Paneth cells of the human small intestine express an antimicrobial peptide gene.

Mucosal surfaces of several organ systems are important interfaces for host defense against microbes. Recent evidence suggests that antimicrobial peptides contribute to the defense of these surfaces. Defensins are one family of antimicrobial peptide, but their known distribution in humans has been limited to four members found in cells of myeloid origin. We sought to determine if the human defensin family was more complex. We found that the family of human defensins is diverse and is not restricted to expression in leukocytes. Southern blot and genomic clone analyses reveal that numerous defensin-related sequences are present in the human genome. A gene for a new human defensin family member was characterized. This gene, designated human defensin-5, is highly expressed in Paneth cells of the small intestine. This is the first example of an antimicrobial peptide gene expressed in an epithelial cell in humans. The data support the hypotheses that epithelial defensins equip the human small bowel with a previously unrecognized defensive capability which would augment other antimicrobial defenses.     

Immunocytochemical identification and localization of immunoglobulin A within Paneth cells of the rat small intestine.

Light microscopic immunocytochemistry was used to identify Paneth cells by their lysozyme content and to detect immunoglobulin antigens within a subpopulation of these cells. Antisera specific for the heavy chains of rat or human immunoglobulin A and for immunoglobulin light chain antigens produced specific staining of rat Paneth cells. The distribution of immunoglobulin staining varied between adjacent Paneth cells in the same crypt and between Paneth cells in adjacent crypts, as well as between Paneth cell populations of different animals. No staining of rat Paneth cells was detected using antisera specific for the heavy chain of immunoglobulins G or M. The specific staining of Paneth cells for immunoglobulin A and light chain antigens was blocked by absorption of each antiserum with its respective purified antigen. Absorption of these antisera with purified rat lysozyme did not affect staining and thereby eliminated the possibility of immunologic cross-reactivity between lysozyme and immunoglobulin antigens. It is suggested, in light of current concepts of Paneth cell function, that the immunoglobulin staining of Paneth cells may reflect their ability to phagocytize immunoglobulin A-coated microorganisms or immune complexes containing immunoglobulin A.     

Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) activity within columnar, goblet, and Paneth cells of rat small intestine

The distribution of nicotinamide adenine dinucleotide phosphatase (NADPase) activity was examined in epithelial cells of rat small intestine. Segments of ileum were fixed with glutaraldehyde and tissue chopper sections were incubated for up to 4 hr at pH 5.0 in cytochemical media prepared with NADP as substrate. NADPase activity was found primarily within the Golgi saccules of columnar, goblet, and Paneth cells. Columnar and goblet cells showed most of the NADPase activity within the saccules which were intermediate between the cis and trans faces of the Golgi stack. Paneth cells, however, showed the heaviest staining within saccules between the intermediate and innermost saccule at the trans aspect of the Golgi stack. Both columnar cells and Paneth cells also contained spotty, and sometimes heavy, deposits of reaction product within an occasional focal area of the GERL system and within an occasional lysosome. Control experiments indicated that the Golgi-associated NADPase activity was enhanced if cells were pretreated for about 12 hr with EGTA prior to incubation. No similar enhancement was apparent if the tissues were pretreated with DMSO. Furthermore, NADPase activity within the Golgi saccules could be inhibited completely by incubating intestinal epithelial cells with NADP in the presence of sodium fluoride or L(+)-tartrate.     

Influence of high-calorie (cafeteria) diets on the population of Paneth cells in the small intestine of the rat

A high-calorie (cafeteria) diet is known to cause changes in the intestinal morphology and functioning that seem to be related to calorie overfeeding. Among the cell lineages found in the small intestine epithelium, the Paneth cell (PC) population is known to be influenced by factors related mainly to the intestinal microbiota. The role of PCs in the intestinal cell concert remains unclear, because experimental evidence suggests PC involvement in local processes other than protection against pathogens. Participation of PC in digestive mechanisms has been proposed on this basis. We have analyzed the effect of high-carbohydrate (HC) and high-fat (HF) cafeteria diets on the PC population in the small intestine of the adult rat. For 8 weeks, both HC and HF diets caused a gain in body weight, but whereas the HC-fed rats showed reduced counts of intestinal crypts per 5-mum section, the HF-fed group showed the opposite. In control rats, the number of crypts per section showed a slight tendency to decrease along the duodenum - ileum axis, whereas the number of PCs per crypt was increased towards the ileum. As a result, the number of PCs per section (calculated from these data) remained constant along the three segments of the intestine. The hypercaloric diets did not modify the general tendencies seen in the crypt and PC counts, but reduced the number of PCs per section in the duodenum by 50%. HC-fed, but not HF-fed, rats showed a similar reduction in jejunum also. These changes do not correlate particularly with any of the predictable effects of diet composition, so that a multifactorial control of PC density is proposed.     

Neurotensin modulates pacemaker activity in interstitial cells of Cajal from the mouse small intestine

Neurotensin, a tridecapeptide localized in the gut to discrete enteroendocrine cells of the small bowel mucosa, is a hormone that plays an important role in gastrointestinal secretion, growth, and motility. Neurotensin has inhibitory and excitatory effects on peristaltic activity and produces contractile and relaxant responses in intestinal smooth muscle. Our objective in this study is to investigate the effects of neurotensin in small intestinal interstitial cells of Cajal (ICC) and elucidate the mechanism. To determine the electrophysiological effects of neurotensin on ICC, whole-cell patch clamp recordings were performed in cultured ICC from the small intestine. Exposure to neurotensin depolarized the membrane of pacemaker cells and produced tonic inward pacemaker currents. Only neurotensin receptor1 was identified when RT-PCR and immunocytochemistry were performed with mRNA isolated from small intestinal ICC and c-Kit positive cells. Neurotensin-induced tonic inward pacemaker currents were blocked by external Na⁺- free solution and in the presence of flufenamic acid, an inhibitor of non-selective cation channels. Furthermore, neurotensin-induced action is blocked either by treatment with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, a phospholipase C inhibitor, or thapsigargin, a Ca²⁺-ATPase inhibitor in ICC. We found that neurotensin increased spontaneous intracellular Ca²⁺ oscillations as seen with fluo4/AM recording. These results suggest that neurotensin modulates pacemaker currents via the activation of non-selective cation channels by intracellular Ca²⁺-release through neurotensin receptor1.     

Mass spectrometric analysis of combinatorial peptide libraries derived from the tandem repeat unit of MUC2 mucin.

Four 19-member synthetic peptide libraries, based on the TX1TX2T epitope motif of the mucin-2 gastrointestinal glycoprotein (MUC2) and ranging in peptide length from dipeptides to 15-mers (XT, TXT, TQTXT and KVTPTPTPTGTQTXT), were synthesized by combinatorial solid phase peptide synthesis using the portioning-mixing combinatorial approach, and analysed by electrospray ionization mass spectrometry at different (1000-10000) resolutions. Most of the components of the individual libraries could be easily identified in a single-stage molecular mass screening experiment. The resolving power of the instrument becomes an important factor above 800-1000 Da molecular mass, when predominantly multiply charged molecular ions are formed. Approaches to the identification of isobars (glutamine/lysine), isomers leucine/isoleucine) and sequence variations by tandem mass spectrometry, and/or by high-performance liquid chromatography-mass spectrometry are outlined.     

Participation of the goblet cells of the small intestine in the excretion of Fe, Zn and Pb cations

A comparative study of the excess of Zn, Fe and Pb on the amount of goblet cells (GC) and localization of these cations in the chick small intestine epithelium was carried out. In chicks with additional dose of Zn (2 g/kg) and Pb (0.2 g/kg), compared to control chicks the amount of GC in the epithelium of various intestinal parts increased by 1.5-1.8 times. Under a high dose of Pb (2.0 g/kg), the amount of GC increased by 3-4 times, and degenerative changes in the epithelium were observed. A histochemical electron microscopical study of duodenal sections revealed an accumulation of Fe, Zn and Pb within GC and in their excretion, in addition to brush border and glycocalyx. The involvement of intestinal GC in cation excretion is proven.     

A novel core protein as well as polymorphic epithelial mucin carry peanut agglutinin binding sites in human gastric carcinoma cells: sequence analysis and examination of gene expression.

The peanut agglutinin (PNA)-binding site is protein-bound Gal beta 1-->3GalNAc, and is a tumor-associated carbohydrate marker expressed in many human carcinomas. PNA-binding glycoproteins isolated from KATO-III human gastric carcinoma cells were deglycosylated by trifluoromethanesulfonic acid, and rabbit antibodies against the core proteins were used to screen a lambda gt11 expression library constructed from these cells. Two different core proteins were identified by this approach. One was polymorphic epithelial mucin (PEM), initially found in breast carcinomas. PEM mRNA was expressed in normal tissues of the stomach, colon, and lung, but not in the small intestine, thyroid, and spleen. High levels of PEM mRNA were detected in some nude mouse-transplanted carcinomas, i.e. colorectal, pancreatic, stomach, and lung carcinomas. The other core protein was a novel one called MGC-24, which has a molecular mass of 24 kDa, is rich in hydroxyl amino acids and cysteine, and lacks repeating motifs. The mature MGC-24 glycoprotein behaved as a high-molecular-mass one upon SDS-polyacrylamide gel electrophoresis even after neuraminidase treatment. Treatment with endo-alpha-N-acetylgalactosaminidase in the absence of neuraminidase significantly changed the staining pattern by anti-MGC-24, confirming that MGC-24 carried PNA-binding sites. MGC-24 mRNA was intensely expressed in normal tissues of the colon, small intestine and thyroid, and in some nude mouse-transplanted colorectal and pancreatic adenocarcinomas.     

The distribution of endocrine cells along the mouse small intestine. Bombesin and somatostatin producing cells

The topographical distribution and incidence of endocrine cells in the crypt and villus epithelium and along the length of the mouse intestine was studied. Cells containing somatostatin and bombesin like reactivity were stained by immunocytochemical techniques using polyclonal antiserum. Most of the somatostatin cells were found in the duodenum, jejunum and ileum, and these cells were generally more frequent on the villus compared to the crypts. This may indicate that the somatostatin cells develop late in the endocrine cell lineage. Bombesin like cells were rare in occurrence, and were only present in measureable numbers in the ileum, where they were observed in the crypt and villi. The application of ELISA assays to determine the specificity of the antisera for these peptides is also discussed.     

Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep.

Crude soluble mucus from sheep small intestine was freed of nearly all the nucleic acid contaminants by precipitation with protamine sulphate and treatment with nucleases. After removal of non-covalently bound proteins by equilibrium density-gradient centrifugation in CsCl, a high-Mr glycoprotein was isolated by repeated h.p.l.c. from the partially purified mucin. The high degree of purity of the high-Mr mucin was borne out by (a) the observation of a single boundary on analytical ultracentrifugation in the presence of 5M-guanidinium chloride and (b) the observation of apparent monodispersity on sedimentation-equilibrium analysis. The Mr of the highly purified mucin, determined by sedimentation equilibrium, was 5.0 (+/- 0.1) X 10(6) and was concentration-independent. Finally, only goblet cells and the mucus blanket lining the intestinal epithelial cells were immunofluorescent when guinea-pig anti-(highly purified mucin) serum was used in an indirect immunofluorescence assay. The above antiserum reacted with apparently equal strength with goblet cells and with free mucin in abomasum, caecum and colon. The chemical composition of the glycoprotein was 66% carbohydrate and 34% protein, 45% of the latter being composed of valine and threonine. The glycoprotein migrated anodally on immunoelectrophoresis and contained 7.1% (w/w) sulphate. Neutral hexoses accounted for nearly half of the total carbohydrate content, followed by galactosamine and glucosamine. Whereas fucose and sialic acid were present in only small amounts, uronic acid was not detectable in the highly purified mucus glycoprotein.     

Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas.

To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.     

Induction of mucin and MUC5AC expression by the protease activity of Aspergillus fumigatus in airway epithelial cells

Allergic bronchopulmonary mycosis, characterized by excessive mucus secretion, airflow limitation, bronchiectasis, and peripheral blood eosinophilia, is predominantly caused by a fungal pathogen, Aspergillus fumigatus. Using DNA microarray analysis of NCI-H292 cells, a human bronchial epithelial cell line, stimulated with fungal extracts from A. fumigatus, Alternaria alternata, or Penicillium notatum, we identified a mucin-related MUC5AC as one of the genes, the expression of which was selectively induced by A. fumigatus. Quantitative RT-PCR, ELISA, and histochemical analyses confirmed an induction of mucin and MUC5AC expression by A. fumigatus extracts or the culture supernatant of live microorganisms in NCI-H292 cells and primary cultures of airway epithelial cells. The expression of MUC5AC induced by A. fumigatus extracts diminished in the presence of neutralizing Abs or of inhibitors of the epidermal growth factor receptor or its ligand, TGF-α. We also found that A. fumigatus extracts activated the TNF-α-converting enzyme (TACE), critical for the cleavage of membrane-bound pro-TGF-α, and its inhibition with low-molecular weight inhibitors or small interfering RNA suppressed the expression of MUC5AC. The protease activity of A. fumigatus extracts was greater than that of other fungal extracts, and treatment with a serine protease inhibitor, but not with a cysteine protease inhibitor, eliminated its ability to activate TACE or induce the expression of MUC5AC mRNA in NCI-H292. In conclusion, the prominent serine protease activity of A. fumigatus, which caused the overproduction of mucus by the bronchial epithelium via the activation of the TACE/TGF-α/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.     

MicroRNA-449a overexpression, reduced NOTCH1 signals and scarce goblet cells characterize the small intestine of celiac patients

MiRNAs play a relevant role in regulating gene expression in a variety of physiological and pathological conditions including autoimmune disorders. MiRNAs are also important in the differentiation and function of the mouse intestinal epithelium. Our study was aimed to look for miRNA-based modulation of gene expression in celiac small intestine, and particularly for genes involved in cell intestinal differentiation/proliferation mechanisms. A cohort of 40 children (20 with active CD, 9 on a gluten-free diet (GFD), and 11 controls), were recruited at the Paediatrics Department (University of Naples Federico II). The expression of 365 human miRNAs was quantified by TaqMan low-density arrays. We used bioinformatics to predict putative target genes of miRNAs and to select biological pathways. The presence of NOTCH1, HES1, KLF4, MUC-2, Ki67 and beta-catenin proteins in the small intestine of CD and control children was tested by immunohistochemistry. The expression of about 20% of the miRNAs tested differed between CD and control children. We found that high miR-449a levels targeted and reduced both NOTCH1 and KLF4 in HEK-293 cells. NOTCH1, KLF4 signals and the number of goblet cells were lower in small intestine of children with active CD and in those on a GFD than in controls, whereas more nuclear beta-catenin staining, as a sign of the WNT pathway activation, and more Ki67 staining, as sign of proliferation, were present in crypts from CD patients than in controls. In conclusion we first demonstrate a miRNA mediated gene regulation in small intestine of CD patients. We also highlighted a reduced NOTCH1 pathway in our patients, irrespective of whether the disease was active or not. We suggest that NOTCH pathway could be constitutively altered in the celiac small intestine and could drive the increased proliferation and the decreased differentiation of intestinal cells towards the secretory goblet cell lineage.     

A study of the intracellular and secreted forms of the MUC2 mucin from the PC/AA intestinal cell line.

In this study we present data on the entire population of MUC2 molecules secreted from and within the cell layer of an intestinal cell line. The molecular size distribution of the extracted molecules and their reactivity with two different MUC2 polypeptide antibodies indicated the presence of precursor and mature forms of the mucin. Oligomerized forms of the mucin were found in both the cell layer and medium; however, precursor forms were confined to the cell layer. Isopycnic density gradient centrifugation gave good resolution of mature and precursor forms of MUC2 as assessed by agarose gel electrophoresis. Three different populations of MUC2 were identified: one at low density (>1.3 g/ml) containing the N-glycosylated, non-O-glycosylated polypeptide; a second at intermediate density (1.3-1.35 g/ml) which may represent partially O-glycosylated intermediates; and a third at high density (1.36-1.48 g/ml) containing the mature MUC2 mucins. Rate-zonal centrifugation and agarose electrophoretic analysis of the low-density fraction indicated that the N-glycosylated MUC2 polypeptide was present as putative monomer and dimer/oligomer species. The combination of isopycnic density gradient centrifugation with agarose electrophoresis provides a new and simple approach that allows us to follow the MUC2 gene product from polypeptide through to the mature glycosylated mucin.