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Iwanaga T, Takahashi-Iwanaga H. Disposal of intestinal apoptotic epithelial cells and their fate via divergent routes. Biomed Res 2022; 43:59-72. [PMID: 35718446 DOI: 10.2220/biomedres.43.59] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Gut epithelial cells are characterized by rapid, constant cell renewal. The disposal of aging epithelial cells around the villus tips of the small intestine occurs so regularly that it has been regarded as a consequence of well-controlled cell death, designated as apoptosis. However, the notion of live cell extrusion in the intestine has been intensively built among researchers, and the disposal processes of effete epithelial cells display species and regional differences. Chemical mediators and mechanical forces rising from surrounding cells contribute to the regulated cell replacement. Cytotoxic intraepithelial lymphocytes and lamina propria macrophages play a leading role in the selection of disposal cells and their extrusion to maintain fully the epithelial homeostasis in tandem with the dynamic reconstruction of junctional devices. Lymphocyte-mediated cell killing is predominant in the mouse and rat, while the disposal of epithelial cells in the guinea pig, monkey, and human is characterized by active phagocytosis by subepithelially gathering macrophages. The fenestrated basement membrane formed by immune cells supports their involvement and explains species differences in the disposal of epithelial cells. Via these fenestrations, macrophages and dendritic cells can engulf apoptotic epithelial cells and debris and convey substantial information to regional lymph nodes. In this review, we attempt to focus on morphological aspects concerning the apoptosis and disposal process of effete epithelial cells; in vitro or ex vivo analyses using cultured monolayer has become predominant in recent studies concerning the exfoliation of apoptotic enterocytes. Furthermore, we give attention to their species differences, which is controversial but crucial to our understanding.
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Affiliation(s)
- Toshihiko Iwanaga
- Department of Anatomy, Hokkaido University Graduate School of Medicine
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Mantani Y, Haruta T, Nakanishi S, Sakata N, Yuasa H, Yokoyama T, Hoshi N. Ultrastructural and phenotypical diversity of macrophages in the rat ileal mucosa. Cell Tissue Res 2021; 385:697-711. [PMID: 33961127 DOI: 10.1007/s00441-021-03457-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2020] [Accepted: 03/25/2021] [Indexed: 12/23/2022]
Abstract
Several types of macrophages have been reported in the intestinal mucosa, but their histological localization remains ambiguous. Here, we obtained detailed information about ultrastructural and phenotypical diversity of macrophage-like cells (MLCs) in the rat ileal mucosa using immunofluorescent analysis and serial block-face scanning electron microscopy (SBF-SEM). The results revealed that the cells immunopositive for CD68, the pan-macrophage marker, included CD163-CD4+, CD163+CD4+, and CD163-CD4- cells in the lamina propria (LP) of the intestinal villus and around the crypt. CD68+CD4+CD163- cells seemed to be preferentially localized in the intestinal villus, whereas CD68+CD163+CD4+ cells were frequently localized around the crypt. SBF-SEM analysis identified three types of MLCs in the ileal mucosa, which were tentatively named types I-III MLC based on aspects of the 3D-ultrastructure, such as the localization, quantity of lysosomes, endoplasmic reticulum, and exoplasm. Type I and II MLCs were localized in the villous LP, while type III MLCs were localized around the crypt, although type II MLCs were a minor population. All three MLC types extended their cellular processes into the epithelium, with type I MLCs showing the greatest abundance of extended processes. Type I MLCs in the upper portion of the intestinal villus showed a higher level of attachment to intraepithelial lymphocytes (IELs) compared to type III MLCs around the crypt. These findings suggest that macrophages of the rat ileal mucosa differed by region along the longitudinal axis of the villous tip-crypt from the perspective of ultrastructure, cellular composition, localization, and interactions with IELs.
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Affiliation(s)
- Youhei Mantani
- Laboratory of Histophysiology, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
| | - Tomohiro Haruta
- Bio 3D Promotion Group, Application Management Department, JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo, 196-8558, Japan
| | - Satoki Nakanishi
- Laboratory of Histophysiology, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan
| | - Nanami Sakata
- Laboratory of Histophysiology, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan
| | - Hideto Yuasa
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, 1-4-3, Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan
| | - Toshifumi Yokoyama
- Laboratory of Animal Molecular Morphology, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan
| | - Nobuhiko Hoshi
- Laboratory of Animal Molecular Morphology, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan
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Nakanishi S, Mantani Y, Haruta T, Yokoyama T, Hoshi N. Three-dimensional analysis of neural connectivity with cells in rat ileal mucosa by serial block-face scanning electron microscopy. J Vet Med Sci 2020; 82:990-999. [PMID: 32493889 PMCID: PMC7399320 DOI: 10.1292/jvms.20-0175] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The comprehensive targets of innervation in the intestinal mucosa are unknown, partly because of the diversity of cell types and the complexity of the neural circuits. Herein, we
investigated the comprehensive targets of neural connectivity and analyzed the precise characteristics of their contact structures in the mucosa of rat ileum. We examined target
cells of neural connections and the characteristics of their contact structures by serial block-face scanning electron microscopy at four portions of the rat ileal mucosa: the
apical and basal portions in the villi, and the lateral and basal portions around/in the crypts. Nerve fibers were in contact with several types of fibroblast-like cells (FBLCs),
macrophage-like cells, eosinophils, lymphocyte-like cells, and other types of cells. The nerve fibers almost always ran more inside of lamina propria than subepithelial FBLC, and
thus contacts with epithelial cells were very scarce. The contact structures of the nerve fibers were usually contained synaptic vesicle-like structures, and we classified them
into patterns based on the number of nerve fiber contacting the target cells at one site, the maximum diameter of the contact structures, and the relationship between nerve fibers
and nerve bundles. The contact structures for each type of cells occasionally dug into the cellular bodies of the target cells. We revealed the comprehensive targets of neural
connectivity based on the characteristics of contact structures, and identified FBLCs, immunocompetent cells, and eosinophils as the candidate targets for innervation in the rat
ileal mucosa.
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Affiliation(s)
- Satoki Nakanishi
- Laboratory of Histophysiology, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan
| | - Youhei Mantani
- Laboratory of Histophysiology, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan
| | - Tomohiro Haruta
- Bio 3D Promotion Group, Application Management Department, JEOL Ltd., 3-1-2, Musashino, Akishima, Tokyo 196-8558, Japan
| | - Toshifumi Yokoyama
- Laboratory of Animal Molecular Morphology, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan
| | - Nobuhiko Hoshi
- Laboratory of Animal Molecular Morphology, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan
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Fox S, Ryan KA, Berger AH, Petro K, Das S, Crowe SE, Ernst PB. The role of C1q in recognition of apoptotic epithelial cells and inflammatory cytokine production by phagocytes during Helicobacter pylori infection. JOURNAL OF INFLAMMATION-LONDON 2015; 12:51. [PMID: 26357509 PMCID: PMC4563842 DOI: 10.1186/s12950-015-0098-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/26/2014] [Accepted: 08/28/2015] [Indexed: 12/11/2022]
Abstract
Background Gastric epithelial cells (GECs) undergo apoptosis during H. pylori infection and phagocytes within the mucosa engulf these cells. The recognition and clearance of apoptotic cells is a multifactorial process, enhanced by the presence of various bridging molecules and opsonins which are abundant in serum. However, it is not clear how recognition or clearance may differ in the context of H. pylori infection induced apoptosis. In addition, efferocytosis of sterile apoptotic cells is known to confer anti-inflammatory properties in the engulfing phagocyte, however it is unknown if this is maintained when phagocytes encounter H. pylori-infected cells. Thus, the ability of macrophages to bind and engulf gastric epithelial cells rendered apoptotic by H. pylori infection and the association of these interactions to the modulation of phagocyte inflammatory responses was investigated in the absence and presence of serum with a particular focus on the role of serum protein C1q. Methods Control (uninfected) or H. pylori-infected AGS cells were co-cultured with THP-1 macrophages in the presence or absence of serum or serum free conditions + C1q protein (40–80 μg/mL). Binding of AGS cells to THP-1 macrophages was assessed by microscopy and cytokine (IL-6 and TNF-α) release from LPS stimulated THP-1 macrophages was quantified by ELISA. Results We show that macrophages bound preferentially to cells undergoing apoptosis subsequent to infection with H. pylori. Binding of apoptotic AGS to THP-1 macrophages was significantly inhibited when studied in the absence of serum and reconstitution of serum-free medium with purified human C1q restored binding of macrophages to apoptotic cells. Co-culture of sterile apoptotic and H. pylori-infected AGS cells both attenuated LPS-stimulated cytokine production by THP-1 macrophages. Further, direct treatment of THP-1 macrophages with C1q attenuated LPS stimulated TNF-α production. Conclusions These studies suggest that C1q opsonizes GECs rendered apoptotic by H. pylori. No differences existed in the ability of infected or sterile apoptotic cells to attenuate macrophage cytokine production, however, there may be a direct role for C1q in modulating macrophage inflammatory cytokine production to infectious stimuli. Electronic supplementary material The online version of this article (doi:10.1186/s12950-015-0098-8) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Sarah Fox
- Department of Pathology, University of California, La Jolla, San Diego, CA USA
| | - Kieran A Ryan
- Division of Gastroenterology and Hepatology, University of Virginia, Charlottesville, VA USA ; National University Ireland, Galway, Ireland
| | - Alice H Berger
- Division of Gastroenterology and Hepatology, University of Virginia, Charlottesville, VA USA ; Broad Institute of MIT and Harvard, Boston, MA USA
| | - Katie Petro
- Division of Gastroenterology and Hepatology, University of Virginia, Charlottesville, VA USA ; Athersys, Inc, Cleveland, OH USA
| | - Soumita Das
- Department of Pathology, University of California, La Jolla, San Diego, CA USA ; Division of Gastroenterology and Hepatology, University of Virginia, Charlottesville, VA USA
| | - Sheila E Crowe
- Department of Pathology, University of California, La Jolla, San Diego, CA USA ; Division of Gastroenterology and Hepatology, University of Virginia, Charlottesville, VA USA
| | - Peter B Ernst
- Department of Pathology, University of California, La Jolla, San Diego, CA USA ; Division of Gastroenterology and Hepatology, University of Virginia, Charlottesville, VA USA
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Liu Y, Rager T, Johnson J, Enmark J, Besner GE. Enriched Intestinal Stem Cell Seeding Improves the Architecture of Tissue-Engineered Intestine. Tissue Eng Part C Methods 2015; 21:816-24. [PMID: 25603285 DOI: 10.1089/ten.tec.2014.0389] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
OBJECTIVE To develop a methodology to separate intestinal stem cell (ISC)-enriched crypts from differentiated epithelial cell (DEC)-containing villi to improve the morphology of tissue-engineered intestine (TEI). METHODS Small intestinal tissues from 5- to 7-day-old transgenic Lgr5-EGFP mice (with fluorescently labeled ISCs) were used to measure the height of villi and the depth of crypts. Based on the significant size difference between crypts and villi, a novel cell filtration system was developed. Filtration of mixed organoid units from full-thickness intestine of transgenic Lgr5-EGFP mice allowed determination of the percentage of ISCs in the different size-based filtration fractions obtained. In vivo, 5-7-day-old Lewis rat pups were used as cell donors to obtain purified crypts and villi, and the dams of the pups served as recipients. Flat and tubular polyglycolic acid (PGA) scaffolds were seeded with either ISC-enriched crypts or DEC-containing villi and implanted intra-abdominally on the anterior abdominal wall. After 1, 3, 7, 14, 21, and 28 days of in vivo incubation, explants were processed for histologic evaluation. RESULTS Small intestine from transgenic Lgr5-EGFP mice contained villi with an average height of 134.89±41.91 μm and crypts with an average depth of 49.59±8.95 μm. After filtration, we found that the 100-200 μm fractions contained relatively pure villi in which DECs were located, whereas the 25-70 μm range fractions contained concentrated crypts in which ISCs were located. In vivo, flat PGA scaffolds implanted with purified crypts formed well-developed mucosa by day 14 postimplantation, whereas flat scaffolds seeded with villi were replaced with fibrous tissue. Tubular scaffolds seeded with the crypt fraction developed a well-formed mucosal layer on the interior surface, with 80.9% circumferential mucosal engraftment and an average villous height of 478±65 μm, which was very close to native intestine (512±98 μm), whereas tubular scaffolds seeded with the villous fraction only had 21.7% circumferential mucosal engraftment and an average villous height of 243±78 μm. CONCLUSION The novel filtration system described can effectively and efficiently isolate ISC-containing crypts. TEI produced from ISC-containing crypts has an improved morphology that is similar to native intestine.
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Affiliation(s)
- Yanchun Liu
- 1 Department of Pediatric Surgery and The Research Institute at Nationwide Children's Hospital , Columbus, Ohio
| | - Terrence Rager
- 1 Department of Pediatric Surgery and The Research Institute at Nationwide Children's Hospital , Columbus, Ohio
| | | | | | - Gail E Besner
- 1 Department of Pediatric Surgery and The Research Institute at Nationwide Children's Hospital , Columbus, Ohio
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Takeuchi T, Gonda T. Distribution of the pores of epithelial basement membrane in the rat small intestine. J Vet Med Sci 2004; 66:695-700. [PMID: 15240945 DOI: 10.1292/jvms.66.695] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The distribution and diameter of the pores of epithelial basement membrane in the intestinal villi and the lymph nodules of ileal Peyer's patches were investigated in the rat small intestine by scanning electron microscopy after the removal of the overlying epithelial cells with OsO(4) maceration. In the duodenum, jejunum and ileum, the pores were mainly distributed at the upper three fourths of the villi, but were scarce around the top of the villi. The diameter of some of the pores in the upper three fourths of the villi was larger than that of those in the lower portion. The protrusion of lymphocytes and the cytoplasmic processes of macrophages were also seen at the orifices of the pores. In ileal Peyer's patches, in contrast, pores were densely distributed in the lower one third of the follicle-associated epithelium (FAE) where M cells were mainly seen. Furthermore, these pores were larger than those found in the upper two thirds. Lymphocytes or cytoplasmic processes of macrophages were frequently seen in the lower one third of FAE. These results suggest that the pores at the basement membrane correspond to the passage of the immunocompetent cells which are in contact with M cells or villous columnar epithelial cells and that the abundance of pores is a sign of aggressive interaction between the particular epithelial cells and the immunocompetent cells at the upper three fourths of intestinal villi and the lower one third of FAE in the rat small intestine.
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Affiliation(s)
- Takashi Takeuchi
- Institute of Experimental Animals, Shimane Medical University, Izumo, Japan
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van Nevel CJ, Decuypere JA, Dierick N, Molly K. the influence oflentinus edodes(shiitake mushroom) preparations on bacteriological and morphological aspects of the small intestine in piglets. Arch Anim Nutr 2003; 57:399-412. [PMID: 14982320 DOI: 10.1080/0003942032000161054] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Among substances intended to replace growth promoting antibiotics in pig nutrition, non-digestible oligosaccharides or polysaccharides could be potential alternative compounds. Therefore, the influence of beta-1,3-1,6 glucans on bacteriological, biochemical and morphological aspects of the small intestine in weaned piglets was investigated. As sources of beta-glucans, Lentinan (extract of Lentinus edodes mycelium) or dried L. edodes mycelium were added to the diet. Four homogenous groups of 5 newly weaned piglets (4 weeks of age) received one of four diets: control diet (C), C supplemented with Avilamycin (50 mg/kg, positive control), C supplemented with 0.1% of Lentinan and C supplemented with 5% of dried L. edodes mycelium powder. A first group of 10 piglets was euthanized after 11 days and the remaining 10 on day 12 of the experiment. The gastrointestinal tract was divided in segments and samples taken from digesta (stomach, proximal and distal jejunum, caecum), mucosal scrapings (jejunum) and ring shaped tissue samples (1 cm) of proximal and distal jejunum. Bacterial counts were made with digesta and mucosal samples, and short-chain fatty acids (SCFA), lactic acid and ammonia concentrations were determined. Tissue samples of both jejunal sites were embedded in paraffin wax for morphometrical (villus length, crypt depth) and histological observations (numbers of intraepithelial lymphocytes (IEL), goblet cells, apoptotic enterocytes on villi, mitotic cells in crypts). Only the diet containing 5% of dried L. edodes consistently resulted in lower viable counts (ca. 1-2 log10 CFU) of total bacteria, E. coli, streptococci and lactic acid bacteria, and luminal and mucosal effects agreed very well. With this diet, acetate and butyrate concentrations in the distal jejunum were doubled, which is favourable in view of the trophic effect on enterocytes and colonocytes. Villus length (V) was increased with both diets containing beta-glucans while crypt depth (C) was not altered, but V/C was higher. IEL counts were decreased by both diets although bacterial numbers, which is only one parameter of bacterial load, were only diminished with the L. edodes feed. The three supplemented feeds lowered the number of apoptotic enterocytes on the villi, but these numbers were very low (control diet : 44 cells per 100 villi), making clear interpretation difficult. The mitotic index was slightly lower with the L. edodes feed, although not statistically significant. Decreased viable counts observed with the latter diet is a favourable effect as it is accepted that a lower bacterial load causes lower turnover rates of the intestinal epithelial cells, while there is also less competition for specific substrates. A higher V/C ratio, a smaller number of IEL in the epithelium and a lower apoptotic index also indicate slower turnover rate of the mucosa when Lentinan and L. edodes diets were fed. The inconsistent effects observed with Lentinan were probably due to the low amount added to the diet. It should be taken into account that the influence of L. edodes mycelium powder was more likely due to the presence of antibacterial compounds (eg. lenthionine, lentinamycin, terpenoids, polyphenols), rather than to an immunostimulating action of beta-glucans with increased release of IgA onto the mucosa surface.
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Affiliation(s)
- C J van Nevel
- Department ofAnimal Production, Ghent University, Melle, Belgium.
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Wang L, Li J, Li Q, Zhang J, Duan XL. Morphological changes of cell proliferation and apoptosis in rat jejunal mucosa at different ages. World J Gastroenterol 2003; 9:2060-4. [PMID: 12970906 PMCID: PMC4656674 DOI: 10.3748/wjg.v9.i9.2060] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the changes of cell proliferation and apoptosis in rat jejunal epithelium at different ages.
METHODS: Cell proliferation and apoptosis of the jejunal mucosal and glandulous epithelia from birth to postnatal 12th month were observed using immunocytochemistry (ICC), and TUNEL method. The height of villus, the thickness of muscle layer and the number of goblet cells in jejunal mucosal and glandulous epithelia were measured by BeiHang analytic software and analyzed by STAT.
RESULTS: (1) Proliferating cell nuclear antigen (PCNA) positive cells of jejunal glandulous recess were found and increased in number from birth to the postnatal 3rd month. The number of PCNA positive cells peaked in the postnatal 3rd month, and decreased from then on. (2) The number of apoptotic cells also peaked in the postnatal 3rd month, showing a similar trend to that of the PCNA positive cells. (3) The height of jejunal villus increased after birth, peaked in the postnatal 3rd month and decreased from then on. The jejunal muscle layer became thicker in the postnatal 3rd week and the postnatal 12th month. The number of goblet cells of the jejunal mucosal and glandulous epithelia had a linear correlation with age.
CONCLUSION: (1) PCNA positive cells are distributed in the jejunal glandulous recess. (2) Apoptotic cell number peaks in the postnatal 3rd month, indicating that cell proliferation and apoptosis are developed with the formation of digestive metabolism as rat grows to maturity. (3) The thickness of jejunal muscle layer increases to a maximum in the postnatal 3rd week, which may be related to the change in diet from milk to solid food. (4) The number of goblet cells increases rapidly in the postnatal 3rd week, probably due to ingestion of solid food.
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Affiliation(s)
- Li Wang
- Life Science College, Hebei Normal University, Shijiazhuang 050016, Hebei Province, China
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Nakano Y, Kawamoto T, Takano Y. Phosphatase activities of rat intestinal enterocytes and their relation to diverse luminal pH, with special references to the possible localization of phytase along the brush border membrane. ARCHIVES OF HISTOLOGY AND CYTOLOGY 2001; 64:483-92. [PMID: 11838708 DOI: 10.1679/aohc.64.483] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Phosphatase activities associated with the intestinal brush border membrane (BBM) of the rat were examined histochemically in relation to the characteristic environment of the intestine, where luminal pH fluctuates drastically between alkaline and acid pH ranges. Special attention was given to intestinal alkaline phosphatase (IALP) and phytase on the BBM. Whole body fresh-frozen sections of young rats and their rapidly frozen and freeze-substituted small intestines, embedded in Technovit 7100, were processed for the histochemical demonstration of phosphatase activity at three different pH values (9.2, 7.3, and 5.2), representing the deviation of luminal pH in vivo. Either an azo-dye method or lead-salt method was employed using naphthol AS-MX phosphate and ATP as substrate, respectively. With the azo-dye method, intense phosphatase reactions were demonstrated along the BBM at all three pH ranges. Phosphatase reactions of the BBM at pH 9.2 and 7.3 were abolished by L(+)-phenylalanine, heat pre-treatment, and EDTA chelation although some reaction remained at pH 7.3 after the treatment with EDTA or L(+)-phenylalanine. Phosphatase reactions of the BBM at pH 5.2 were resistant to L(+)-phenylalanine, L(+)-tartrate, PCMB and EDTA chelation, implying that the characteristics of the enzyme responsible for phosphohydrolysis at acid pH values differed from those at higher pH values. The lead-salt method in which ATP was used as substrate revealed intense reactions--which were dependent on Mg++ and stimulated by Ca++ and resistant to L(+)phenylalanine--to be localized along the BBM at alkaline and neutral pH values, but not at acid pH values. In vitro experiments showed progressive hydrolysis of naphthol AS-MX phosphate by purified phytase at pH 5.2, in a dose-dependent manner, and suggested the possible involvement of phytase in the phosphatase reactions of the BBM at acid pH. These data indicate that the phosphatase reactions at alkaline and neutral pH values, associated with the BBM of the rat intestine, represent IALP and Mg++/ Ca++-ATPase, while those at acid pH appear to correspond to phytase activity, something which has not been demonstrated by histochemical methods despite the availability of extensive data based on biochemical analyses.
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Affiliation(s)
- Y Nakano
- Graduate School of Tokyo Medical and Dental University, Japan
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Heczko U, Carthy CM, O'Brien BA, Finlay BB. Decreased apoptosis in the ileum and ileal Peyer's patches: a feature after infection with rabbit enteropathogenic Escherichia coli O103. Infect Immun 2001; 69:4580-9. [PMID: 11402002 PMCID: PMC98535 DOI: 10.1128/iai.69.7.4580-4589.2001] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Significant changes occur in intestinal epithelial cells after infection with enteropathogenic Escherichia coli (EPEC). However, it is unclear whether this pathogen alters rates of apoptosis. By using a naturally occurring weaned rabbit infection model, we determined physiological levels of apoptosis in rabbit ileum and ileal Peyer's patches (PP) and compared them to those found after infection with adherent rabbit EPEC (REPEC O103). Various REPEC O103 strains were first tested in vitro for characteristic virulence features. Rabbits were then inoculated with the REPEC O103 strains that infected cultured cells the most efficiently. After experimental infection, intestinal samples were examined by light and electron microscopy. Simultaneously, ileal apoptosis was assessed by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase 3 assays and by apoptotic cell counts based on morphology (hematoxylin-and-eosin staining). The highest physiological apoptotic indices were measured in PP germinal centers (median = 14.7%), followed by PP domed villi (8.1%), tips of absorptive villi (3.8%), and ileal crypt regions (0.5%). Severe infection with REPEC O103 resulted in a significant decrease in apoptosis in PP germinal centers (determined by TUNEL assay; P = 0.01), in the tips of ileal absorptive villi (determined by H&E staining; P = 0.04), and in whole ileal cell lysates (determined by caspase 3 assay; P = 0.001). We concluded that REPEC O103 does not promote apoptosis. Furthermore, we cannot rule out the possibility that REPEC O103, in fact, decreases apoptotic levels in the rabbit ileum.
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Affiliation(s)
- U Heczko
- Biotechnology Laboratory and Departments of Microbiology and Immunology, Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
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Gitter AH, Bendfeldt K, Schmitz H, Schulzke JD, Bentzel CJ, Fromm M. Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced by tumor necrosis factor-alpha. Ann N Y Acad Sci 2001; 915:193-203. [PMID: 11193576 DOI: 10.1111/j.1749-6632.2000.tb05242.x] [Citation(s) in RCA: 65] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The barrier function of intestinal epithelia relies upon the continuity of the enterocyte monolayer and intact tight junctions. After incubation with tumor necrosis factor-alpha TNF-alpha, however, the number of strands that form the tight junctions decreases, and apoptosis is induced in intestinal epithelial cells. These morphological changes lead to a rise of transepithelial ion permeability, because the paracellular ion permeability increases and leaks associated with sites of apoptosis increase by number and magnitude. Thus apoptosis and degradation of tight junctions contribute to the increased permeability observed after exposure to TNF-alpha. These mechanisms explain clinical manifestations in the inflamed intestinal wall containing cytokine-secreting macrophages--for example, leak flux diarrhea and invasion of bacterial enterotoxins.
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Affiliation(s)
- A H Gitter
- Institut für Klinische Physiologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, 12200 Berlin, Germany.
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Yamada M, Kobayashi Y, Furuoka H, Matsui T. Comparison of enterotoxicity between autumn crocus (Colchicum autumnale L.) and colchicine in the guinea pig and mouse : enterotoxicity in the guinea pig differs from that in the mouse. J Vet Med Sci 2000; 62:809-13. [PMID: 10993176 DOI: 10.1292/jvms.62.809] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Autumn crocus poisoning of cattle is characterized by severe diarrhea caused by alkaloid colchicine. Previously, we examined pathologically this poisoning in cattle and reported that enterotoxic lesions were closely associated with apoptosis. To examine enterotoxicity of autumn crocus more precisely, a reproductive study was performed using guinea pigs and mice, and pathological findings associated with autumn crocus poisoning were compared with those of colchicine. Each group of guinea pigs given the bulb of autumn crocus or colchicine exhibited severe diarrhea. Histopathological findings in intoxicated guinea pigs were entirely consistent with those in the autumn crocus-poisoned cattle. In contrast, each group of mice administered with the bulb or colchicine did not develop diarrhea. Our results confirmed that the toxicity of autumn crocus bulb is attributable to the toxicity of ingredient colchicine, and revealed that the guinea pig has high reproducibility of autumn crocus poisoning in cattle and colchicine poisoning in humans. It has been reported that the physiological mechanism of the apoptotic process for eliminating the enterocytes in the mouse and rat differs from that of the guinea pig, monkey, cattle and horse. Taking the observation that the former animals do not develop diarrhea, whereas the latter animals do so in the autumn crocus or colchicine poisoning into consideration, it would seem that the species-difference in enterotoxicity of autumn crocus may be closely associated with the physiological mechanism of eliminating the effete enterocytes.
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Affiliation(s)
- M Yamada
- Department of Veterinary Pathology, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan
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13
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Huang FP, Platt N, Wykes M, Major JR, Powell TJ, Jenkins CD, MacPherson GG. A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J Exp Med 2000; 191:435-44. [PMID: 10662789 PMCID: PMC2195813 DOI: 10.1084/jem.191.3.435] [Citation(s) in RCA: 672] [Impact Index Per Article: 26.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
This study identifies a dendritic cell (DC) subset that constitutively transports apoptotic intestinal epithelial cell remnants to T cell areas of mesenteric lymph nodes in vivo. Rat intestinal lymph contains two DC populations. Both populations have typical DC morphology, are major histocompatibility complex class II(hi), and express OX62, CD11c, and B7. CD4(+)/OX41(+) DCs are strong antigen-presenting cells (APCs). CD4(-)/OX41(-) DCs are weak APCs and contain cytoplasmic apoptotic DNA, epithelial cell-restricted cytokeratins, and nonspecific esterase (NSE)(+) inclusions, not seen in OX41(+) DCs. Identical patterns of NSE electrophoretic variants exist in CD4(-)/OX41(-) DCs, intestinal epithelial cells, and mesenteric node DCs but not in other DC populations, macrophages, or tissues. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-positive DCs and strongly NSE(+) DCs are present in intestinal lamina propria. Peyer's patches and mesenteric but not other lymph nodes contain many strongly NSE(+) DCs in interfollicular and T cell areas. Similar DCs are seen in the ileum and in T cell areas of mesenteric nodes in gnotobiotic rats. These results show that a distinct DC subset constitutively endocytoses and transports apoptotic cells to T cell areas and suggest a role for these DCs in inducing and maintaining peripheral self-tolerance.
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Affiliation(s)
- Fang-Ping Huang
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
| | - Nicholas Platt
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
| | - Michelle Wykes
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
| | - James R. Major
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
| | - Timothy J. Powell
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
| | - Christopher D. Jenkins
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
| | - G. Gordon MacPherson
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
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14
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Ramachandran A, Madesh M, Balasubramanian KA. Apoptosis in the intestinal epithelium: its relevance in normal and pathophysiological conditions. J Gastroenterol Hepatol 2000; 15:109-20. [PMID: 10735533 DOI: 10.1046/j.1440-1746.2000.02059.x] [Citation(s) in RCA: 150] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Apoptosis is now recognized as an important process responsible for maintenance of the cellular balance between proliferation and death. Apoptosis is distinct from necrosis in that it is a programmed form of cell death and occurs without any accompanying inflammation. This form of cell death can be induced by a wide range of cellular signals, which leads to activation of cell death machinery within the cell and is characterized by distinct morphological changes. Apoptosis is especially relevant in the gastrointestinal tract, as the mammalian intestinal mucosa undergoes a process of continual cell turnover that is essential for maintenance of normal function. Cell proliferation is confined to the crypts, while differentiation occurs during a rapid, orderly migration up to the villus. The differentiated enterocytes, which make up the majority of the cells, then undergo a process of programmed cell death (apoptosis). Although apoptosis is essential for the maintenance of normal gut epithelial function, dysregulated apoptosis is seen in a number of pathological conditions in the gastrointestinal tract. The cellular mechanisms regulating this tightly regimented process have not been clearly defined and this topic represents an area of active investigation as delineation of this process will lead to a better understanding of normal gut mucosal growth.
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Affiliation(s)
- A Ramachandran
- Department of Gastrointestinal Sciences, Christian Medical College and Hospital, Vellore, India
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15
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Takahashi-Iwanaga H, Iwanaga T, Isayama H. Porosity of the epithelial basement membrane as an indicator of macrophage-enterocyte interaction in the intestinal mucosa. ARCHIVES OF HISTOLOGY AND CYTOLOGY 1999; 62:471-81. [PMID: 10678576 DOI: 10.1679/aohc.62.471] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The epithelial basement membrane of intestinal villi is perforated with numerous small pores, through which free cells in the lamina propria communicate with the enterocytes. This study was a comparative analysis of the pores in the basement membrane by SEM after removal of the gut epithelium with OsO4 maceration. The porosity as represented by the area fraction of the pores varied along the baso-apical axis of villi in patterns specific for each animal species examined: consistent scantiness along the entire length of villi in mice, acute elevation in the second and third distal one-sixths of villi in rats, and gradual augmentation toward the villus tips in guinea pigs. Size distribution analyses of the pores indicated their heterogeneous enlargement in the regions of elevated porosity. Concomitant observation of lamina propria macrophages by histochemical labelings and by conventional TEM showed that the cells specifically clustered beneath the hyperporous basement membrane, with their thick processes penetrating it. The spatially-regulated patterns of perforation of the epithelial basement membrane indicate phase-specific interventions of lamina propria macrophages in the maturation or aging of enterocytes, which steadily proliferate in crypts and exfoliate at the villus tips.
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Affiliation(s)
- H Takahashi-Iwanaga
- Department of Anatomy, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
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16
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McIntosh KR, Drachman DB. Induction of apoptosis in activated T cell blasts by suppressive macrophages: a possible immunotherapeutic approach for treatment of autoimmune disease. Cell Immunol 1999; 193:24-35. [PMID: 10202110 DOI: 10.1006/cimm.1998.1445] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Large suppressive macrophages (LSM) were induced by restimulating spleen cells from rats with experimental autoimmune myasthenia gravis (EAMG) in vitro, with the autoantigen acetylcholine receptor (AChR) in the presence of cyclosporine A. LSM, purified from these cultures, are extremely potent suppressors of AChR-stimulated lymphoproliferative responses and antibody responses in vitro. In the present study, we have analyzed the factors that determine susceptibility of primed lymph node cells (pLNC) to suppression by LSM and examined the fate of these cells. We found three characteristics of pLNC that influenced their susceptibility to suppression. First, pLNC were required to be activated (by antigen in these experiments) in order for suppression to occur. Resting lymphocytes were not affected, even when they were present in cultures where antigen-activated lymphoblasts were being actively suppressed. Second, antigen specificity of the responder cells influenced their susceptibility to suppression by LSM. AChR-specific cells were relatively more susceptible to suppression by AChR-induced LSM than pLNC primed to an unrelated antigen, keyhole limpet hemocyanin. Third, T cell proliferation was suppressed by LSM to a far greater extent than antibody production by B cells. Using enriched T cell blasts generated from AChR-stimulated T cell lines, we found that LSM rapidly suppressed [3H]TdR uptake and induced DNA fragmentation assessed by the TUNEL assay (within 8 h of coculture) and induced morphological signs of apoptosis of T cells (within 24 h). Few, if any, blasts remained by 48 h of coculture. The ability to suppress an activated immune response permanently, without affecting nonactivated, bystander lymphocytes, holds promise that LSM, or their cellular products, could be used for immunotherapy of autoimmune diseases such as myasthenia gravis.
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Affiliation(s)
- K R McIntosh
- Department of Neurology, Johns Hopkins University, Baltimore, Maryland, 21287-7519, USA
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17
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Yoshikai Y. The interaction of intestinal epithelial cells and intraepithelial lymphocytes in host defense. Immunol Res 1999; 20:219-35. [PMID: 10741862 DOI: 10.1007/bf02790405] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Intestinal intraepithelial lymphocytes (i-IEL) are located at the basolateral surfaces of intestinal epithelial cells (i-EC) and play important roles in the homeostasis of intestinal microenvironment. i-IEL comprise unique T cell populations including CD4-CD8alphaalpha+ T cells expressing T cell receptor (TCR)alphabeta or TCRgammadelta and CD4+ CD8alphaalpha+ T cells expressing TCR alphabeta. We show here that CD4+ CD8alphaalpha+ i-IEL belongs to Th1 type T cells capable of responding to self-MHC class I on i-EC and that a significant fraction of i-IEL expressed Fas ligand (Fas-L) and induced apoptosis in the i-EC via Fas-dependent pathway. i-IEL may recognize and eliminate the effete i-EC for homeostatic regulation of intestinal epithelia. The interaction of i-EC and i-IEL through E-cadherin/alphaEbeta7 integrin is important for homing and maintenance of i-IEL in intestine. Listeria monocytogenes are also known to interact with E-cadherin on i-EC and invade into the epithelial cells. Invasion of L. monocytogenes into i-EC activated NFkappa-B and subsequently up-regulated the expression of IL-15 gene, which has a NFkappa-B binding site at the promoter region. i-IEL, especially gammadelta T cells, were significantly activated to produce Th1 type cytokines at the early stage after oral infection with L. monocytogenes in mice and rats. The activation of i-IEL coincided with a peak response of IL-15 production by i-EC after infection. Taken together, mutual interaction of i-IEL and i-EC may be important not only for homeostatic regulation but also host defense against microbial infection in intestine.
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Affiliation(s)
- Y Yoshikai
- Laboratory of Host Defense, Research Institute for Disease for Mechanism and Control, Nagoya University School of Medicine, Japan.
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18
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Kraehenbuhl JP, Pringault E, Neutra MR. Review article: Intestinal epithelia and barrier functions. Aliment Pharmacol Ther 1997; 11 Suppl 3:3-8; discussion 8-9. [PMID: 9467973 DOI: 10.1111/j.1365-2036.1997.tb00803.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The mucosal epithelia of the digestive tract acts as a selective barrier, permeable to ions, small molecules and macromolecules. These epithelial cells aid the digestion of food and absorption of nutrients. They contribute to the protection against pathogens and undergo continuous cell renewal which facilitates the elimination of damaged cells. Both innate and adaptive defence mechanisms protect the gastrointestinal-mucosal surfaces against pathogens. Interaction of microorganisms with epithelial cells triggers a host response by activating specific transcription factors which control the expression of chemokines and cytokines. This host response is characterized by the recruitment of macrophages and neutrophils at the site of infection. Disruption of epithelial signalling pathways that recruit migratory immune cells results in a chronic inflammatory response. The adaptive defence mechanism relies on the collaboration of epithelial cells (resident sampling system) with antigen-presenting and lymphoid cells (migratory sampling system); in order to obtain samples of foreign antigen, these samples must be transported across the barriers without affecting the integrity of the barrier. These sampling systems are regulated by both environmental and host factors. Fates of the antigen may differ depending on the way in which they cross the epithelial barrier, i.e. via interaction with motile dendritic cells or epithelial M cells in the follicle-associated epithelium.
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Affiliation(s)
- J P Kraehenbuhl
- Swiss Institute for Experimental Cancer Research and Institute of Biochemistry, University of Lausanne
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19
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Que F, Gores GJ. Apoptosis and the gastrointestinal system. ADVANCES IN PHARMACOLOGY (SAN DIEGO, CALIF.) 1997; 41:409-28. [PMID: 9204154 DOI: 10.1016/s1054-3589(08)61067-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Affiliation(s)
- F Que
- Department of Surgery, Mayo Clinic and Foundation, Rochester, Minnestota 55905, USA
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20
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Szabolcs M, Michler RE, Yang X, Aji W, Roy D, Athan E, Sciacca RR, Minanov OP, Cannon PJ. Apoptosis of cardiac myocytes during cardiac allograft rejection. Relation to induction of nitric oxide synthase. Circulation 1996; 94:1665-73. [PMID: 8840859 DOI: 10.1161/01.cir.94.7.1665] [Citation(s) in RCA: 142] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
BACKGROUND Apoptosis is a distinct form of programmed cell death characterized by activation of endonucleases that cleave nuclear DNA, condensation and fragmentation of nuclear chromatin, blebbing of intact membranes, and cell shrinkage and fragmentation. The mechanisms responsible are unclear, but nitric oxide (NO) generated by inducible NO synthase (iNOS) has been demonstrated to induce apoptosis in macrophages in vitro. This study investigated whether apoptosis occurs during cardiac allograft rejection and examined the relationship of apoptosis to iNOS expression. METHODS AND RESULTS Heterotopic abdominal transplantation from Lewis to Wistar-Furth rats was used as a model of cardiac allograft rejection; Lewis-to-Lewis grafts served as controls. Apoptosis was identified by DNA ladders after electrophoresis on agarose gels and by in situ labeling of DNA fragments; cell types were determined by immunohistochemistry. The number of apoptotic cardiac myocytes increased sharply from day 3 (0.31/mm2 ventricular tissue) to day 5 (1.27/mm2) after transplantation. At day 5, allografts showed a significant increase (P < .01) in apoptotic cardiac myocytes, macrophages, and endothelial cells compared with syngeneic grafts. The expression of iNOS mRNA, protein, and enzyme activity paralleled in time and extent the apoptosis of cardiac myocytes. iNOS immunostaining of infiltrating macrophages and cardiac muscle fibers increased significantly in the allografts at days 3 to 5 and was accompanied by immunostaining of both cell types for nitrotyrosine, which is indicative of peroxynitrite formation. CONCLUSIONS Apoptosis of myocardial cells occurs during cardiac allograft rejection. Apoptosis during rejection parallels the expression of iNOS, which suggests that apoptosis may be triggered by NO and peroxynitrite.
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Affiliation(s)
- M Szabolcs
- Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
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Ichimura E, Fukuda T, Oyama T, Kashiwabara K, Sakurai S, Sano T, Nakajima T. Formalin fixation by boiling: is it suitable for the TUNEL staining? Pathol Int 1995; 45:971-2. [PMID: 8808304 DOI: 10.1111/j.1440-1827.1995.tb03424.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
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