Showing papers on "Epithelium published in 1994"

E-cadherin null mutant embryos fail to form a trophectoderm epithelium

Abstract: The cell adhesion molecule E-cadherin mediates the compaction process of mouse preimplantation embryos and is important for the maintenance and function of epithelial cell layers. To determine precisely the role of E-cadherin in epithelial biogenesis we monitored the developmental potential of embryos homozygously negative for E-cadherin that were derived from E-cadherin heterozygous transgenic mice. The homozygous negative embryos died around the time of implantation, although they did undergo compaction like their littermate controls, largely due to the presence of residual maternal E-cadherin. At the blastocyst stage, E-cadherin-negative embryos failed to form a trophectodermal epithelium or a blastocyst cavity. These results demonstrate the pivotal role of E-cadherin in one of the most basic morphogenetic events in the development of multicellular organisms, the biogenesis of an epithelium.

CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.

Abstract: A major limitation in the study of vectorial ion transport, secretion, and differentiated function in the human airway epithelium has been the lack of suitable cell culture systems. Progress in this direction has been made through the transformation of primary cultured epithelial cells. However, these transformants tend to lose differentiated properties with increasing serial passage, particularly following crisis. The suc­ cessful establishment of a postcrisis SV40 large T-antigen transformed epithelial cell line derived from human bronchial epithelium is described. This cell line, 16HBEI40-, retains differentiated epithelial mor­ phology and functions. Cell cultures show the presence of tight junctions and cilia, and monolayers gener­ ate transepithelial resistance, as measured in Ussing chambers, and retain iJ-adrenergic stimulation of cAMP-dependent chloride ion transport, measured either by ,6CI- efflux or as short-circuit current in Ussing chambers. The cells also increase chloride transport in response to bradykinin or calcium iono­ phore. In addition, 16HBE140-cells express levels of both the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein readily detectable by Northern and Western hybridization analysis, respectively. These cells provide a valuable resource for studying the modulation of CFTR and its role in regulation of chloride ion transport in human airway epithelium as well as other aspects of human airway cell biology. The human airway epithelium is pseudostratified, consisting of highly organized layers of polar cells with specific dif­ ferentiated functions. It includes ciliated columnar cells, basal cells, and secretory goblet cells that are linked by tight junctions. The tight junctions provide a barrier between the airway lumen and the underlying tissues and divide the epi­ thelial cells into apical and basolateral domains. Both of these plasma membrane compartments contain different populations of proteins that allow for directional flux of ions

Salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial M cells of the Peyer's patches.

Abstract: Salmonella species are known to initiate infection of mammalian hosts by penetrating the intestinal epithelium of the small bowel. These bacteria preferentially interact with Peyer's patches which are collections of lymphoid follicles making up the gut-associated lymphoid tissue. We infected murine ligated intestinal loops with invasive and noninvasive Salmonella typhimurium strains for 30, 60, 120, and 180 min and examined the infected tissue by transmission electron microscopy. Within 30 min, we found that invasive S. typhimurium exclusively entered M cells found within the follicle-associated epithelium (FAE) of the Peyer's patches. Initially, interactions between invasive bacteria and enterocytes adjacent to the M cells were not found. Invasion of M cells was associated with the ability of the bacteria to invade tissue culture cells. S. typhimurium mutants, which were noninvasive for tissue culture cells, could not be found in ligated loops associated with M cells or enterocytes after incubations of 30, 60, 120, or 180 min. At 60 min, internalized invasive S. typhimurium were cytotoxic for the M cells. Destruction of an M cell formed a gap in the FAE which allowed organisms to invade enterocytes adjacent to the dead cell. Later in the infection process (120 and 180 min), the presence of bacteria beneath the FAE correlated with changes in the cytoarchitecture of the lymphoid follicle. In addition, replicating Salmonella began to enter both the apical and basolateral surfaces of enterocytes adjacent to infected M cells.

Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis

Abstract: The regulation of cell number in adult tissues is determined by the balance of cell production and cell loss. In the gastrointestinal tract, where there are well defined zones of proliferation and migration of both epithelial cells and associated fibroblasts, it is widely held that cell loss occurs by shedding into the gut lumen. Since the evidence for this is not compelling, we investigated the distribution and amount of apoptosis in the normal mammalian gut. In the stomach, small intestine and colon of rodents and man, there is a small number of apoptotic bodies in the epithelium and in the immediate sub-epithelial connective tissue. Engulfment by adjacent epithelial cells and sub-epithelial macrophages accounts for the removal of apoptotic bodies. Apoptotic bodies are not randomly distributed but are found towards the distal end of the known cellular migration routes of both epithelial and mesenchymal cells. Furthermore, consideration of the absolute numbers of apoptotic bodies, their rapid clearance and the dimensions of the small intestinal villi and colonic crypts indicates that the cell loss in the normal murine intestine can largely be explained on the basis of the observed apoptosis. Despite being inconspicuous in histological material, apoptosis probably accounts for the bulk of cell loss in the gut and is a central feature of the regulation of cell number in adult tissues.

Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia

Abstract: A new member of the metallothionein (MT) gene family was discovered that lies about 20 kb 5' of the MT-III gene in both mouse and human. The MT-IV proteins are highly conserved in both species and have a glutamate insertion at position 5 relative to the classical MT-I and MT-II proteins. Murine MT-IV mRNA appears to be expressed exclusively in stratified squamous epithelia associated with oral epithelia, esophagus, upper stomach, tail, footpads, and neonatal skin. The MT derived from tongue epithelium contains both zinc and copper. Many of these epithelia develop parakeratosis during zinc deficiency in the rat. In situ hybridization reveals intense labeling of MT-IV mRNA in the differentiating spinous layer of cornified epithelia, whereas MT-I is expressed predominantly in the basal, proliferative layer; thus, there is a switch in MT isoform synthesis during differentiation of these epithelia. We suggest that MT-IV plays a special role in regulating zinc metabolism during the differentiation of stratified epithelia.

Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction.

Abstract: Extracellular matrix (ECM) profoundly influences the growth and differentiation of the mammary gland epithelium, both in culture and in vivo. Utilizing a clonal population of mouse mammary epithelial cells that absolutely requires an exogenous ECM for function, we developed a rapid assay to study signal transduction by ECM. Two components of the cellular response to a basement membrane overlay that result in the expression of the milk protein beta-casein were defined. The first component of this response involves a rounding and clustering of the cells that can be physically mimicked by plating the cells on a nonadhesive substratum. The second component is biochemical in nature, and it is associated with beta 1 integrin clustering and increased tyrosine phosphorylation. The second component is initiated in a morphology-independent manner, but the proper translation of this biochemical signal into a functional response requires cell rounding and cell clustering. Thus, physical and biochemical signal transduction events contribute to the ECM-dependent regulation of tissue-specific gene expression in mouse mammary epithelial cells.

Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung.

Abstract: Mouse lung development begins when two lung buds sprout from the epithelium of the embryonic gut. Patterning of the airways is then accomplished by the outgrowth and repetitive branching of the two lung buds, a process called branching morphogenesis. One of the four fibroblast growth factor (FGF) receptor genes, FGFR2, is expressed in the epithelium of a number of embryonic organs including the lung buds. To block the function of FGFR2 during branching morphogenesis of the lung without affecting its function in other embryonic tissues, the human surfactant protein C promoter was used to target expression of a dominant negative FGFR2 exclusively to lung bud epithelium in transgenic mice. Newborn mice expressing the transgene were completely normal except that instead of normally developed lungs they had two undifferentiated epithelial tubes that extended from the bifurcation of the trachea down to the diaphragm, a defect that resulted in perinatal death. Thus, the dominant negative FGF receptor completely blocked airway branching and epithelial differentiation, without prohibiting outgrowth, establishing a specific role for FGFs in branching morphogenesis of the mammalian lung.

Rapid dendritic cell recruitment is a hallmark of the acute inflammatory response at mucosal surfaces.

Abstract: Immunohistochemical analysis of challenge sites such as skin and the peritoneal cavity has identified neutrophils as virtually the sole cellular participants in acute bacterial inflammation, peak influx occurring 24-48 h in advance of mononuclear cell populations associated with adaptive immunity. This study challenges the general applicability of this paradigm. We demonstrate here that the earliest detectable cellular response after inhalation of Moraxella catarrhalis organisms is the recruitment of putative class II major histocompatibility complex-bearing dendritic cell (DC) precursors into the airway epithelium, the initial wave arriving in advance of the neutrophil influx. Unlike the neutrophils which rapidly transit into the airway lumen, the DC precursors remain within the epithelium during the acute inflammatory response where they differentiate, and develop the dendriform morphology typical of resident DC found in the normal epithelium. During the ensuing 48-h period, these cells then migrate to the regional lymph nodes. No comparable DC response was observed after epidermal or intraperitoneal challenge, and it may be that mucosal surfaces are unique in their requirement for rapid DC responses during acute inflammation. We hypothesize that the role of the DC influx during acute inflammation may be surveillance for opportunistic viruses, and that this covert protective mechanism is operative at a restricted number of mucosal tissue sites.

CYP3A gene expression in human gut epithelium.

Abstract: CYP3A4, a major Phase I xenobiotic metabolizing enzyme present in liver, is also present in human small bowel epithelium where it appears to catalyse significant 'first pass' metabolism of some drugs. To determine whether CYP3A4 or the related enzymes CYP3A3, CYP3A5, and CYP3A7 are present in other regions of the digestive tract, we used CYP3A-specific antibodies to examine histological sections and epithelial microsomes obtained from a human organ donor. CYP3A-related proteins were detected in epithelia throughout the digestive tract and in gastric parietal cells, in pericentral hepatocytes, and in ductular cells of the pancreas. Immunoblot analysis suggested that the major CYP3A protein present in liver, jejunum, colon, and pancreas was CYP3A4 or CYP3A3, whereas CYP3A5 was the major protein present in stomach. Both CYP3A4 and CYP3A5 mRNA were detectable in all regions of the digestive tract using the polymerase chain reaction (PCR); however, only CYP3A4 could be detected by Northern blot analysis. CYP3A7 mRNA was consistently detected only in the liver by PCR and CYP3A3 mRNA was not detected in any of the tissues. We conclude that CYP3A4 and CYP3A5 are present throughout the human digestive tract and that differences in the expression of these enzymes may account for inter-organ differences in the metabolism of CYP3A substrates.

Patterns of matrix metalloproteinase expression in cycling endometrium imply differential functions and regulation by steroid hormones.

Abstract: Matrix metalloproteinases are a highly regulated family of enzymes, that together can degrade most components of the extracellular matrix. These proteins are active in normal and pathological processes involving tissue remodeling; however, their sites of synthesis and specific roles are poorly understood. Using in situ hybridization, we determined cellular distributions of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1, an inhibitor of matrix metalloproteinases, in endometrium during the reproductive cycle. The mRNAs for all the metalloproteinases were detected in menstrual endometrium, but with different tissue distributions. The mRNA for matrilysin was localized to epithelium, while the others were detected in stromal cells. Only the transcripts for the 72-kD gelatinase and tissue inhibitor of metalloproteinases-1 were detected throughout the cycle. Transcripts for stromelysin-2 and the 92-kD gelatinase were only detected in late secretory and menstrual endometrium, while those for matrilysin, the 72-kD gelatinase, and stromelysin-3 were also consistently detected in proliferative endometrium. These data indicate that matrix metalloproteinases are expressed in cell-type, tissue, and reproductive cycle-specific patterns, consistent with regulation by steroid hormones, and with specific roles in the complex tissue growth and remodeling processes occurring in the endometrium during the reproductive cycle.

Interleukin-8 expression in Helicobacter pylori infected, normal, and neoplastic gastroduodenal mucosa.

Abstract: AIMS--To investigate the expression of interleukin-8 (IL-8) in Helicobacter pylori infected normal and neoplastic gastroduodenal mucosa, and in established gastric cancer cell lines. METHODS--Immunofluorescence techniques were used to localise IL-8 in cryosections of gastric (n = 25) and duodenal (n = 17) endoscopic biopsy specimens an in resected gastric tumour tissue samples from 16 patients. Two gastric cancer cell lines (Kato 3 and MKN 45) were examined for IL-8 protein expression by immunofluorescence and for the presence of IL-8 mRNA by reverse transcription followed by the polymerase chain reaction (RT-PCR). RESULTS--IL-8 was localised to the epithelium in histologically normal gastric mucosa, with particularly strong expression in the surface cells. IL-8 expression was also a feature of surface epithelium in the duodenal bulb, but was much reduced in the second part of the duodenum. In chronic H pylori-associated gastritis gastritis gastric epithelial IL-8 expression was increased and expression of IL-8 within the lamina propria was evident. By contrast, large areas of IL-8 negative epithelium were observed in the body mucosa of a subject with Menetrier's disease. In gastric carcinoma the tumour cells were positive for IL-8. IL-8 was also detected by immunofluorescence in unstimulated Kato 3 and MKN 45 cells, and constitutive IL-8 gene expression in these cell lines was confirmed by detection of IL-8 mRNA by RT-PCR. CONCLUSIONS--Immunoreactive IL-8, a potent neutrophil chemotactic and activating factor, is present in the epithelium of both normal and inflamed gastric mucosa with increased expression in the latter. There is site dependent variation in epithelial IL-8 expression within the gastroduodenal mucosa. The expression of the pro-inflammatory cytokine IL-8 in gastric carcinoma cells may influence peritumoural cellular infiltrates.

Cell number and distribution in human and rat airways.

Abstract: Morphometric procedures were used to determine the number of cells, cell volume, cell diameter, and surface areas of the airways in human and rat lungs. Nuclear sizes of epithelial cells from human bronchi were significantly larger than other lung cell nuclei. The average volume of human ciliated cell nuclei was 310 +/- 30 microns 3 and 167 +/- 12 microns 3 in bronchi and bronchioles, respectively. The smaller nuclei of human bronchioles were comparable to those of alveolar cells. In the pseudostratified epithelium of human bronchi, basal cells had a large surface area in contact with the basement membrane (51.3 +/- 4.6 microns 2 per cell) when compared with ciliated (1.1 +/- 0.1 microns 2), goblet (7.6 +/- 1.2 microns 2), or other secretory cells (12.0 +/- 2.1 microns 2). In the first four airway generations distal to the trachea, basal cells account for 30% of the cells in human airway epithelium and 2% of the cells in rat airway epithelium. Total airway surface area from trachea to bronchioles was 2,471 +/- 320 and 27.2 +/- 1.7 cm2 in human and rat lungs, respectively. These direct measurements of airway surface area are less than half of the estimates based on current lung models. The total number of airway epithelial cells were 10.5 x 10(9) for human and 0.05 x 10(9) for rat lungs. For both species, there were 18 times more alveolar cells than bronchial epithelial cells.

Role of mesenchymal-epithelial interactions in normal and abnormal development of the mammary gland and prostate.

Abstract: Development of the mammary gland (MG) and prostate occurs via mesenchymal-epithelia interactions. Epithelial MG buds are induced in ventral epidermis by mammary mesenchyme, which ultimately specifies the functional expression of the ability to produce milk. Mammary ductal branching is induced by embryonic mammary mesenchyme and is promoted by the mammary fat pad postnatally. These influences of connective tissue on the differentiation of mammary epithelium (ME) begin prenatally, but in adulthood, the connective tissue environment of adult ME profoundly influences epithelial growth, ductal branching, epithelial differentiation, and the ability of adult ME to produce milk. In a similar fashion, prostatic development occurs via mesenchymal-epithelial interactions in which urogenital sinus mesenchyme (UGM) induces epithelial morphogenesis, regulates epithelial proliferation, and evokes the expression of epithelial androgen receptors and prostate-specific secretory proteins. Although prostatic development is induced by androgens, androgenic effects on epithelial development are elicited via androgen receptors of UGM. As in MG, mesenchymal-epithelial interactions in the prostate begin during fetal periods, but continue into adulthood. The responsiveness of adult epithelial cells from various glands to stroma raises the possibility that carcinomas also may be regulated by connective tissue. Indeed, UGM can induce a rat prostatic carcinoma (Dunning tumor) to undergo striking changes in differentiation, which are accompanied by a reduction in growth rate and an apparent loss of tumorigenesis. Although the mechanism of mesenchymal-epithelial interactions remains unknown, the communication between the epithelium and stroma undoubtedly is multifactorial, involving the extracellular matrix, soluble growth or differentiation, and angiogenesis.

Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

Abstract: Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut.

Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization.

Abstract: We have used in situ hybridization to localize expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the human gastrointestinal tract and associated organs. The stomach exhibits a low level of CFTR expression throughout gastric mucosa. In the small intestine, expression is relatively high in the mucosal epithelium, with a decreasing gradient of expression along the crypt to tip axis. The cells of the Brunner's glands express high levels of CFTR mRNA. In addition, there is a small subpopulation of highly positive cells scattered along the epithelium in the duodenum and jejunum, but not in the ileum. These cells do not represent endocrine cells, as determined by lack of colocalization with an endocrine-specific marker. The distribution of CFTR mRNA in the colon is similar to the small intestine, with highest level of expression in the epithelial cells at the base of the crypts. In the pancreas, CFTR is expressed at high levels in the small, intercalated ducts and at lower levels in the interlobular ducts. CFTR transcripts are expressed at uniformly high levels in the epithelium of the gallbladder. Throughout the gastrointestinal tract, CFTR expression is increased in mucosal epithelial cells that are near lymph nodules.

Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro.

Abstract: Endometriosis is a common gynecological disorder of unclear pathogenesis. We have established an in vitro model to investigate phenotypic similarities and differences between normal endometrial and endometriosis cells. Highly purified cultures of epithelial and stromal cells were isolated from normal endometrium and endometriosis implants. Morphological features as well as immunocytochemical markers confirm these isolates as epithelial and stromal cells. Potential hormone responsiveness was established by the documentation of estrogen receptor mRNA in epithelial and stromal cells isolated from both tissue types. Expression of this receptor protein was verified in stromal cells by competitive radioligand binding, revealing comparable receptor numbers and dissociation constants. CA-125 is selectively secreted in similar concentrations by epithelial cells isolated from both tissue types. PRL secretion is selectively exhibited by progestin-stimulated stromal cells from both tissue types. Our findings demonstrate that highly purified epithelial and stromal cells cultured from normal endometrial and endometriosis tissues express the same phenotypic and functional markers as their in vivo counterparts. These cultures provide useful models to identify endometriosis-specific cell products that contribute to the pathogenesis of this disorder.

The polymorphic epithelial mucin MUC1 in human endometrium is regulated with maximal expression in the implantation phase.

Abstract: After ovulation, progesterone stimulates a temporally regulated secretory transformation in human endometrial epithelium. Using a combination of immunohistochemistry, and Western and Northern blotting, we demonstrate that 1) the polymorphic epithelial mucin MUC1 is secreted by human endometrial epithelium; 2) low levels of both mRNA and core protein are present in the preovulatory phase of the menstrual cycle; 3) mRNA levels increase several-fold after ovulation, consistent with transcriptional regulation by progesterone; 4) there is an increase in translation product in postovulatory endometrium; and 5) the tandem repeat domain of the MUC-1 polypeptide is glycosylated in endometrium.

Demonstration and Characterization of the Angiogenic Properties of Cervical Dysplasia

Abstract: Cervical dysplasia, or cervical intraepithelial neoplasia (CIN), is a premalignant precursor to cervical cancer. This study was designed to determine whether dysplastic lesions are angiogenic. Tissue sections from 23 surgical specimens were immunohistochemically stained for factor VIII antigen, a marker for endothelial cells. The results demonstrate that a region of neovascularization develops along the basement membrane subtending dysplastic epithelium when compared to adjacent normal epithelium. Comparison of microvessel counts underlying low grade lesions (condyloma and CIN I) with microvessel counts of CIN III lesions shows a statistically significant increase in the more advanced lesions. In a subset of the high grade lesions, large vascular structures are also noted in the upper layers of the epithelium, suggesting that a second stage of neovascularization consists of extension of stromal vascular papillae into the dysplastic lesions toward the surface of the epithelium. There is no statistical correlation between the amount of inflammation and the angiogenic ratio for each lesion, implying that angiogenesis is not secondary to the inflammatory response evoked by the lesion. The human papillomavirus type present in four CIN III lesions was determined by in situ hybridization; the amount of angiogenesis appears to be independent of the human papillomavirus type.

Increased expression of the monocyte chemoattractant protein-1 in bronchial tissue from asthmatic subjects.

Abstract: The expression of the monocyte chemoattractant protein (MCP-1), a member of the chemokine family of low molecular weight cytokines, was assessed by immunohistochemistry in bronchial biopsies from 12 asthmatic and 12 normal subjects. Both a monoclonal antibody (F9) and a polyclonal antibody were employed to detect MCP-1, while the mouse myeloma protein (MOPC21) was used as a negative control. Strong positive reactions for MCP-1 were seen in the bronchial epithelium. Subepithelial macrophages, blood vessels, and bronchial smooth muscle were also stained. Hue-saturation-intensity color image analysis was used to quantify reactions of the monoclonal antibody in the epithelial and subepithelial layers. With the monoclonal antibody, asthmatic biopsies showed 51.8 +/- 3.7% (mean +/- SEM) of the epithelium staining positively, whereas normal subjects reacted much less, with 6.4 +/- 1.9% of the epithelium staining (P < 0.0001); there was no overlap between the two groups. Likewise, staining was increased in the subepithelium of asthmatic airway biopsies, with 11.5 +/- 3.1% and 2.0 +/- 1.0% staining positively in asthmatic and normal subepithelium, respectively, (P < 0.002). There was a significant correlation between staining of the epithelium and subepithelium (r = 0.77, P < 0.001). The polyclonal anti-MCP-1 antibody also gave strong reactions in the epithelium and subepithelium, with 34.0 +/- 7.8% of the asthmatic and 1.6 +/- 1.0% of the normal bronchial epithelium staining positively (P < 0.0001). These increased levels of MCP-1 in the asthmatic airways suggest that they may play a role in macrophage recruitment and activation and thereby contribute to the inflammatory pathology of bronchial asthma.

Extrarenal tissue distribution of CHIP28 water channels by in situ hybridization and antibody staining

Abstract: This study is an extension of in situ hybridization experiments showing expression of mRNA encoding CHIP28 in selected epithelial or endothelia in spleen, colon, lung, and eye (H. Hasegawa, R. Zhang, A. Dohrman, and A. S. Verkman. Am. J. Physiol. 264 (Cell Physiol. 33): C237-C245, 1993). Additional tissues from rat were screened by in situ hybridization, and tissues from rat and humans were stained with a polyclonal anti-CHIP28 antibody. Northern blot showed the 2.8-kilobase mRNA encoding CHIP28 in kidney, lung, and heart. In situ hybridization showed strong hybridization in epithelial cells in choroid plexus, iris, ciliary body, and lens and in epithelial and subepithelial layers of trachea. Except for colonic crypts, specific hybridization was not observed in the gastrointestinal tract, liver, thyroid gland, and muscle. Immunoblot of tissues from exsanguinated rats showed immunoreactive CHIP28 protein in kidney, lung, trachea, and heart. In fixed frozen rat and/or human tissues, the anti-CHIP28 antibody stained epithelial cells in kidney proximal tubule and thin limb of Henle, lung alveolus, bronchial mucosa and glands, choroid plexus, ciliary body, iris, lens surface, colonic crypt, sweat gland, pancreatic acini, gallbladder epithelium, and placental syncytial trophoblast cells. Endothelial cells were stained in many tissues. These studies indicate a wide and selective CHIP28 tissue distribution, suggesting an important role for CHIP28 in fluid transport. The absence of CHIP28 in many nonrenal membranes believed to be water permeable suggests the existence of non-CHIP28 water transporters.

IL-4 directly modulates function of a model human intestinal epithelium.

Abstract: Intestinal epithelia are in intimate contact with subepithelial and intraepithelial lymphocytes. When stimulated, mucosal lymphocytes generate inflammatory cytokines such as IL-4 and IFN-gamma. We have shown that IFN-gamma directly regulates epithelial function. It is unknown whether IL-4 might influence epithelial function and, if so, whether such influences are similar to or differ from those exerted by IFN-gamma. In this study, we examine the effect of human IL-4 on barrier function, ion transport, and immune accessory ligand expression on T84 cells, a crypt-like epithelial cell line. Basolateral exposure of epithelial monolayers to IL-4 attenuated epithelial barrier function by greater than 65% in a dose (50% of effective dose = 1 U/ml)- and time (t1/2 = 24 h)-dependent fashion, and was inhibitable by neutralizing anti-IL-4 and anti-IL-4R Ab. Stimulated Cl- secretion, as measured by epithelial short circuit current, was diminished by as much as 70% by IL-4. Epithelial preexposure to IL-4 brought about a greater than twofold increase in beta 2 integrin-dependent neutrophil adhesion to epithelial, but retarded neutrophil migration into and across epithelial monolayers. ELISAs revealed that epithelial exposure to IL-4 had no effect on cell surface expression of MHC class I, MHC class II, or ICAM-1. These results indicate that IL-4, like IFN-gamma, may serve to regulate intestinal epithelial function, but that resulting phenotypes may be cytokine specific. We speculate from these data that activation of the basolateral receptor for IL-4 potentially provides a new strategy for damping the cellular component of active inflammation in the intestine.

Expression and polarized targeting of a rab3 isoform in epithelial cells.

Abstract: Pathways of polarized membrane traffic in epithelial tissues serve a variety of functions, including the generation of epithelial polarity and the regulation of vectorial transport. We have identified a candidate regulator of polarized membrane traffic in epithelial cells (i.e., rab3B), which is a member of the rab family of membrane traffic regulators. Rab3B is highly homologous to a brain-specific rab3 isoform (rab3A) that targets in a polarized fashion to the presynaptic nerve terminal, where it probably regulates exocytosis. The coding region for human rab3B was cloned from epithelial mRNA using a reverse-transcription polymerase chain reaction strategy. This cDNA clone hybridized to a single mRNA species in Northern blots of poly(A)+ RNA isolated from epithelial cell lines. A rab3B-specific antibody that was raised against recombinant fusion protein recognized a 25-kD band in immunoblots of cell lysates prepared from cultured epithelial cells (e.g., T84 and HT29-CL19A), but not from a variety of nonepithelial cells (e.g., PC12 neuroendocrine cells). Immunofluorescence analysis confirmed that rab3B protein is preferentially expressed in cultured epithelial cells as well as in a number of native epithelial tissues, including liver, small intestine, colon, and distal nephron. Rab3B localized to the apical pole very near the tight junctions between adjacent epithelial cells within all of these cell lines and native epithelial tissues, as determined by immunofluorescence and immunoelectron microscopic analysis. Moreover, this pattern of intracellular targeting was regulated by cell contact; namely, rab3B was reversibly retrieved from the cell periphery as epithelial cell contact was inhibited by reducing the extracellular Ca2+ concentration. Our results indicate that neurons and epithelial cells express homologous rab3 isoforms that target in a polarized fashion within their respective tissues. The pattern and regulation of rab3B targeting in epithelial cells implicates this monomeric GTPase as a candidate regulator of apical and/or junctional protein traffic in epithelial tissues.

Porphyromonas gingivalis invades human pocket epithelium in vitro

Abstract: The present study examined the adhesive and invasive potential of Porphyromonas gingivalis interacting with human pocket epithelium in vitro. Pocket epithelial tissue, obtained during periodontal surgery of patients with advanced periodontal disease, generated a stratified epithelium in culture. P. gingivalis strains W50 and FDC 381 (laboratory strains), OMGS 712, 1439, 1738, 1739 and 1743 (clinical isolates) as well as Escherichia coli strain HB101 (non-adhering control) were tested with respect to epithelial adhesion and invasion. Adhesion was quantitated by scintillation spectrometry after incubation of radiolabeled bacteria with epithelial cells. The invasive ability of P. gingivalis was measured by means of an antibiotic protection assay. The epithelial multilayers were infected with the test and control strains and subsequently incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). The number of internalized bacteria surviving the antibiotic treatment was assessed after plating lyzed epithelial cells on culture media. All tested P. gingivalis strains adhered to and entered pocket epithelial cells. However, considerable variation in their adhesive and invasive potential was observed. E. coli strain HB101 did not adhere or invade. Transmission electron microscopy revealed that internalization of P. gingivalis was preceded by formation of microvilli and coated pits on the epithelial cell surfaces. Intracellular bacteria were most frequently surrounded by endosomal membranes; however, bacteria devoid of such membranes were also seen. Release of outer membrane vesicles (blebs) by internalized P. gingivalis was observed. These results support and extend previous work from this laboratory which demonstrated invasion of a human oral epithelial cell-line (KB) by P. gingivalis.

Bacterial symbionts induce host organ morphogenesis during early postembryonic development of the squid Euprymna scolopes

Abstract: The mutualistic association between the squid Euprymna scolopes and the bacterium Vibrio fischeri is an emerging experimental system for the study of the influence of bacteria on animal development. Taking advantage of the ability to raise both this host and its microbial partner independently under laboratory conditions, we describe the effects of bacterial interactions on morphogenesis of the juvenile host symbiotic organ. Our results show that bacteria are essential for normal postembryonic development of the symbiotic organ, which involves changes in both the surface epithelium and the epithelial tissue within the organ where the bacterial culture will take up residence. Cell death induced by exposure to symbiotic V. fischeri results in the regression of a complex ciliated surface epithelium, a tissue that apparently functions to facilitate inoculation of the juvenile organ with the appropriate specific bacterial species. Regression of this tissue begins within hours of exposure to symbiosis-competent bacteria and progresses over the next 5 days, at which time full regression is complete, resulting in a symbiotic organ whose epithelial surface resembles that of the fully mature organ. Moreover, symbiosis-competent bacteria induce modification of the epithelial cells of the crypts that will house these symbionts; these cells undergo significant changes in shape and size in response to interactions with symbiotic V. fischeri. In contrast, we find that when these tissues are not exposed to the proper bacterial symbionts they remain in a state of arrested morphogenesis, a condition that can be rescued by interactions with symbionts. The results of these studies are the first experimental data demonstrating that a specific bacterial symbiont can play an inductive role in animal development.

Ciliostasis and loss of cilia induced by Mycoplasma hyopneumoniae in porcine tracheal organ cultures.

Abstract: In vivo- and in vitro-grown Mycoplasma hyopneumoniae organisms were inoculated onto newborn piglet tracheal organ cultures to provide a model for interaction of this organism with ciliated respiratory epithelium. Ciliostasis and loss of cilia in tracheal rings were induced by M. hyopneumoniae grown in vivo and with low-passage cultures when grown in vitro. Levels of calmodulin or dehydrogenase enzymes in tracheal ring epithelium were not altered even though ciliostasis and loss of cilia induced by M. hyopneumoniae were extensive. The capacity for inducing epithelial damage diminished with in vitro passage of the organism. Attempts to induce higher-passage cultures to attach to cilia, cause ciliostasis, or cause ciliary damage by supplementation of mycoplasmal medium with porcine lung extract failed. Epithelial damage induced by M. hyopneumoniae in tracheal rings was averted by using porcine immune serum or by separating the organisms from ciliated epithelium with a 0.1-microns-pore-size membrane. Attachment, or at least close association, of M. hyopneumoniae to ciliated epithelium appeared to be necessary to induce ciliostasis and loss of cilia in this model.

Regulation of intestinal epithelial barrier function by TGF-beta 1. Evidence for its role in abrogating the effect of a T cell cytokine.

Abstract: Maintenance of the integrity of the single-cell-thick intestinal epithelium as an in vivo barrier between environmental Ags and mucosal immunocytes is pivotal for health. The T cell cytokine IFN-gamma consistently disrupts this epithelial barrier in vitro, but the substances in mucosa that may be responsible for sustaining or enhancing barrier function have not been clearly identified. Therefore, we characterized the effect on the epithelial barrier of TGF-beta 1 and three prominent neuropeptides (VIP, substance P, somatostatin) by using a model system in which barrier function of a mature polar human colonic epithelial (T84) cell monolayer is reflected in 1) the electrical potential difference across the apical to basolateral surface of each cell, 2) the transmonolayer permeability to macromolecules such as horseradish peroxidase, and 3) lactate dehydrogenase release into the medium indicating epithelial cell cytolysis. Whereas T84 monolayers exposed to TGF-beta 1 alone demonstrated a modest increase in electrical resistance and barrier integrity, TGF-beta 1 showed a striking ability to reduce the capacity of IFN-gamma to disrupt epithelial barrier function. Characterization studies demonstrated that this TGF-beta 1 effect was prolonged (e.g., days) after a single exposure, progressive over the dose range 0.1 to 2.5 ng/ml, reversible with increased concentrations of IFN-gamma, and more pronounced when TGF-beta 1 exposure was to basolateral rather than to apical epithelial membranes. Macromolecular (horseradish peroxidase) penetration of epithelium was not simultaneously altered by TGF-beta 1 and epithelial cellular injury was minimal as gauged by lactate dehydrogenase release. Additional studies using a human pathogen demonstrated that TGF-beta 1 delayed and decreased the barrier disruption caused by exposure to Cryptosporidium parvum. TGF-beta 1 may be the first of a new class of cytokines that maintains and/or enhances barrier function of human enterocytes, in part by countering the effect of a T cell cytokine.

Bcl-2 protein expression during murine development.

Abstract: Bcl-2 functions as a death repressor molecule in an evolutionarily conserved cell death pathway. To further explore the role of Bcl-2 in development, we assessed its pattern of expression during murine embryogenesis. Immunohistochemical analysis demonstrates that Bcl-2 is widely expressed early in mouse fetal development in tissues derived from all three germ layers and that this expression becomes restricted with maturation. Within epithelium, the E12.5 lung bud demonstrates a proximal to distal gradient of Bcl-2 expression which is enhanced by E18.5. Bcl-2 is expressed throughout the intestinal epithelium through E14.5, but by E18.5 only cells in the crypts and lower villi express Bcl-2. In the mesoderm-derived kidney, Bcl-2 is expressed in both the ureteric bud and metanephric cap tissue at E12.5. Tubular structures also express Bcl-2, although overall levels drop as the kidney matures. Retinal neuroepithelial cells uniformly express Bcl-2 until cells begin to differentiate and then display the topographic distribution maintained into adulthood. The developing limb provides a clear example where Bcl-2 is restricted to zones of cell survival; Bcl-2 is expressed in the digital zones but not in the interdigital zones of cell death. The wide distribution of Bcl-2 in the developing mouse suggests that many immature cells require a death repressor molecule or that Bcl-2 may have roles beyond regulating developmental cell death.