The antisecretory factor: synthesis and intracellular localisation in porcine tissues

The antisecretory factor, AF, is a 41-kDa protein, cloned and sequenced from a human pituitary library. AF is a potent inhibitor of experimental intestinal hypersecretion in rats and pigs. An antiserum against the C-terminal of the truncated, recombinantly produced AF protein was raised in rabbits. The affinity-purified antiserum was used to study the expression of AF in mucosal membranes and in the pituitary gland of the pig; distinctly stained cells were found in lymphoid cells in the connective tissue of all parts of the gastrointestinal, respiratory and urinary tracts. Cytoplasmic AF was demonstrated in endocrine and epithelial cells in the pituitary gland. In situ hybridisation with a digoxigenin-labelled mRNA probe also demonstrated specific cytoplasmic staining in epithelial and lymphoid cells in all of these tissues. The cells stained by either method were similarly distributed topographically within the tissues. The results suggest that a specific defined cell population in these various tissues possesses the capability of both synthesising and storing the AF protein within the cellular cytoplasmic compartment.     

Isolation of pure human mucosal epithelium for RNA analysis

Techniques for isolating the desired cell populations from complex tissues are essential for characterizing cells through mRNA analysis. We established a procedure for isolating pure mucosal epithelium from the human alimentary tract. To do this, we made rotating hooks that hold mucosal strips and detach the epithelial sheets from the irregular mucosae surface in medium containing EDTA. An additional step using a cell strainer was required to reduce contamination by lymphoid cells. Sheets of epithelial cells were detached successfully from mucosal samples derived from five different parts of the human alimentary tract. Contamination by lymphoid cells or fibroblasts was monitored by competitive RT-PCR and was no more than 0.5% of the total cells. Total RNA yields were 12.5-17 micrograms for each separation, and the integrity of the RNA was as good as that of RNAs extracted from mucosa immediately after resection. In conclusion, our method permits isolation of RNAs from a pure population of epithelial cells that can be used for mRNA-based gene expression analyses.     

Role of Janus Kinase 3 in Mucosal Differentiation and Predisposition to Colitis

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Previously, we characterized the functions of Jak3 in cytoskeletal remodeling, epithelial wound healing, and mucosal homeostasis. However, the role of Jak3 in mucosal differentiation and inflammatory bowel disease was not known. In this report, we characterize the role of Jak3 in mucosal differentiation, basal colonic inflammation, and predisposition toward colitis. Using the Jak3 knock-out (KO) mouse model, we show that Jak3 is expressed in colonic mucosa of mice, and the loss of mucosal expression of Jak3 resulted in reduced expression of differentiation markers for the cells of both enterocytic and secretory lineages. Jak3 KO mice showed reduced expression of colonic villin, carbonic anhydrase, secretory mucin muc2, and increased basal colonic inflammation reflected by increased levels of pro-inflammatory cytokines IL-6 and IL-17A in colon along with increased colonic myeloperoxidase activity. The inflammations in KO mice were associated with shortening of colon length, reduced cecum length, decreased crypt heights, and increased severity toward dextran sulfate sodium-induced colitis. In differentiated human colonic epithelial cells, Jak3 redistributed to basolateral surfaces and interacted with adherens junction (AJ) protein β-catenin. Jak3 expression in these cells was essential for AJ localization of β-catenin and maintenance of epithelial barrier functions. Collectively, these results demonstrate the essential role of Jak3 in the colon where it facilitated mucosal differentiation by promoting the expression of differentiation markers and enhanced colonic barrier functions through AJ localization of β-catenin.     

Prostanoid receptors in intestinal epithelium: selective expression, function, and change with inflammation

The tissue concentration of PGE(2)is heightened during mucosal inflammation. Nevertheless, the cellular targets of this prostanoid and its effects on epithelial cell physiology are incompletely understood. We used a panel of specific immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGE(2)on cells of histologically normal and inflamed human colonic mucosa, and then examined the physiological consequences for the epithelial component of intestine, with special attention to its barrier function. Prostanoid receptors were selectively expressed on a limited number of human colonic mucosal cells, and differed markedly between normal and inflamed tissue. In non-inflamed mucosa, EP(2)and EP(3)were expressed on epithelia at the apex of crypts; while EP(4)was expressed on surface and lateral crypt epithelia. Dual immunostaining and in situ hybridization with digoxygenin-labelled RNA probes largely confirmed the epithelial localization of EP(4). On the other hand, during inflammation, lateral crypt (non-surface) epithelial cells newly and significantly expressed prostanoid receptors EP(2)and EP(3)(p<0.05, by computer-assisted densitometry). Functionally, exogenous E series prostanoids applied to epithelial monolayers in nM concentrations brought about a 24% increase in the level of barrier function; an associated rise in intracellular cAMP (EC(50)of 281); and protection of epithelium from the effects of T cell cytokines. A major perturbation in the number and distribution of functional eicosonoid receptors on epithelia occurs in chronic inflammation of human colonic mucosa.     

Transforming growth factor-β1 preserves epithelial barrier function: Identification of receptors,biochemical intermediates,and cytokine antagonists

Freshly isolated human mucosal T lymphocytes in vitro can markedly diminish an important property of intestinal epithelium—its barrier function. On the other hand, cytokines and their cellular receptors, which maintain homeostasis of epithelia, limit epithelial permeability, and preserve barrier function, are not well characterized. Using a described human colonic epithelial cell monolayer system, we found that transforming growth factor-β1 (TGF-β1) preserved 75% or more of epithelial barrier function, quantitated electrophysiologically, even in the presence of cytokines generated by a high density of barrier-disruptive mucosa-derived mononuclear cells. In opposing the TGF-β1 effect, cytokines able to reduce barrier function were spontaneously secreted by mucosal T cells and were increased in their barrier effect after T-lymphocyte activation. Further, neutralization of individual cytokines with specific monoclonal antibodies abrogated the lymphocyte-induced reduction in epithelial barrier function, and identified interferon gamma (IFN-γ), interleukin (IL)-4, and IL-10, but not IL-6, as the primary cytokines whose barrier effects were curtailed by TGF-β1. Receptors (RI and RII) for TGF-β1 were found to be localized primarily to the apical and basal membranes of surface epithelium in colonic crypts. These findings provide the scientific basis for new strategies to pharmacologically enhance the barrier function of epithelia in mucosal organs regularly exposed to environmental antigens and to T-lymphocyte products. J. Cell. Physiol. 181:55–66, 1999. © 1999 Wiley-Liss, Inc.     

Effects of BAMBI on luteinized follicular granulosa cell proliferation and steroid hormone production in sheep

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) regulates mammalian ovarian follicle growth and maturation; however, its effect on luteinized granulosa cells (LGCs) in sheep ovarian follicles remains unknown. Here we explored the regulatory role of LGC functions and steroid hormone synthesis by BAMBI. Multiple sequence alignment revealed that the sheep BAMBI gene sequence was relatively conserved. Sheep LGCs were strongly positive for BAMBI. LGC proliferation increased when BAMBI was silenced and decreased when BAMBI was overexpressed. After BAMBI overexpression, the expression of CASP3, CASP8, CASP9, and BAX significantly increased, whereas that of BCL2 and the ratio of BCL2/BAX expression decreased. The opposite was observed after BAMBI silencing. CDKN1A, CCND1, and CCND2 were downregulated with BAMBI overexpression and upregulated with BAMBI silencing. Expression of steroid hormone-related genes (CYP11A1, STAR, and 3BHSD), except CYP19A1, significantly increased after BAMBI overexpression. Moreover, estrogen and progesterone secretion increased after BAMBI overexpression and decreased after BAMBI interference. The effect of the exogenous addition of bone morphogenetic protein 2 (BMP2) on GCs was similar to that of BAMBI overexpression. In conclusion, BAMBI can regulate the proliferation and steroid hormone synthesis of sheep LGCs, and BMP2 can affect LGCs as an activator of BAMBI. These findings provide a basis for further research on the physiological role of BAMBI.     

Neurokinin-1 receptor (NK-1R) expression is induced in human colonic epithelial cells by proinflammatory cytokines and mediates proliferation in response to substance P

We have previously shown that the receptor for substance P (SP), neurokinin-1 receptor (NK-1R), is a marker of human mucosal but not peripheral mononuclear cells. In the present study, we investigate NK-1R expression in the human colonic mucosa in vivo, particularly in the epithelial cells. We investigate the influence of proinflammatory Th1 cytokines and SP on expression and function of NK-1R in colonic epithelial cells in vitro. Using in situ hybridization to detect NK-1R mRNA, and immunohistochemistry to detect NK-1R protein, colonic epithelial cells were found to express NK-1R in vivo. In contrast, colon epithelial cell lines (Caco-2, HT29, SW620, T84) were negative for NK-1R mRNA and protein. However, stimulation with a proinflammatory cytokine cocktail containing IFN-gamma, TNF-alpha, and IL-1beta, caused induction of NK-1R expression. Expression of NK-1R in human colonic epithelial cells in vivo may therefore reflect cytokine conditioning by the mucosal microenvironment. SP did not alter ion transport in monolayers of cytokine-treated T84 cells. While SP stimulated epithelial ion transport in colonic mucosae ex vivo, this was not a direct effect of SP on the epithelial cells, and appeared to be neurally mediated. However, SP (10(-10)-10(-8) M) elicited a dose-dependent proliferative effect on cytokine-stimulated, but not unstimulated, SW620 cells. Proliferation of the epithelial cells in response to SP was mediated specifically via cytokine-induced NK-1R, since an NK-1R-specific antagonist (Spantide 1) completely blocked SP-mediated proliferation in the cytokine-treated cells. Our results therefore demonstrate that proinflammatory cytokines induce expression of NK-1R in human colonic epithelial cell lines, and that SP induces proliferation of the epithelial cells via cytokine-induced NK-1R.     

Ultrastructural and cytoarchitectural features of lymphoreticular organs in the colon and rectum of adult BALB/c mice

The structure and function of colonic mucosal lymphoid organs remain largely unexplored, especially in the rectum hidden within the pelvic vault. Two-month-old female BALB/c mice were anesthetized, and the entire colon was removed from cecum to anus. Distal colonic patches were then prepared for electron microscopy or were quick-frozen and sectioned for immunoperoxidase localization of B cells and T cell subsets. Aggregated lymphoid follicles were distributed irregularly along the entire colon with an average of 1.4 patches per centimeter of colon length. There were large collections of follicles opposite the ileocecal valve (cecal patches), variable numbers of patches throughout the colon, and at least one patch within 10 mm of the anus (rectal patch). Follicles were adjacent to branching crypts lined by epithelium infiltrated by lymphoid cells and containing few goblet cells. In electron micrographs, M cells were identified by their short, irregular microvilli; intraepithelial lymphoid cells; reduced lysosomal dense bodies; and an expanded tubulovesicular network. Small germinal centers were seen. Cytoarchitectural components of colonic lymphoid follicles and Peyer's patch follicles were remarkably similar, despite differences in surrounding mucosa and luminal microbial exposure. The presence of organized lymphoid tissue with M cells and germinal centers suggests that transepithelial particle transport and antigen recognition can take place in the rectum. Whether such tissue has the capacity for uptake of luminal microorganisms is of particular interest, not only because colonic follicles may be sites for local initiation of immune responses but also because they may be important entry points for systemic infection.     

Properties of the esophageal epithelium in relation to organizative patterns of lymphoid infiltrations in the red-eared turtle

The esophagus of the turtle, like the mucosal surfaces in other species, contains variously sized areas of lymphoid infiltration. The tunica propria and the surface epithelial layer of this area are invaded by the lymphoid cells. The features of the layer of epithelial cells which cover the lymphoid infiltrations are of a special kind: they do not possess vibratile cilia and are able to take up materials flowing into the lumen. The present paper contains further information concerning lymphoid infiltration obtained by histological and histochemical methods. The epithelial layer covering the lymphoid infiltrations is composed of cells with irregularly distributed microvilli, ciliated cells and mucous-secreting cells. After administration of silica and colloidal carbon, the microvillar epithelial cells proved to have these substances inside them, thereby accounting for the pinocytotic activity. The absorbing epithelial cells were not damaged by silica which is a macrophage-toxic agent, while the underlying macrophages are damaged. These results are compared with the features of lymphoid infiltration associated cells in various organs and animals; the hypothesis is proposed that these cells in the esophagus of turtles may originate from the covering epithelial cells.     

Colonic epithelial cells express specific ligands for mucosal macrophage immunosuppressive receptors siglec-7 and -9

Immune cells are known to express specific recognition molecules for cell surface glycans. However, mechanisms involved in glycan-mediated cell-cell interactions in mucosal immunity have largely been left unaccounted for. We found that several glycans preferentially expressed in nonmalignant colonic epithelial cells serve as ligands for sialic acid-binding Ig-like lectins (siglecs), the immunosuppressive carbohydrate-recognition receptors carried by immune cells. The siglec ligand glycans in normal colonic epithelial cells included disialyl Lewis(a), which was found to have binding activity to both siglec-7 and -9, and sialyl 6-sulfo Lewis(x), which exhibited significant binding to siglec-7. Expression of these siglec-7/-9 ligands was impaired upon carcinogenesis, and they were replaced by cancer-associated glycans sialyl Lewis(a) and sialyl Lewis(x), which have no siglec ligand activity. When we characterized immune cells expressing siglecs in colonic lamina propriae by flow cytometry and confocal microscopy, the majority of colonic stromal immune cells expressing siglec-7/-9 turned out to be resident macrophages characterized by low expression of CD14/CD89 and high expression of CD68/CD163. A minor subpopulation of CD8(+) T lymphocytes also expressed siglec-7/-9. Siglec-7/-9 ligation suppressed LPS-induced cyclooxygenase-2 expression and PGE(2) production by macrophages. These results suggest that normal glycans of epithelial cells exert a suppressive effect on cyclooxygenase-2 expression by resident macrophages, thus maintaining immunological homeostasis in colonic mucosal membranes. Our results also imply that loss of immunosuppressive glycans by impaired glycosylation during colonic carcinogenesis enhances inflammatory mediator production.     

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberkühn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.     

FOXP3+ cell density in lymphoid follicles from histologically normal mucosa is a strong prognostic factor in early stage colon cancer

There are few clearly established prognostic factors available to guide the use of adjuvant chemotherapy in early stage colon cancer patients. Some of the most promising candidates include the invasion of extramural blood vessels by tumour cells and the densities of FOXP3+ T regulatory cells (Tregs) in tumour and adjacent normal colonic mucosal tissue. The aim of our study was to evaluate the prognostic significance of these markers in AJCC stage II colon cancer, with particular reference to lymphoid follicles in the mucosa. Histopathological review for the presence of vascular and serosal invasion was conducted on a series of 165 stage II colon cancers treated by surgery alone. Immunohistochemical staining for FOXP3 was performed on tumour tissue and histologically normal colonic mucosa from the surgical margin. Image analysis software was used to evaluate the density of FOXP3+ cells in the tumour core, invading margin and lymphoid follicles from the colonic mucosa. For survival analysis, cases were classified into high- or low-density of FOXP3+ cells according to the median value. The mean density of FOXP3+ Tregs in lymphoid follicles was twofold and fivefold higher than in the invading margin and tumour core, respectively. Multivariate analysis identified extramural vascular invasion (HR, 2.47; 95% CI: 1.00-6.07; P?=?0.05) and high FOXP3+ cell density in lymphoid follicles (HR, 4.22; 95% CI: 1.49-11.91; P?=?0.007) as independent factors for worse survival, whereas a high frequency of lymphoid follicles in histologically normal colonic mucosa was associated with better survival (HR, 0.31; 95% CI: 0.12-0.79; P?=?0.014). Our data suggest that host factors related to the immune system have major prognostic significance in early stage colon cancer. The density of FOXP3+ cells within lymphoid follicles and the frequency of these structures in normal colonic mucosa represent novel and independent prognostic factors.     

Transepithelial transport and mucosal defence II: secretion of IgA

In this second article on mucosal defence and transepithelial transport, Jean-Pierre Kraehenbuhl and Marian Neutra discuss the part played by a special class of antibody, polymeric IgA, in the protection of mucosal surfaces lining the digestive, respiratory and genital tracts, and the implications for mucosal vaccines. Polymeric IgA crosslinks luminal antigens or pathogens, thus preventing their interaction with epithelial cells. Following stimulation by antigen in the organized mucosal lymphoid tissue, effector B lymphocytes enter the circulation and migrate to distant mucosal or glandular sites, where they differentiate into polymeric-IgA-producing plasma cells. These antibodies reach the environment by transport across the epithelial cells of mucosal and glandular tissues.     

Hepatocyte growth factor enhances IL-1β stimulated IL-8 secretion by Caco-2 epithelial cells

Hepatocyte growth factor (HGF) can induce proliferation and migration of intestinal epithelial cells and has also been shown to be important in wound healing of inflamed mucosal tissues. HGF is known to be expressed along with interleukin-1 (IL-1) by inflamed mucosal tissues, yet the effect of HGF on IL-1-induced proinflammatory cytokine responses by colonic epithelial cells is unknown. In this report, we have examined the effect of HGF on IL-1-induced secretion of IL-8 by the Caco-2 colonic epithelial cell line. HGF stimulation alone had no effect on the secretion of IL-8 by the Caco-2 cells. However, culture of the cells with HGF and suboptimal levels of IL-1 resulted in a significant enhancement of IL-8 secretion compared to cells cultured with IL-1 alone. A similar effect was seen with HGF and IL-1 simulation of monocyte chemoattractant protein-1 secretion by the rat IEC-6 intestinal epithelial cell line. The enhancing effect of HGF was seen regardless of whether the culture medium contained serum or not. Simultaneous stimulation with HGF and IL-1 was required for the enhancing effect as cells pretreated with HGF for 24 h and then stimulated with IL-1 alone secreted IL-8 levels similar to that of cells stimulated with IL-1 alone. These results suggest that in addition to wound healing, HGF may play a role in the IL-1-induced chemokine response of epithelial cells in inflamed mucosal tissues.     

Gut- and bronchus-associated lymphoid tissue

Bronchus-associated and gut-associated lymphoid tissues (BALT and GALT) have both functional and morphologic similarities and are involved in seeding lung, gut, and other mucosal sites with predominantly IgA-containing B cells. Both types of lymphoid tissue are engaged in the regulation and the controlled amplification of immune responses, which vary from positive mucosal responses in both mucosae and peripheral tissues to local mucosal responses and systemic tolerance. Their further involvement in provision of cells destined to reside in the epithelial compartment of the body appears likely but requires further investigation. Their role in the provision of precursors of mucosal mast cells must also be explored further, but some participation in this event appears likely. The mucosa-associated lymphoid tissue (MALT) system appears to be integrated with the systemic immune system but may be considered as separate from it in several functional ways.     

Current Concepts of the Pathogenesis and Pathology of Inflammatory Lesions of the Intestine

The histopathologic lesions of regional enteritis and ulcerative colitis, particularly in their early stages, are distinct and distinguishable, irrespective of the sites that are involved. Regional enteritis is characterized by lymphangiectasis, lymphedema, lymphoid hyperplasia, and granulomatous inflammation of the submucosal and subserosal layers of intestine, whereas chronic ulcerative colitis is an exudative, ulcerative disorder of the mucosal layer that commences with “crypt abscesses” and only in its later stages progresses to deeper coats of the wall.Electron microscopy of a rectal biopsy from a juvenile patient with chronic ulcerative colitis for five years disclosed a labyrinthine system of clefts and compartments between columnar, mucosal epithelial cells. Regenerated colonic epithelial cells were of primitive, germinal type and featured a “vesicular” rather than a “goblet” pattern of mucus secretion. Clusters of small “clavate fimbriae” projected from the tips of microvilli. Each of these newly recognized substructures measured 30 to 60 mμ. in diameter, and was enclosed by a tri-laminar “unit membrane”, derived from the surface plasma membrane of the cell.     

Carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1), apically expressed on human colonic M cells,are potential receptors for microbial adhesion

In the human gut mucosa, specialized M cells deliver intact foreign macromolecules and commensal bacteria from the lumen to organized mucosal lymphoid tissues triggering immune responses. M cells are also major sites of adhesion and invasion for enteric pathogens. The molecular features of M cell apical surfaces that promote microbial normal attachment are still largely unknown. We have demonstrated previously that in the human colonic epithelium, carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1) are integral components of the apical glycocalyx which participate in epithelial–microbial interactions. In this study, based on the reactivity of specific monoclonal antibodies and on immunoelectron microscopy, we show that M cells of human colonic solitary lymphoid follicles express CEA and CEACAM1 on the apical surface. Recently these highly glycosylated molecules have been characterized as protein receptors for different bacteria. This leads us to propose a role for CEA and CEACAM1 in the adherence of enteric bacteria to the apical membrane of colonic M cells. We also hypothesize that, unlike colonic enterocytes, M cells lack the defense mechanism that eliminates CEA and CEACAM1 upon microbial binding and which is based on vesiculation of microvillus plasma membrane.     

江山乌猪肠道黏膜菌群组成及其功能的性别差异

肠道黏膜微生物在调控宿主生理功能方面发挥重要作用,其结构组成受到多种因素影响。性别被认为是塑造肠道微生物的因素之一。然而,性别对肠道黏膜菌群的差异影响还不清楚。【目的】以江山乌猪为研究对象,探究性别差异对其肠道黏膜微生物组成及功能的影响。【方法】选取性成熟的雌性和雄性江山乌猪各8头,利用16S rRNA基因高通量测序技术分析回肠和结肠黏膜菌群。【结果】在回肠黏膜中,雄性江山乌猪菌群的Chao1指数和Shannon指数显著高于雌性(P<0.05),在结肠黏膜中,不同性别江山乌猪菌群Chao1指数和Shannon指数无显著差异(P>0.05)。菌群差异分析显示,回肠黏膜中,沙雷氏菌属(Serratia)和埃希氏志贺菌属(Escherichia_Shigella)在雌性组中的相对丰度显著高于雄性组(P<0.05),雄性组中Oscillospiraceae UCG-005、拟普雷沃氏菌属(Alloprevotella)、布劳特氏菌属(Blautia)和Prevotellaceae_NK3B31_group相对丰度显著高于雌性组(P<0.05);结肠黏膜中,雌性组中unclassified_Muribaculaceae、Rikenellaceae_RC9_gut_group和Prevotellaceae UCG-003相对丰度显著高于雄性组(P<0.05),Oscillospiraceae UCG-005、Lachnospiraceae_NK4A136_group和unclassified_Lachnospiraceae在雄性组中相对丰度更高(P<0.05)。功能预测发现,雄性乌猪回肠黏膜菌群显著富集了氨基酸代谢、碳水化合物代谢和能量代谢等功能途径(P<0.05);结肠黏膜菌群主要富集了膜转运相关的ABC转运蛋白和信号转导相关的双组分系统等功能途径(P<0.05)。【结论】不同性别江山乌猪肠黏膜菌群结构及功能具有明显差异。这些结果揭示了不同性别江山乌猪肠道黏膜菌群的差异特征,为了解和挖掘我国地方畜禽品种肠道微生物资源提供部分参考。