfhydryl groups under denaturing conditions. Taken together, the results suggest that interchain disulfide bonds in rat dIgA are unstable, presumably due to the influence of nearby free sulfhydryl groups, and that non-covalent forces are critical for stabilizing the dIgA complex. The results also indicate that J chain is entirely non-covalently associated with the H chains, an apparently unique feature of rat dIgA. A model for interchain disulfide bonding in rat dIgA is proposed.