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M R Neutra

Publications and source records attributed to M R Neutra.

At least 73 records · Page 4Linked to original sources
PubMed1992

Regulation of gene expression in gastric epithelial cell populations of fetal, neonatal, and adult transgenic mice.

Little is known about lineage relationships and differentiation programs of various epithelial cells present in mouse gastric units. We have previously used rat liver fatty acid binding protein/human growth hormone (L-FABP/hGH) transgenes to define epithelial cell lineages relationships in the small intestine of fetal and adult mice and to examine regulation of their terminal differentiation programs along the crypt-to-villus and duodenal-to-ileal axes. We have now used these transgenes to explore similar issues in the stomach. Immunocytochemical studies of fetal and adult transgenic L-FABP/hGH animals and their normal littermates revealed that the intact endogenous mouse L-FABP gene (Fabpl) is not expressed in gastric epithelium. Nucleotides-596 to +21 of the rat L-FABP gene direct "inappropriate" expression of hGH in the gastric epithelium as early as fetal day 15. From 1 to 13 mo, L-FABP-596 to +21/hGH expression occurs only in surface mucous cells of zymogenic and mucous gastric units; the reporter is not detectable in the enteroendocrine, parietal and chief cell populations of zymogenic glands. Electron microscopic immunocytochemistry revealed that hGH is directed to apical secretory granules in surface and pit mucous cells expressing the transgene. hGH levels vary widely among surface mucous cells both within single pits and between gastric units in a given animal. The heterogeneity noted in reporter expression suggests that there are marked differences in the regulatory environments of individual cells of a single type within a given gastric unit. This raises the possibility that cell differentiation programs in the stomach may not be as tightly coupled to cellular translocation as in the small intestine. Finally, the lack of expression of L-FABP-596 to +21/hGH in gastrin- and serotonin-immunoreactive cells of the stomach contrasts with its efficient expression in comparable cell types located in the duodenum; providing a model system for examining differential regulation of gene expression in terminally differentiated cell types represented in both gastric and intestinal epithelium.

Animals↗
PubMed1991

New model for analysis of mucosal immunity: intestinal secretion of specific monoclonal immunoglobulin A from hybridoma tumors protects against Vibrio cholerae infection.

Secretory immunoglobulin A (sIgA) plays a role in defense against Vibrio cholerae and other microorganisms that infect mucosal surfaces, but it is not established whether sIgA alone can prevent disease. We report here a strategy for identifying the antigen specificities of monoclonal sIgA antibodies that are capable of providing such protection. IgA hybridomas were generated from Peyer's patch lymphocytes after oral immunization with V. cholerae Ogawa 395. A clone was selected that produced dimeric monoclonal IgA antibodies directed against an Ogawa-specific lipopolysaccharide carbohydrate antigen exposed on the bacterial surface. Hybridoma cells were used to produce subcutaneous "backpack" tumors in syngeneic mice, resulting in secretion of monoclonal sIgA onto mucosal surfaces. Neonatal mice bearing anti-lipopolysaccharide hybridoma backpack tumors were specifically protected against oral challenge with 100 50% lethal doses of virulent Ogawa 395 organisms. Thus, the IgA hybridoma backpack tumor method identifies protective epitopes in the mucosal system and demonstrates that a single monoclonal sIgA can be sufficient to protect against intestinal disease.

Animals↗
PubMed1991

Biogenesis of the apical endosome-lysosome complex during differentiation of absorptive epithelial cells in rat ileum.

Absorptive cells of the neonatal rat ileum have an elaborate apical endocytic complex consisting of tubular and vesicular endosomes, multivesicular bodies (MVB), and a giant lysosomal vacuole. This system develops rapidly over the last 3 days (20-22) of gestation. We followed the assembly of this complex by ultrastructural analysis and immunocytochemistry using antigenic markers for microvilli, endosomal tubules and lysosomal membranes. At 19 days gestation, low levels of lactase appeared on microvilli but specialized apical endosomal tubules and lysosomes were absent. At 20 days, expression of microvillar lactase increased and the endosomal marker entubin appeared, in parallel with the appearance of specialized apical endosomal tubules. The compartments of the apical endosome-lysosome system were assembled sequentially after differentiation of the apical plasma membrane domains; first endosomal tubules and vesicles, followed by MVB, and ending with the assembly of the giant lysosome shortly after birth. During early stages of the assembly process, membrane components of the tubular endosomes and lysosomes appeared in the apical plasma membrane before being restricted to their respective intracellular compartments.

Animals↗
PubMed1991

Transepithelial transport of HIV-1 by intestinal M cells: a mechanism for transmission of AIDS.

This study was designed to determine whether human immunodeficiency virus type 1 (HIV-1) might enter the host by penetrating epithelial barriers through antigen-transporting M cells in lymphoid follicle-associated epithelia. Interaction of HIV-1 with epithelial cells was examined using mucosal explants from Peyer's patches of mice and rabbits. HIV-1 adhered to the luminal membranes of M cells of both species, and was endocytosed and delivered to intraepithelial spaces containing lymphocytes and macrophages. These observations suggest that M cells, which are numerous in the human rectal mucosa, may efficiently deliver HIV-1 to target cells in mucosal lymphoid tissue, and that such transport may contribute to sexual transmission of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Interaction of cholera toxin with cloned human goblet cells in monolayer culture.

Exposure of the intestinal mucosa to Vibrio cholerae enterotoxin (CT) results in mucus secretion from intestinal goblet cells. On the other hand, there is evidence that elevation of intracellular adenosine 3',5'-cyclic monophosphate levels is not sufficient to induce rapid mucin secretion. To determine whether CT has direct effects on human goblet cells and whether CT alone can elicit rapid exocytosis of apical mucin granules, purified CT was applied to monolayer cultures of well-differentiated HT-29-18 N2 cells, a goblet cell subclone derived from the human colon carcinoma line HT-29. CT bound with high affinity (dissociation constant = 10.5 +/- 1.9 nM) to receptors on these cells (approximately 60,000/apical membrane). Binding of radiolabeled CT was inhibited by excess CT or B subunit but not by A subunit. Preincubation of goblet cells with CT blocked the subsequent CT-specific ribosylation of a 45-kDa protein in membrane fractions of the cells and increased the activity of adenylate cyclase by 2- and 10-fold after 1 and 20 h, respectively. Light micrographs revealed that goblet cells incubated with CT, like control cells, contained abundant apical mucin granules. In contrast, goblet cells incubated with Ca2+ inophore A23187 and phorbol ester were rapidly depleted of mucin granules. Thus CT has direct physiological effects on HT-29 goblet cells, but these do not lead to rapid mucin secretion. These results raise the possibility that CT may accelerate mucin secretion in intact mucosa by an indirect mechanism perhaps mediated by mucosal nerves or other cell types.

Cell Line↗
PubMed1989

Lipoxygenase metabolites of arachidonic acid do not induce mucus secretion from rabbit intestinal goblet cells in vitro.

Lipoxygenase metabolites of arachidonic acid are effective mucus secretagogues in the respiratory tract but their efficacy in the intestinal tract was unknown. Mucosal explants and sheets of epithelial cells isolated from rabbit small and large intestine were exposed to leukotrienes B4, C4, and D4 and monohydroxyeicosatetraenoic acids 5-HETE, 12-HETE, and 15-HETE. Light and electron microscopic inspection of goblet cells in treated tissues failed to detect evidence of recent compound exocytosis of mucin granules or other morphological evidence of secretory activity. These results indicate that lipoxygenase metabolites are not directly responsible for the increased mucus secretion observed in ulcerative colitis.

Animals↗
PubMed1989

Binding and transepithelial transport of immunoglobulins by intestinal M cells: demonstration using monoclonal IgA antibodies against enteric viral proteins.

M cells of intestinal epithelia overlying lymphoid follicles endocytose luminal macromolecules and microorganisms and deliver them to underlying lymphoid tissue. The effect of luminal secretory IgA antibodies on adherence and transepithelial transport of antigens and microorganisms by M cells is unknown. We have studied the interaction of monoclonal IgA antibodies directed against specific enteric viruses, or the hapten trinitrophenyl (TNP), with M cells. To produce monospecific IgA antibodies against mouse mammary tumor virus (MMTV) and reovirus type 1, Peyer's patch cells from mucosally immunized mice were fused with myeloma cells, generating hybridomas that secreted virus-specific IgA antibodies in monomeric and polymeric forms. One of two anti-MMTV IgA antibodies specifically bound the viral surface glycoprotein gp52, and 3 of 10 antireovirus IgA antibodies immunoprecipitated sigma 3 and mu lc surface proteins. 35S-labeled IgA antibodies injected intravenously into rats were recovered in bile as higher molecular weight species, suggesting that secretory component had been added on passage through the liver. Radiolabeled or colloidal gold-conjugated mouse IgA was injected into mouse, rat, and rabbit intestinal loops containing Peyer's patches. Light microscopic autoradiography and EM showed that all IgA antibodies (antivirus or anti-TNP) bound to M cell luminal membranes and were transported in vesicles across M cells. IgA-gold binding was inhibited by excess unlabeled IgA, indicating that binding was specific. IgG-gold also adhered to M cells and excess unlabeled IgG inhibited IgA-gold binding; thus binding was not isotype-specific. Immune complexes consisting of monoclonal anti-TNP IgA and TNP-ferritin adhered selectively to M cell membranes, while TNP-ferritin alone did not. These results suggest that selective adherence of luminal antibody to M cells may facilitate delivery of virus-antibody complexes to mucosal lymphoid tissue, enhancing subsequent secretory immune responses or facilitating viral invasion.

Animals↗
PubMed1989

Membrane domains of intestinal epithelial cells: distribution of Na+,K+-ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation.

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.

Aminopeptidases↗
PubMed1989

Use of transgenic mice to study the routing of secretory proteins in intestinal epithelial cells: analysis of human growth hormone compartmentalization as a function of cell type and differentiation.

The intestinal epithelium is a heterogeneous cell monolayer that undergoes continuous renewal and differentiation along the crypt-villus axis. We have used transgenic mice to examine the compartmentalization of a regulated endocrine secretory protein, human growth hormone (hGH), in the four exocrine cells of the mouse intestinal epithelium (Paneth cells, intermediate cells, typical goblet cells, and granular goblet cells), as well as in its enteroendocrine and absorptive (enterocyte) cell populations. Nucleotides -596 to +21 of the rat liver fatty acid binding protein gene, when linked to the hGH gene (beginning at nucleotide +3) direct efficient synthesis of hGH in the gastrointestinal epithelium of transgenic animals (Sweetser, D. A., D. W. McKeel, E. F. Birkenmeier, P. C. Hoppe, and J. I. Gordon. 1988. Genes & Dev. 2:1318-1332). This provides a powerful in vivo model for analyzing protein sorting in diverse, differentiating, and polarized epithelial cells. Using EM immunocytochemical techniques, we demonstrated that this foreign polypeptide hormone entered the regulated basal granules of enteroendocrine cells as well as the apical secretory granules of exocrine Paneth cells, intermediate cells, and granular goblet cells. This suggests that common signals are recognized by the "sorting mechanisms" in regulated endocrine and exocrine cells. hGH was targeted to the electron-dense cores of secretory granules in granular goblet and intermediate cells, along with endogenous cell products. Thus, this polypeptide hormone contains domains that promote its segregation within certain exocrine granules. No expression of hGH was noted in typical goblet cells, suggesting that differences exist in the regulatory environments of granular and typical goblet cells. In enterocytes, hGH accumulated in dense-core granules located near apical and lateral cell surfaces, raising the possibility that these cells, which are known to conduct constitutive vesicular transport toward both apical and basolateral surfaces, also contain a previously unrecognized regulated pathway. Together our studies indicate that transgenic mice represent a valuable system for analyzing trafficking pathways and sorting mechanisms of secretory proteins in vivo.

Animals↗
PubMed1989

Cholinergic responsiveness of goblet cells during intestinal maturation.

In adult rat intestine, cholinergic stimulation accelerates discharge of mucus from crypt, but not villus, goblet cells. It was not known whether goblet cells in fetal or suckling rats are cholinergically sensitive. Rat pups (20 days of gestation to 30 postnatal days) were given subcutaneous injections of carbachol and the intestines were fixed 5 min later. Accelerated mucus secretion was assessed by light microscopy of semithin plastic sections. Goblet cells in ileal and colonic crypts did not show an adult-like response until 20-25 days after birth. Ileal and colonic mucosal explants in vitro showed age-dependent responses identical to those observed in vivo. The onset of cholinergic sensitivity occurred well after the formation of crypts and was always limited to crypt goblet cells.

Aging↗
PubMed1988

Human intestinal goblet cells in monolayer culture: characterization of a mucus-secreting subclone derived from the HT29 colon adenocarcinoma cell line.

HT29-18N2 (N2) cells, a subclone of the HT29 human colon carcinoma cell line, are shown in this report to be a model system for the study of human goblet cell differentiation and mucin secretion. Grown in the absence of glucose, these cells formed homogeneous epithelial monolayers of columnar cells with typical goblet cell morphology. Differentiation occurred on uncoated glass; laminin, fibronectin, or collagen type I or IV did not enhance differentiation. HT29-18N2 cells grown on uncoated or matrix-coated permeable filters formed differentiated monolayers, but mucin granules within some of these cells polarized along intraepithelial lumens. Polyclonal antibodies raised against purified human colonic mucin, and also a monoclonal antibody against a protease-sensitive epitope of human colonic mucin, stained secretory granules of all differentiated goblet cells within N2 cell monolayers but did not stain predifferentiated goblet cells lacking large secretory granules. Monoclonal antibodies against specific carbohydrate sequences of human mucins also failed to stain N2 cells before differentiation, but recognized varying fractions of differentiated N2 goblet cells. Autoradiographic visualization of radiolabeled glycoproteins demonstrated transport and secretion of N2 cell mucin granules. Cholinergic stimulation of differentiated N2 cell monolayers resulted in depletion of intracellular mucin granules.

Adenocarcinoma↗
PubMed1988

Membrane domains and macromolecular transport in intestinal epithelial cells.

Epithelial cell plasma membranes are organized as biochemically and functionally distinct domains and subdomains. We have explored the composition and maintenance of specific membrane regions during endocytosis and transepithelial vesicular transport in specialized cells of the intestinal epithelium. A unique membrane glycoprotein has been identified in apical endocytic subdomains and endosomal tubules of absorptive cells in suckling rat ileum. This endocytic system is involved in sorting and transepithelial transport of peptide growth factors as well as delivery of milk macromolecules to lysosomes. In M cells of follicle-associated epithelium, we have shown that membrane-bound proteins are efficiently transported to a specialized subdomain of basolateral membrane. The apical membranes of these cells bear immunoglobulin binding sites and transport monoclonal IgA antibodies to the basolateral side, where they may interact with cells of the mucosal immune system.

Absorption↗
PubMed1987

Transport of membrane-bound macromolecules by M cells in follicle-associated epithelium of rabbit Peyer's patch.

M cells in Peyer's patch epithelium conduct transepithelial transport of luminal antigens to cells of the mucosal immune system. To determine the distribution of specific lectin-binding sites on luminal membranes of living M cells and to follow the transport route of membrane-bound molecules, lectin-ferritin conjugates and cationized ferritin were applied to rabbit Peyer's patch mucosa in vivo and in vitro. The degree to which binding enhances transport was estimated by comparing quantitatively the transport of an adherent probe, wheat germ agglutinin-ferritin, to that of a nonadherent BSA-colloidal gold probe. When applied to fixed tissue, the lectins tested bound equally well to M cells and columnar absorptive cells. On living mucosa, however, ferritin conjugates of wheat germ agglutinin and Ricinus communis agglutinins I and II bound more avidly to M cells. Absorptive cells conducted little uptake and no detectable transepithelial transport. Lectins on M cell membranes were endocytosed from coated pits, rapidly transported in a complex system of tubulocisternae and vesicles, and remained adherent to M cell basolateral membranes. Cationized ferritin adhered to anionic sites and was similarly transported, but was released as free clusters at M cell basolateral surfaces. When applied simultaneously to Peyer's patch mucosa, wheat germ agglutinin-ferritin was transported about 50 times more efficiently than was bovine serum albumin-colloidal gold.

Animals↗
PubMed1987

Macromolecules can pass through occluding junctions of rat ileal epithelium during cholinergic stimulation.

Crypt, but not villus, goblet cells in the ileum accelerate their secretion of mucus within 5 min following cholinergic stimulation. This study was done to determine whether the macromolecular permeability and structure of occluding junctions in the ileum are altered during accelerated secretion. Rats were injected intravenously with horseradish peroxidase followed by carbachol (250 micrograms/kg, subcutaneous) and the intestinal mucosa was fixed 3-12 min later. In control mucosa (saline-injected), peroxidase filled lateral intercellular spaces up to the occluding junctions of both crypt and villus epithelium, but did not enter occluding junctions or pass into the lumen. In 3 of 8 carbachol-stimulated rats, peroxidase was present within occluding junctions in crypt epithelium and in the crypt lumen, although all intermembrane junctional fusion sites appeared intact. Villus epithelial occluding junctions, in contrast, continued to exclude peroxidase. In freeze-fracture replicas of crypt cells prepared after carbachol stimulation, we detected no structural changes in strand networks of occluding junctions that could account for increased paracellular permeability.

Animals↗
PubMed1987

Identification of an endosomal antigen specific to absorptive cells of suckling rat ileum.

A membrane fraction enriched in apical endosomal tubules was isolated from absorptive cells of suckling rat ileum and used as an immunogen to generate anti-endosome monoclonal antibodies. By immunofluorescence, one of these antibodies bound exclusively to the region of the apical endocytic complex in ileal absorptive cells, but not to other cell types. Immunoblot analysis showed the antigen as a diffuse 55-61-kD band which was highly enriched in the endosome fraction over whole-cell homogenate. The antigen appears to be an intramembrane glycoprotein: it partitioned primarily in the detergent phase after TX-114 extraction, and shifted to 44 kD after chemical deglycosylation. EM immunocytochemistry showed that the antibody bound to the luminal side of endosomal tubule membranes, a portion of endosomal vesicle membranes, and in endocytic pits of apical plasma membranes. However, it did not bind to multivesicular bodies, the giant lysosome, or other organelles. Immunocytochemistry after uptake with adsorbed or soluble tracer proteins showed that the antigen labeled portions of both prelysosomal pathways previously described in these cells (Gonnella, P.A., and M. R. Neutra, 1984, J. Cell Biol., 99:909-917). The function of this glycoprotein is not known, but inasmuch as it has been detected only in absorptive cells of suckling rat ileum, it may serve a function specific to these cells. Nevertheless, this endosomal antigen, designated glycoprotein (gp) 55-61, will serve as a useful marker for exploring membrane dynamics in early stages of the endocytic pathway.

Animals↗