Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences.

Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an understanding of the possible roles of cell-extracellular matrix interactions and the requirements for meeting polarity needs of the epithelium. When cells are cultured on floating collagen gels they assemble a basal lamina-like structure composed of laminin, collagen (IV), and heparan sulfate proteoglycan at the interface of the cells with the stromal collagen. To assess the role of these components, an exogenous basement membrane containing these molecules was generated using the mouse endodermal cell line, PFHR-9. This matrix was isolated as a thin sheet attached to the culture dish, and mammary cells were then plated onto it. It was found that cultures on attached PFHR-9 matrices expressed slightly higher levels of beta-casein than did cells on plastic tissue culture dishes, and also accumulated a large number of fat droplets. However, the level of beta-casein was approximately fourfold lower than that in cultures on floating collagen gels. Moreover, the beta-casein made in cells on attached matrices was not secreted but was instead rapidly degraded intracellularly. If, however, the PFHR-9 matrices with attached cells were floated in the culture medium, beta-casein expression became equivalent to that in cells cultured on floating stromal collagen gels, and the casein was also secreted into the medium. The possibility that floatation of the cultures was necessary to allow access to the basolateral surface of cells was tested by culturing cells on nitrocellulose filters in Millicell (Millipore Corp., Bedford, MA) chambers. These chambers permit the monolayers to interact with the medium and its complement of hormones and growth factors through the basal cell surface. Significantly, under these conditions alpha 1-, alpha 2-, and beta-casein synthesis was equivalent to that in cells on floating gels and matrices, and, additionally, the caseins were actively secreted. Similar results were obtained independently of whether or not the filters were coated with matrices.(ABSTRACT TRUNCATED AT 400 WORDS)

Polymeric Ig receptor: defender of the fort or Trojan horse?

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.

Gene transfer into respiratory epithelial cells by targeting the polymeric immunoglobulin receptor.

A system for targeting foreign DNA to epithelial cells in vitro has been developed by exploiting receptor-mediated endocytosis. The polymeric immunoglobulin receptor transports dimeric immunoglobulin A and immunoglobulin M through epithelial cells, including those of the respiratory tract, by binding the immunoglobulins at the basolateral surface and transporting them across the cell. Fab fragments of antibodies directed against the extracellular portion of the receptor, secretory component, are similarly transported. Anti-human secretory component Fab fragments were covalently linked to a polycation, and complexed to various expression plasmids. When bound to an expression plasmid containing the Escherichia coli lacZ gene ligated to the Rous sarcoma virus promoter, the complexes transfected HT29.74 human colon carcinoma cells induced to express polymeric immunoglobulin receptor, but not those lacking the receptor. Primary cultures of human tracheal epithelial cells grown on collagen gels, which induce the expression of polymeric immunoglobulin receptor, were also transfected with the complexes. From 5 to 66% of the respiratory epithelial cells had beta-galactosidase activity after treatment, comparable to the percentage of cultured human tracheal epithelial cells that express polymeric immunoglobulin receptor (8-35%). The addition of excess human secretory component (Fab ligand) to the culture medium at the time of transfection blocked the delivery of DNA. The expression plasmid, either alone, complexed to the polycation, or complexed to a carrier based on an irrelevant Fab fragment, was not effective in transfecting either cell type. This DNA carrier system introduces DNA specifically into epithelial cells that contain pIgR in vitro.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

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.

Cryptic intron activation within the large exon of the mouse polymeric immunoglobulin receptor gene: cryptic splice sites correspond to protein domain boundaries.

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.

Isolation of a 110 000 molecular weight protein in the purification of the nuclear chick intestinal 1,25-dihydroxyvitamin D-3 receptor.

A single protein band of molecular weight 110 000 has been obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D-3) receptor from crude nuclear extracts of chick intestinal mucosa, prepared in the presence of the protease inhibitors phenylmethylsulfonyl fluoride and epsilon-aminocaproic acid. The nuclear extract was subjected to a six-step purification scheme, involving polymin P and ammonium sulfate fractionation, DNA-cellulose affinity chromatography, Sephacryl S-200 gel filtration, blue dextran-Sepharose and a final DNA-cellulose chromatographic step. The receptor was obtained in about 1% yield and was purified approx. 3700-fold from the nuclear extract, as assessed by specific activity. Single peaks were observed with 3H-1,25-(OH)2D-3-labeled crude nuclear extracts on Sephacryl S-200 gel filtration (Stokes' radius = 35.5 A) and sucrose density gradient centrifugation (3.5 S). Although the identity of the Mr 110 000 protein will remain inconclusive until methods for further characterization are available, it may represent evidence for a higher molecular weight form of the 1,25-(OH)2D-3 receptor than that observed previously.

Unstable inter-H chain disulfide bonding and non-covalently associated J chain in rat dimeric IgA.

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.

Transcriptional regulation of the human polymeric immunoglobulin receptor gene by interferon-gamma.

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.

Interferon-gamma induces polymeric immunoglobulin receptor mRNA in human intestinal epithelial cells by a protein synthesis dependent mechanism.

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.

Binding characteristics of complementary fibronectin fragments on artificial substrata.

Various properties have been evaluated for the binding to tissue culture substrata of proteolytic fragments of human plasma or cellular fibronectins containing complementary sequences from the individual and alternatively spliced chains, since related fragments are known to yield differing adhesive responses from cells. These studies utilize ELISA methods and a polyclonal antiserum directed to human pFN for direct measurement or an occupancy test utilizing anti-albumin. Very related fragments (with or without an extra type III homology unit or extra domaina or b) have significantly different properties in substratum binding and such differences provide a partial explanation for alteration of cellular adhesive responses on such fragments.

IgA and mucosal defense.

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.

Targeted deletion of MyD88 in intestinal epithelial cells results in compromised antibacterial immunity associated with downregulation of polymeric immunoglobulin receptor, mucin-2, and antibacterial peptides.

Intestinal epithelial cells (IECs) form a physical and immunological barrier that separates the vast gut microbiota from host tissues. MyD88-dependent Toll-like receptor signaling is a key mediator of microbial-host cross-talk. We examined the role of epithelial MyD88 expression by generating mice with an IEC-targeted deletion of the Myd88 gene (MyD88(ΔIEC)). Loss of epithelial MyD88 signaling resulted in increased numbers of mucus-associated bacteria; translocation of bacteria, including the opportunistic pathogen Klebsiella pneumoniae, to mesenteric lymph nodes; reduced transmucosal electrical resistance; impaired mucus-associated antimicrobial activity; and downregulated expression of polymeric immunoglobulin receptor (the epithelial IgA transporter), mucin-2 (the major protein of intestinal mucus), and the antimicrobial peptides RegIIIγ and Defa-rs1. We further observed significant differences in the composition of the gut microbiota between MyD88(ΔIEC) mice and wild-type littermates. These physical, immunological, and microbial defects resulted in increased susceptibility of MyD88(ΔIEC) mice to experimental colitis. We conclude that MyD88 signaling in IECs is crucial for maintenance of gut homeostasis.

Regulation of the polymeric immunoglobulin receptor and IgA transport: new advances in environmental factors that stimulate pIgR expression and its role in mucosal immunity.

Secretory IgA (SIgA) antibodies represent the first line of antigen-specific immune defense protecting the mucosal surfaces against environmental pathogens and antigens, and maintaining homeostasis with the commensal microbiota. The polymeric immunoglobulin receptor (pIgR) has the dual role of transporting locally produced dimeric IgA across mucosal epithelia, and serving as the precursor of secretory component, a glycoprotein that enhances the immune functions of SIgA. The complex regulation of pIgR expression and transcytosis by host and microbial factors is finely tuned to optimize the role of SIgA in mucosal immunity. Disruption of this regulatory network in disease states similar to inflammatory bowel disease can result in profound consequences for mucosal homeostasis and systemic sequelae. Future research into the function and regulation of pIgR and SIgA may offer new insights into the prevention and treatment of infectious and inflammatory diseases that originate at mucosal surfaces.

Regulation of the polymeric immunoglobulin receptor by the classical and alternative NF-κB pathways in intestinal epithelial cells.

The polymeric immunoglobulin receptor (pIgR) transports IgA antibodies across intestinal epithelial cells (IECs). Expression of pIgR is upregulated by proinflammatory signaling pathways via activation of nuclear factor-κB (NF-κB). Here, we examined the contributions of the RelA-dependent classical and RelB-dependent alternative pathways of NF-κB to pIgR regulation in the HT-29 human IEC line following stimulation with tumor necrosis factor (TNF), lipopolysaccharide (LPS; Toll-like receptor 4 (TLR4) ligand), and polyinosinic: polycytidylic acid (pIC; TLR3 ligand). Whereas induction of proinflammatory genes such as interleukin-8 (IL-8) required only RelA, pIgR expression was regulated by complex mechanisms that involved both RelA and RelB. Upregulation of pIgR expression by ligation of the lymphotoxin-ß receptor suggested a direct role for the alternative NF-κB pathway. Inhibition of mitogen-activated protein kinases reduced the induction of IL-8, but enhanced the induction of pIgR by TNF and TLR signaling. Regulation of pIgR through unique signaling pathways could allow IECs to sustain high levels of IgA transport while limiting the proinflammatory responses.

Signature biomarkers in Crohn's disease: toward a molecular classification.

In an effort to develop a molecular classification scheme for Crohn's disease (CD), mucosal biopsies from 69 CD patients and 28 normal controls were analyzed for expression of the RelA subunit of nuclear factor (NF)-kappaB, A20 (a negative regulator of NF-kappaB), polymeric immunoglobulin receptor (pIgR), tumor necrosis factor (TNF), and interleukin (IL)-8. Principal component analysis was used to classify individuals into three subsets based on patterns of biomarker expression. Set 1 included normal subjects and CD patients with mild disease and good responses to therapy, thus defining "normal" biomarker expression. CD patients in set 2, characterized by low expression of all five biomarkers, had moderate to severe disease and poor responses to immunosuppressive and anti-TNF therapy. Patients in set 3, characterized by low expression of RelA, A20, and pIgR, normal TNF and elevated IL-8, had acute inflammation that responded well to therapy. Classification of CD patients by these biomarkers may predict disease behavior and responses to therapy.