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Zhou JY, Lu Q, Hu Y, Fujii S, Espenschied ST, Engelhart MJ, Lewis KJ, Karell PE, Han Y, Shin H, Schmidt RE, Silver DJ, Ivanov AI, Yilmaz OH, Stappenbeck TS. Intestinal stem cells enhance local mucosal immunity through apoptotic body phagocytosis. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.10.29.620856. [PMID: 39554082 PMCID: PMC11565879 DOI: 10.1101/2024.10.29.620856] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2024]
Abstract
Modulation of immune tone at mucosal surfaces is critical to maintain homeostasis while facilitating the handling of emerging threats. One dynamic component of immune modulation is the phagocytosis and clearance of apoptotic bodies known as efferocytosis that inhibits inflammation by promoting its resolution. Here, we evaluated the effects of apoptotic body phagocytosis by intestinal epithelial stem and progenitor cells (ISCs). Unexpectedly, instead of immunomodulation through efferocytosis, this process elevated local immune system activity. To achieve this result, ISCs actively engaged apoptotic bodies in a unique fashion, leading to their engulfment and ultimate delivery to lysosomes for processing. We found that ISCs were capable of actively recruiting inert material such as apoptotic bodies by using actin-based intrinsic biomechanical processes. Uptake of apoptotic bodies was facilitated by complement factor C3 produced by apoptotic bodies themselves. ISCs in turn generated signals heightening T cell activity that was driven in part by ISC-generated TNF. Taken together, uptake of apoptotic bodies by ISCs produced a local inflammatory alert to specific immune cells. This altered paradigm for the response to phagocytosed apoptotic bodies fits the needs of active mucosal surfaces and demonstrates that efferocytosis as currently defined is not a universal response of all cell types.
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Reyes EA, Castillo-Azofeifa D, Rispal J, Wald T, Zwick RK, Palikuqi B, Mujukian A, Rabizadeh S, Gupta AR, Gardner JM, Boffelli D, Gartner ZJ, Klein OD. Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis. J Clin Invest 2023; 133:e163591. [PMID: 37643009 PMCID: PMC10575728 DOI: 10.1172/jci163591] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Accepted: 08/22/2023] [Indexed: 08/31/2023] Open
Abstract
The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell-derived TNF as an upstream regulator of mucin homeostasis.
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Affiliation(s)
- Efren A. Reyes
- Department of Orofacial Sciences and Program in Craniofacial Biology, and
- Department of Pharmaceutical Chemistry and TETRAD Program, UCSF, San Francisco, California, USA
| | - David Castillo-Azofeifa
- Department of Orofacial Sciences and Program in Craniofacial Biology, and
- Department of Regenerative Medicine, Genentech, Inc., South San Francisco, California, USA
| | - Jérémie Rispal
- Department of Orofacial Sciences and Program in Craniofacial Biology, and
| | - Tomas Wald
- Department of Orofacial Sciences and Program in Craniofacial Biology, and
| | - Rachel K. Zwick
- Department of Orofacial Sciences and Program in Craniofacial Biology, and
| | - Brisa Palikuqi
- Department of Orofacial Sciences and Program in Craniofacial Biology, and
| | - Angela Mujukian
- F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA
| | - Shervin Rabizadeh
- F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA
- Department of Pediatrics, Cedars-Sinai Guerin Children’s, Los Angeles, California, USA
| | | | - James M. Gardner
- Department of Surgery, and
- Diabetes Center, UCSF, San Francisco, California, USA
- Chan-Zuckerberg Biohub, San Francisco, California, USA
- The Center for Cellular Construction, San Francisco, California, USA
| | - Dario Boffelli
- Department of Pediatrics, Cedars-Sinai Guerin Children’s, Los Angeles, California, USA
| | - Zev J. Gartner
- Department of Pharmaceutical Chemistry and TETRAD Program, UCSF, San Francisco, California, USA
| | - Ophir D. Klein
- Department of Orofacial Sciences and Program in Craniofacial Biology, and
- Department of Pediatrics, Cedars-Sinai Guerin Children’s, Los Angeles, California, USA
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Basuony GA, M.A.Basyoni M, Negm MSI, Mostafa EAM, El-Wakil ES, Shemis MA, Gouda AE, Saftawy EAE. Influence of Blastocystis hominis on the small intestine and lactase enzyme activity. J Parasit Dis 2022; 46:243-253. [PMID: 35299913 PMCID: PMC8901820 DOI: 10.1007/s12639-021-01442-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Accepted: 08/07/2021] [Indexed: 11/29/2022] Open
Abstract
Blastocystis hominis is a cosmopolitan protozoan that has been associated with several gastrointestinal disturbances involving lactose intolerance. However, the underlying pathogenic factors remain indistinct. 20 Swiss albino mice were utilized and assembled into four groups, each of five mice: group-I: received neither infection nor lactose (healthy control), group-II: received a single dose of 10,000 cysts of Blastocystis and lactose diets in a dose of 12.5 g/day/mouse for 7 consecutive days starting from day 14 p.i., group-III: non-infected mice with oral doses of lactose (12.5 g/day/mouse) for 7 consecutive days (positive control), group-IV: infected mice on lactose free diet (negative control). We investigated the histopathological changes using H&E stain.s Also, lactase enzyme activity was measured using spectrophotometry and the production of TNF-α and apoptotic events were explored via immunohistochemistry and compared in the small intestine of all groups. The active inflammatory changes in the infected animals were moderate in the form of loss of villous architecture, increased ILC (P-value > 0.001) besides scattered forms of the parasite as compared to non-infected mice. There was a reduction in lactase enzyme activity p.i. The TNF-α levels were induced p.i. as compared to non-infected mice (P-value > 0.001). The expression of Bax protein was upgraded, while Bcl-2 expression decreased significantly with a reverse in Bax/Bcl2 ratio in infected animals. Blastocystis infection appears to humble lactase enzyme activity via the induction of apoptosis in the epithelial cells of the small intestinal brush border in a TNF-α associative pathway.
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Affiliation(s)
- Gehad A. Basuony
- Parasitology Department, Kasr Al-Ainy School of Medicine, Cairo University, Cairo, Egypt
| | - Maha M.A.Basyoni
- Parasitology Department, Kasr Al-Ainy School of Medicine, Cairo University, Cairo, Egypt
| | | | | | | | - Mohamed A. Shemis
- Biochemistry and Molecular Biology Department, Theodore Bilharz Research Institute, Giza, Egypt
| | - Abdullah E. Gouda
- Biochemistry and Molecular Biology Department, Theodore Bilharz Research Institute, Giza, Egypt
| | - Enas A. El Saftawy
- Parasitology Department, Kasr Al-Ainy School of Medicine, Cairo University, Cairo, Egypt ,Medical Parasitology Department, College of Medicine, Armed Forces College of Medicine (AFCM), Cairo, Egypt
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Shastri S, Shinde T, Perera AP, Gueven N, Eri R. Idebenone Protects against Spontaneous Chronic Murine Colitis by Alleviating Endoplasmic Reticulum Stress and Inflammatory Response. Biomedicines 2020; 8:biomedicines8100384. [PMID: 32998266 PMCID: PMC7601570 DOI: 10.3390/biomedicines8100384] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2020] [Revised: 09/22/2020] [Accepted: 09/24/2020] [Indexed: 12/18/2022] Open
Abstract
Endoplasmic reticulum (ER) stress in intestinal secretory goblet cells has been linked to the development of ulcerative colitis (UC). Emerging evidence suggests that the short chain quinone drug idebenone displays anti-inflammatory activity in addition to its potent antioxidant and mitochondrial electron donor properties. This study evaluated the impact of idebenone in Winnie mice, that are characterized by spontaneous chronic intestinal inflammation and ER stress caused by a missense mutation in the mucin MUC2 gene. Idebenone (200 mg/kg) was orally administered daily to 5-6 weeks old Winnie mice over a period of 21 days. Idebenone treatment substantially improved body weight gain, disease activity index (DAI), colon length and histopathology score. Immunohistochemistry revealed increased expression of MUC2 protein in goblet cells, consistent with increased MUC2 mRNA levels. Furthermore, idebenone significantly reduced the expression of the ER stress markers C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6) and X-box binding protein-1 (XBP-1) at both mRNA and protein levels. Idebenone also effectively reduced pro-inflammatory cytokine levels in colonic explants. Taken together, these results indicate that idebenone could represent a potential therapeutic approach against human UC by its strong anti-inflammatory activity and its ability to reduce markers of ER stress.
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Affiliation(s)
- Sonia Shastri
- Gut Health Laboratory, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston 7250, Tasmania, Australia; (T.S.); (A.P.P.)
- Correspondence: (S.S.); (R.E.); Tel.: +61-4-4992-4236 (S.S.); +61-3-6226-5017 (R.E.)
| | - Tanvi Shinde
- Gut Health Laboratory, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston 7250, Tasmania, Australia; (T.S.); (A.P.P.)
- Centre for Food Innovation, Tasmanian Institute of Agriculture, University of Tasmania, Launceston 7250, Tasmania, Australia
| | - Agampodi Promoda Perera
- Gut Health Laboratory, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston 7250, Tasmania, Australia; (T.S.); (A.P.P.)
| | - Nuri Gueven
- School of Pharmacy and Pharmacology, College of Health and Medicine, University of Tasmania, Hobart 7005, Tasmania, Australia;
| | - Rajaraman Eri
- Gut Health Laboratory, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston 7250, Tasmania, Australia; (T.S.); (A.P.P.)
- Correspondence: (S.S.); (R.E.); Tel.: +61-4-4992-4236 (S.S.); +61-3-6226-5017 (R.E.)
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Inhibition of histone deacetylase 6 attenuates intestinal inflammation and apoptosis in a rodent model of hemorrhagic shock. J Trauma Acute Care Surg 2019; 86:874-880. [DOI: 10.1097/ta.0000000000002169] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
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Tumour Necrosis Factor Alpha in Intestinal Homeostasis and Gut Related Diseases. Int J Mol Sci 2019; 20:ijms20081887. [PMID: 30995806 PMCID: PMC6515381 DOI: 10.3390/ijms20081887] [Citation(s) in RCA: 139] [Impact Index Per Article: 23.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2019] [Revised: 04/05/2019] [Accepted: 04/13/2019] [Indexed: 02/06/2023] Open
Abstract
The intestinal epithelium constitutes an indispensable single-layered barrier to protect the body from invading pathogens, antigens or toxins. At the same time, beneficial nutrients and water have to be absorbed by the epithelium. To prevent development of intestinal inflammation or tumour formation, intestinal homeostasis has to be tightly controlled and therefore a strict balance between cell death and proliferation has to be maintained. The proinflammatory cytokine tumour necrosis factor alpha (TNFα) was shown to play a striking role for the regulation of this balance in the gut. Depending on the cellular conditions, on the one hand TNFα is able to mediate cell survival by activating NFκB signalling. On the other hand, TNFα might trigger cell death, in particular caspase-dependent apoptosis but also caspase-independent programmed necrosis. By regulating these cell death and survival mechanisms, TNFα exerts a variety of beneficial functions in the intestine. However, TNFα signalling is also supposed to play a critical role for the pathogenesis of inflammatory bowel disease (IBD), infectious diseases, intestinal wound healing and tumour formation. Here we review the literature about the physiological and pathophysiological role of TNFα signalling for the maintenance of intestinal homeostasis and the benefits and difficulties of anti-TNFα treatment during IBD.
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Holly MK, Smith JG. Paneth Cells during Viral Infection and Pathogenesis. Viruses 2018; 10:v10050225. [PMID: 29701691 PMCID: PMC5977218 DOI: 10.3390/v10050225] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2018] [Revised: 04/17/2018] [Accepted: 04/24/2018] [Indexed: 02/07/2023] Open
Abstract
Paneth cells are major secretory cells located in the crypts of Lieberkühn in the small intestine. Our understanding of the diverse roles that Paneth cells play in homeostasis and disease has grown substantially since their discovery over a hundred years ago. Classically, Paneth cells have been characterized as a significant source of antimicrobial peptides and proteins important in host defense and shaping the composition of the commensal microbiota. More recently, Paneth cells have been shown to supply key developmental and homeostatic signals to intestinal stem cells in the crypt base. Paneth cell dysfunction leading to dysbiosis and a compromised epithelial barrier have been implicated in the etiology of Crohn’s disease and susceptibility to enteric bacterial infection. Our understanding of the impact of Paneth cells on viral infection is incomplete. Enteric α-defensins, produced by Paneth cells, can directly alter viral infection. In addition, α-defensins and other antimicrobial Paneth cell products may modulate viral infection indirectly by impacting the microbiome. Here, we discuss recent insights into Paneth cell biology, models to study their function, and the impact, both direct and indirect, of Paneth cells on enteric viral infection.
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Affiliation(s)
- Mayumi K Holly
- Department of Microbiology, University of Washington, Box 357735, 1705 NE Pacific St., Seattle, WA 98195, USA.
| | - Jason G Smith
- Department of Microbiology, University of Washington, Box 357735, 1705 NE Pacific St., Seattle, WA 98195, USA.
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8
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Saleh H, El-Shorbagy HM. Mechanism underlying methyl eugenol attenuation of intestinal ischemia/reperfusion injury. Appl Physiol Nutr Metab 2017. [DOI: 10.1139/apnm-2017-0043] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Intestinal ischemia/reperfusion (I/R) injury is associated with a high risk of mortality in the clinical situation. Many factors are involved in I/R, including reactive oxygen species, cytokine release, and apoptosis. We aimed to determine whether a pure methyl eugenol (ME) given before intestinal ischemia, protects against intestinal I/R injury and the possible mechanism involved in this protection. Rat received ME (100 mg/kg) for 30 days then underwent intestinal I/R with 30 min ischemia and 60 min reperfusion. Serum lactate dehydrogenase (LDH) level, tissue malondialdehyde (MDA), as well as some antioxidant biomarkers were assessed, while the serum level of tumor necrosis factor alpha (TNF-α) was determined by ELISA. The change in TNF-α and interleukin 6 (IL-6) gene expressions were evaluated and confirmed by assessing protein level of TNF-α in the intestinal tissue by immunohistochemistry. Apoptosis was evaluated using DNA-laddering assay and by detecting caspase-3 immunohistochemically. Administration of ME prior to I/R injury resulted in a modulation of the production of MDA, LDH, and nitric oxide and restoration of the tested oxidative stress biomarkers. Pretreatment with ME downregulated messenger RNA of TNF-α and IL-6 inflammatory cytokines and their protein expressions in I/R rats. Marked inhibition of the apoptotic DNA and improvement of the architectures of small intestine were observed after pretreatment with ME. ME exhibits a protective effect against intestinal I/R via amelioration of the oxidative stress and inflammatory cytokines gene expression. Therefore, the supplementation of ME prior to intestinal I/R might be helpful in the attenuation of I/R complications.
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Affiliation(s)
- Hanan Saleh
- Zoology Department, Faculty of Science, Cairo University, Giza 12631, Egypt
- Zoology Department, Faculty of Science, Cairo University, Giza 12631, Egypt
| | - Haidan M. El-Shorbagy
- Zoology Department, Faculty of Science, Cairo University, Giza 12631, Egypt
- Zoology Department, Faculty of Science, Cairo University, Giza 12631, Egypt
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9
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Zeiser R, Socié G, Blazar BR. Pathogenesis of acute graft-versus-host disease: from intestinal microbiota alterations to donor T cell activation. Br J Haematol 2016; 175:191-207. [PMID: 27619472 DOI: 10.1111/bjh.14295] [Citation(s) in RCA: 86] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2016] [Revised: 06/30/2016] [Accepted: 06/30/2016] [Indexed: 01/03/2023]
Abstract
Acute graft-versus-host disease (aGVHD) is a major life-threatening complication of allogeneic haematopoietic cell transplantation (allo-HCT). Here we discuss the aGVHD pathophysiology initiated by multiple signals that cause alloreactive T-cell activation. The outcome of such donor T-cell activation is influenced by T-cell receptor-signal strength, anatomical location, co-stimulatory/co-inhibitory signals and differentiation stage (naive, effector/memory) of T-cells. Additionally, cross-priming of T cells to antigens expressed by pathogens can contribute to aGVHD-mediated tissue injury. In addition to the properties of donor T-cell activation, highly specialized tissue resident cell types, such as innate lymphoid cells, antigen-presenting cells, immune regulatory cells and various intestinal cell populations are critically involved in aGVHD pathogenesis. The role of the thymus and secondary lymphoid tissue injury, non-haematopoietic cells, intestinal microflora, cytokines, chemokines, microRNAs, metabolites and kinases in aGVHD pathophysiology will be highlighted. Acute GVHD pathogenic mechanisms will be connected to novel therapeutic approaches under development for, and tested in, the clinic.
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Affiliation(s)
- Robert Zeiser
- Department of Haematology, Oncology and Stem Cell Transplantation, Freiburg University Medical Centre, Freiburg, Germany.
| | - Gerard Socié
- Haematology Stem cell transplant Unit, Saint Louis Hospital, APHP, Paris, France
| | - Bruce R Blazar
- Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA.
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Li N, Wang XM, Jiang LJ, Zhang M, Li N, Wei ZZ, Zheng N, Zhao YJ. Effects of endoplasmic reticulum stress on the expression of inflammatory cytokines in patients with ulcerative colitis. World J Gastroenterol 2016; 22:2357-2365. [PMID: 26900298 PMCID: PMC4735010 DOI: 10.3748/wjg.v22.i7.2357] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/24/2015] [Revised: 09/05/2015] [Accepted: 11/30/2015] [Indexed: 02/06/2023] Open
Abstract
AIM: To explore the changes of X-box binding protein 1 splicing (XBP1s) and inflammatory cytokine expression in patients with ulcerative colitis (UC) in response to endoplasmic reticulum stress (ERS).
METHODS: Reverse transcription polymerase chain reaction and quantitative polymerase chain reaction were performed to detect the forms of XBP1s and the expression of interleukin (IL)-2, interferon (IFN)-γ, and IL-17α. Differences between patients with UC and normal subjects were then determined.
RESULTS: Mononuclear cells of the peripheral blood of normal subjects and UC patients with were stimulated with no drugs (control), phytohemagglutinin (PHA), thapsigargin (TG), or both PHA and TG. XBP1s in patients with UC exhibited splicing, which was greater with co-stimulation than single stimulation. Co-stimulation increased the expression level of IL-2, IFN-γ, and IL-17α.
CONCLUSION: The T lymphocytes of both normal subjects and patients with UC responded to ERS by activating the XBP1s-mediated signalling pathway, upregulating the expression of inflammatory cytokines, and increasing the occurrence of inflammation. The mononuclear cells in the peripheral blood of patients with UC were more sensitive to ERS than those in the peripheral blood of normal subjects.
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11
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Buttó LF, Schaubeck M, Haller D. Mechanisms of Microbe-Host Interaction in Crohn's Disease: Dysbiosis vs. Pathobiont Selection. Front Immunol 2015; 6:555. [PMID: 26635787 PMCID: PMC4652232 DOI: 10.3389/fimmu.2015.00555] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2015] [Accepted: 10/16/2015] [Indexed: 12/11/2022] Open
Abstract
Crohn’s disease (CD) is a systemic chronic inflammatory condition mainly characterized by discontinuous transmural pathology of the gastrointestinal tract and frequent extraintestinal manifestations with intermittent episodes of remission and relapse. Genome-wide association studies identified a number of risk loci that, catalyzed by environmental triggers, result in the loss of tolerance toward commensal bacteria based on dysregulated innate effector functions and antimicrobial defense, leading to exacerbated adaptive immune responses responsible for chronic immune-mediated tissue damage. In this review, we discuss the inter-related role of changes in the intestinal microbiota, epithelial barrier integrity, and immune cell functions on the pathogenesis of CD, describing the current approaches available to investigate the molecular mechanisms underlying the disease. Substantial effort has been dedicated to define disease-associated changes in the intestinal microbiota (dysbiosis) and to link pathobionts to the etiology of inflammatory bowel diseases. A cogent definition of dysbiosis is lacking, as well as an agreement of whether pathobionts or complex shifts in the microbiota trigger inflammation in the host. Among the rarely available animal models, SAMP/Yit and TNFdeltaARE mice are the best known displaying a transmural CD-like phenotype. New hypothesis-driven mouse models, e.g., epithelial-specific Caspase8−/−, ATG16L1−/−, and XBP1−/− mice, validate pathway-focused function of specific CD-associated risk genes highlighting the role of Paneth cells in antimicrobial defense. To study the causal role of bacteria in initiating inflammation in the host, the use of germ-free mouse models is indispensable. Unraveling the interactions of genes, immune cells and microbes constitute a criterion for the development of safe, reliable, and effective treatment options for CD.
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Affiliation(s)
- Ludovica F Buttó
- Chair of Nutrition and Immunology, Technische Universität München , Freising-Weihenstephan , Germany
| | - Monika Schaubeck
- Chair of Nutrition and Immunology, Technische Universität München , Freising-Weihenstephan , Germany
| | - Dirk Haller
- Chair of Nutrition and Immunology, Technische Universität München , Freising-Weihenstephan , Germany
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12
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Delgado ME, Grabinger T, Brunner T. Cell death at the intestinal epithelial front line. FEBS J 2015; 283:2701-19. [PMID: 26499289 DOI: 10.1111/febs.13575] [Citation(s) in RCA: 74] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Revised: 09/23/2015] [Accepted: 10/21/2015] [Indexed: 12/25/2022]
Abstract
The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family.
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Affiliation(s)
- Maria Eugenia Delgado
- Chair of Biochemical Pharmacology, Department of Biology, University of Konstanz, Germany
| | - Thomas Grabinger
- Chair of Biochemical Pharmacology, Department of Biology, University of Konstanz, Germany
| | - Thomas Brunner
- Chair of Biochemical Pharmacology, Department of Biology, University of Konstanz, Germany
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13
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Lu W, de Leeuw E. Functional intersection of Human Defensin 5 with the TNF receptor pathway. FEBS Lett 2014; 588:1906-12. [PMID: 24681099 DOI: 10.1016/j.febslet.2014.03.028] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2013] [Revised: 03/06/2014] [Accepted: 03/14/2014] [Indexed: 12/21/2022]
Abstract
Defensins are cationic antimicrobial peptides that contribute to regulation of host cell function also. Here, we report on the regulation of cell death by Human Defensin 5, the major antimicrobial peptide of ileal Paneth cells. We find that Human Defensin 5-mediated cellular effects depend on functional expression of Tumor Necrosis Factor receptors and downstream mediators of TNF signaling. Our data indicate the involvement of interactions between Human Defensin 5 and the extra-cellular domain of Tumor Necrosis Factor receptor 1. Human Defensin-5 also induces apoptosis intrinsically by targeting the mitochondrial membrane.
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Affiliation(s)
- Wuyuan Lu
- Institute of Human Virology, University of Maryland Baltimore School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA
| | - Erik de Leeuw
- Institute of Human Virology, University of Maryland Baltimore School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA.
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Abstract
PURPOSE OF REVIEW To provide an overview of the emerging role of cellular stress responses in inflammatory bowel disease (IBD). RECENT FINDINGS The unfolded protein response (UPR) is a primitive cellular pathway that is engaged when responding to endoplasmic reticulum stress and regulates autophagy. Highly secretory cells such as Paneth cells and goblet cells in the intestines are particularly susceptible to endoplasmic reticulum stress and are exceedingly dependent upon a properly functioning UPR to maintain cellular viability and homeostasis. Primary genetic abnormalities within the components of the UPR (e.g. XBP1, ARG2, ORMDL3), genes that encode proteins reliant upon a robust secretory pathway (e.g. MUC2, HLAB27) and environmental factors that create disturbances in the UPR (e.g. microbial products and inflammatory cytokines) are important factors in the primary development and/or perpetuation of intestinal inflammation. SUMMARY Endoplasmic reticulum stress is an important new pathway involved in the development of intestinal inflammation associated with IBD and likely other intestinal inflammatory disorders.
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Abstract
The cytokine interleukin-17 (IL-17) has received considerable attention since the discovery of a distinct CD4(+) T helper (T(H)) cell subset that produces it, known as the T(H)17 cell subset. Despite the fact that most of the recent literature describes IL-17 as a T cell-secreted cytokine, much of the IL-17 released during an inflammatory response is produced by innate immune cells. In this Review, we explore the many innate immune cell populations that are an early source of IL-17 in response to stress, injury or pathogens. These early sources have been shown to have a central role in the initiation of IL-17-dependent immune responses, even before the first CD4(+)T cell sees its cognate antigen and initiates the T(H)17 cell developmental programme.
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16
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Dekaney CM, Gulati AS, Garrison AP, Helmrath MA, Henning SJ. Regeneration of intestinal stem/progenitor cells following doxorubicin treatment of mice. Am J Physiol Gastrointest Liver Physiol 2009; 297:G461-70. [PMID: 19589945 PMCID: PMC2739827 DOI: 10.1152/ajpgi.90446.2008] [Citation(s) in RCA: 99] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/21/2008] [Accepted: 07/07/2009] [Indexed: 01/31/2023]
Abstract
The intestinal epithelium is in a constant state of renewal. The rapid turnover of cells is fed by a hierarchy of transit amplifying and stem/progenitor cells destined to give rise to the four differentiated epithelial lineages of the small intestine. Doxorubicin (Dox) is a commonly used chemotherapeutic agent that preferentially induces apoptosis in the intestinal stem cell zone (SCZ). We hypothesized that Dox treatment would initially decrease "+4" intestinal stem cell numbers with a subsequent expansion during mucosal repair. Temporal assessment following Dox treatment demonstrated rapid induction of apoptosis in the SCZ leading to a decrease in the number of intestinal stem/progenitor cells as determined by flow cytometry for CD45(-) SP cells, and immunohistochemistry of cells positive for putative +4 stem cell markers beta-cat(Ser552) and DCAMKL1. Between 96 and 168 h postinjection, overall proliferation in the crypts increased concomitant with increases in both absolute and relative numbers of goblet, Paneth, and enteroendocrine cells. This regeneration phase was also associated with increases of CD45(-) SP cells, beta-cat(Ser552)-positive cells, crypt fission, and crypt number. We used Lgr5-lacZ mice to assess behavior of Lgr5-positive stem cells following Dox and found no change in this cell population. Lgr5 mRNA level was also measured and showed no change immediately after Dox but decreased during the regeneration phase. Together these data suggest that, following Dox-induced injury, expansion of intestinal stem cells occurs during mucosal repair. On the basis of available markers this expansion appears to be predominantly the +4 stem cell population rather than those of the crypt base.
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MESH Headings
- Animals
- Antibiotics, Antineoplastic/administration & dosage
- Antibiotics, Antineoplastic/toxicity
- Apoptosis/drug effects
- Cell Lineage
- Cell Proliferation/drug effects
- Doublecortin-Like Kinases
- Doxorubicin/administration & dosage
- Doxorubicin/toxicity
- Female
- Injections, Intraperitoneal
- Intestinal Mucosa/drug effects
- Intestinal Mucosa/metabolism
- Intestinal Mucosa/pathology
- Intestine, Small/drug effects
- Intestine, Small/metabolism
- Intestine, Small/pathology
- Jejunum/drug effects
- Jejunum/pathology
- Leukocyte Common Antigens/analysis
- Mice
- Mice, Inbred C57BL
- Mice, Transgenic
- Protein Serine-Threonine Kinases/metabolism
- RNA, Messenger/metabolism
- Receptors, G-Protein-Coupled/genetics
- Receptors, G-Protein-Coupled/metabolism
- Regeneration/drug effects
- Stem Cells/drug effects
- Stem Cells/metabolism
- Stem Cells/pathology
- Time Factors
- beta Catenin/metabolism
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Affiliation(s)
- Christopher M Dekaney
- Department of Surgery, The University of North Carolina, Chapel Hill, North Carolina 27599-7223, USA.
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17
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Zheng X, Mao Y, Cai J, Li Y, Liu W, Sun P, Zhang JH, Sun X, Yuan H. Hydrogen-rich saline protects against intestinal ischemia/reperfusion injury in rats. Free Radic Res 2009; 43:478-84. [PMID: 19353364 DOI: 10.1080/10715760902870603] [Citation(s) in RCA: 128] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Hydrogen gas was reported to reduce reactive oxygen species and alleviate cerebral, myocardial and hepatic ischemia/reperfusion (I/R) injuries. This paper studied the effect of hydrogen-rich saline, which was easier for clinical application, on the intestinal I/R injury. Model of intestinal I/R injury was induced in male Sprague-Dawley rats. Physiological saline, hydrogen-rich saline or nitrogen-rich saline (5 ml/kg) was administered via intravenous infusion at 10 min before reperfusion, respectively. The intestine damage was detected microscopically and was assessed by Chiu score system after I/R injury. In addition, serum DAO activity, TNF-alpha, IL-1beta and IL-6 levels, tissue MDA, protein carbonyl and MPO activity were all increased significantly by I/R injury. Hydrogen-rich saline reduced these markers and relieved morphological intestinal injury, while no significant reduction was observed in the nitrogen-rich saline-treated animals. In conclusion, hydrogen-rich saline protected the small intestine against I/R injury, possibly by reduction of inflammation and oxidative stress.
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Affiliation(s)
- Xingfeng Zheng
- Chinese PLA Institute of Burn Surgery & Department of Burn Surgery, Changhai Hospital, Second Military Medical University, Shanghai, PR China
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18
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Ciccia F, Bombardieri M, Principato A, Giardina A, Tripodo C, Porcasi R, Peralta S, Franco V, Giardina E, Craxi A, Pitzalis C, Triolo G. Overexpression of interleukin-23, but not interleukin-17, as an immunologic signature of subclinical intestinal inflammation in ankylosing spondylitis. ACTA ACUST UNITED AC 2009; 60:955-65. [PMID: 19333939 DOI: 10.1002/art.24389] [Citation(s) in RCA: 175] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
OBJECTIVE Subclinical gut inflammation is common in spondylarthritis, but the immunologic abnormalities underlying this process are undefined. Perturbation of the interleukin-23 (IL-23)/Th17 axis has emerged as a fundamental trigger of chronic inflammation. This study was undertaken to investigate the expression and tissue distribution of IL-23/Th17-related molecules in Crohn's disease (CD) and in subclinical gut inflammation in ankylosing spondylitis (AS). METHODS Quantitative gene expression analysis of Th1/Th2 and IL-23/Th17 responses was performed in intestinal biopsy samples obtained from 12 patients with CD, 15 patients with AS, and 13 controls. IL-23 tissue distribution and identification of IL-23-producing cells were evaluated by immunohistochemistry. RESULTS We demonstrated a strong and significant up-regulation of IL-23p19 transcripts in the terminal ileum in patients with AS and patients with CD. IL-23 was abundantly produced by infiltrating monocyte-like cells in inflamed mucosa from AS and CD patients. Notably, we also identified Paneth cells as a major source of IL-23 in patients with AS, patients with CD, and normal controls. Unlike CD, in AS patients, IL-23 was not associated with up-regulation of IL-17 and the IL-17-inducing cytokines IL-6 and IL-1beta. Finally, while the Th1-related cytokines interferon-gamma, IL-12p35, and IL-27p28 were overexpressed only in CD patients, IL-4, IL-5, and STAT-6 were also significantly increased in AS patients. CONCLUSION Our findings indicate that overexpression of IL-23, but not IL-17, is a pivotal feature of subclinical gut inflammation in AS. Identification of resident Paneth cells as a pivotal source of IL-23 in physiologic and pathologic conditions strongly suggests that IL-23 is a master regulator of gut mucosal immunity, providing a pathophysiologic significance to the reported association between IL-23 receptor polymorphisms and intestinal inflammation.
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19
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Iwai T, Ichikawa T, Goso Y, Ikezawa T, Saegusa Y, Okayasu I, Saigenji K, Ishihara K. Effects of indomethacin on the rat small intestinal mucosa: immunohistochemical and biochemical studies using anti-mucin monoclonal antibodies. J Gastroenterol 2009; 44:277-84. [PMID: 19280111 DOI: 10.1007/s00535-009-0007-0] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/26/2008] [Accepted: 10/31/2008] [Indexed: 02/04/2023]
Abstract
BACKGROUND The luminal surface of the gastrointestinal tract is covered by a viscoelastic gel layer that acts as a protective barrier against the intraluminal environment. Because the situation of the small intestine has not been elucidated to the same degree as other sections, in this study, we investigated the effects of indomethacin on the rat small intestinal mucosa. METHODS Male Wistar rats were given indomethacin 10 mg/kg s-c and sacrificed 1, 3, 7, or 14 days later. The small intestine was opened along the anti-mesenteric side, and examined macroscopically. Total mucin content in the small intestinal epithelium was measured and immunoreactivity was examined using anti-mucin monoclonal antibodies HCM31 and PGM34. RESULTS Indomethacin caused punched out and linear ulcers located mostly along the mesenteric margin of the distal jejunum with sparing of the ileum. Histological examination showed sialomucin recognized by HCM31 increased on day 3 especially in the regenerating epithelium around the ulcer edge. Furthermore, the surface mucous gel layer displayed a multilaminated pattern, consisting of non-sulfated sialomucin-rich layers and sulfated mucin-rich layers, where both mucins had the common core protein, MUC2. Biochemical measurements also showed the total mucin content of the jejunum increased transiently and HCM31-positive mucin increased approximately 4 times greater than baseline on day 3, but no marked changes were observed in the ileum, with few ulcers observed. CONCLUSIONS Indomethacin administration causes quantitative and qualitative change in jejunal mucin. In particular, sialomucin plays an important role in regenerating epithelium during the healing process following indomethacin-induced mucosal damage.
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Affiliation(s)
- Tomohisa Iwai
- Department of Gastroenterology, Kitasato University School of Medicine, Sagamihara, Japan
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20
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Abstract
Crohn disease and ulcerative colitis are chronic inflammatory diseases of the intestinal tract commonly denoted as inflammatory bowel diseases. It has been proposed that these diseases result from aberrant mucosal immune responses to nonpathogenic microbial residents of the intestines. Recently, it was established that continuous interactions between the innate and the adaptive intestinal immune cells and the microbiota are directly involved in maintaining the physiological noninflammatory state of the intestinal mucosa. In light of the complexity of this mucosal homeostasis, it is astonishing that the inflammatory bowel diseases are relatively rare. Recently, altered functions of the innate immune system have been identified. As such, both hyperresponsiveness and hyporesponsiveness of innate cells have been implicated in the pathogenesis of inflammatory bowel diseases.
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21
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Takahashi N, Vanlaere I, de Rycke R, Cauwels A, Joosten LAB, Lubberts E, van den Berg WB, Libert C. IL-17 produced by Paneth cells drives TNF-induced shock. ACTA ACUST UNITED AC 2008; 205:1755-61. [PMID: 18663129 PMCID: PMC2525583 DOI: 10.1084/jem.20080588] [Citation(s) in RCA: 144] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Tumor necrosis factor (TNF) has very potent antitumor activity, but it also provokes a systemic inflammatory response syndrome that leads to shock, organ failure, and death. Here, we demonstrate that interleukin (IL)-17, a proinflammatory cytokine known to be produced mainly by activated T cells, has a critical role in this process. Antiserum against IL-17 or deletion of Il17r protected mice against a lethal TNF challenge. Serum levels of TNF-induced IL-6 and nitric oxide metabolites were significantly reduced in mice deficient in the IL-17R. TNF-induced leukocyte influx in the small intestine was reduced, and there was no injury to the small intestine. Surprisingly, electron microscopy showed that IL-17 was constitutively present in Paneth cells of the crypts. Upon TNF challenge, the intracellular pool of IL-17 in these cells was drastically reduced, suggesting rapid release of IL-17 from the granules of Paneth cells. Our findings assign a novel role for IL-17 in an acute inflammation and identify Paneth cells as a source of the IL-17 that plays a role in this process. These data indicate that innate immune cytokine responses in the local mucosa may participate in rapidly amplifying responses to systemic inflammatory challenges.
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Affiliation(s)
- Nozomi Takahashi
- Rheumatology Research and Advanced Therapeutics, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, Netherlands
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22
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Keshav S, McKnight AJ, Arora R, Gordon S. Cloning of intestinal phospholipase A2 from intestinal epithelial RNA by differential display PCR. Cell Prolif 2008. [DOI: 10.1111/j.1365-2184.1997.tb00917.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Affiliation(s)
- S. Keshav
- Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
| | - A. J. McKnight
- Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
| | - R. Arora
- Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
| | - S. Gordon
- Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
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23
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Vidrich A, Buzan JM, Barnes S, Reuter BK, Skaar K, Ilo C, Cominelli F, Pizarro T, Cohn SM. Altered epithelial cell lineage allocation and global expansion of the crypt epithelial stem cell population are associated with ileitis in SAMP1/YitFc mice. THE AMERICAN JOURNAL OF PATHOLOGY 2005; 166:1055-67. [PMID: 15793286 PMCID: PMC1602382 DOI: 10.1016/s0002-9440(10)62326-7] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Crohn's disease is characterized by cycles of mucosal injury and ulceration followed by epithelial regeneration and restoration of normal epithelial function. In this study, we examined whether ileitis in SAMP1/YitFc mice, a recombinant-inbred line that spontaneously develops ileitis resembling human Crohn's disease, was associated with alterations in normal patterns of epithelial differentiation or changes in epithelial regeneration after experimental injury. Increased numbers of Paneth, goblet, and intermediate cells were present focally in the ileum of SAMP1/YitFc mice by 4 weeks of age, before any histological evidence of acute or chronic inflammation. This increase in secretory cells became more pronounced at sites of ileitis with increasing age and inflammation. Additionally, there was mispositioning of Paneth and intermediate cells along the crypt-to-villus unit. A concomitant reduction in the number of absorptive enterocytes was observed. In contrast to the ileal-specific changes in lineage allocation, crypt stem cell numbers began to increase in both the ileum and proximal jejunum at the onset of inflammation in SAMP1/YitFc mice. These data suggest that the alterations in epithelial cell differentiation and increases in the size of the crypt stem cell population observed in SAMP1/YitFc mice are regulated by distinct mechanisms. We speculate that these epithelial alterations may play a role in the pathogenesis of ileitis in this murine model of Crohn's disease.
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Affiliation(s)
- Alda Vidrich
- Digestive Health Center of Excellence, University of Virginia Health System, Charlottesville, VA 22908, USA
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24
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25
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Ayabe T, Ashida T, Kohgo Y, Kono T. The role of Paneth cells and their antimicrobial peptides in innate host defense. Trends Microbiol 2004; 12:394-8. [PMID: 15276616 DOI: 10.1016/j.tim.2004.06.007] [Citation(s) in RCA: 79] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The intestinal epithelium is the largest surface area that is exposed to various pathogens in the environment, however, in contrast to the colon the number of bacteria that colonize the small intestine is extremely low. Paneth cells, one of four major epithelial cell lineages in the small intestine, reside at the base of the crypts and have apically oriented secretory granules. These granules contain high levels of antimicrobial peptides that belong to the alpha-defensin family. Paneth cells secrete these microbicidal granules that contain alpha-defensins when exposed ex vivo to bacteria or their antigens, and recent evidence reveals that antimicrobial peptides, particularly alpha-defensins, that are present in Paneth cells contribute to intestinal innate host defense.
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Affiliation(s)
- Tokiyoshi Ayabe
- Department of Internal Medicine III, Asahikawa Medical College, 2-1-1-1 Midorigaoka-Higashi, Asahikawa 078-8510, Japan.
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26
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Fukuzawa H, Sawada M, Kayahara T, Morita-Fujisawa Y, Suzuki K, Seno H, Takaishi S, Chiba T. Identification of GM-CSF in Paneth cells using single-cell RT-PCR. Biochem Biophys Res Commun 2004; 312:897-902. [PMID: 14651956 DOI: 10.1016/j.bbrc.2003.11.009] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Paneth cells, granule-containing cells located at the bottom of the intestinal crypts, have a role in innate mucosal immunity. We identified the exclusive expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in Paneth cells using single-cell reverse transcription-polymerase chain reaction and cDNA array. Cytosolic total RNA was aspirated from single Paneth cells and other villous epithelial cells (non-Paneth cells) of rats using capillary micropipettes. In addition to lysozyme, secretory phospholipase A2, defensin, TNF-alpha, and xanthine dehydrogenase genes, cDNA array analysis revealed that the GM-CSF gene is specifically present in Paneth cells, whereas GM-CSF receptor beta-chain mRNA is expressed in Paneth cells and other epithelial cells. There was intense immunohistochemical staining of GM-CSF in Paneth cells but not in other epithelial cells. Treatment of IEC6 cells with GM-CSF enhanced expression of CD80 and CD86. Thus, GM-CSF in Paneth cells might have an important role in mucosal immunity through increasing the expression of costimulatory molecules in epithelial cells.
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Affiliation(s)
- Hiroaki Fukuzawa
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawara-cho, Sakyo-ku, 606-8507, Kyoto, Japan
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27
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Lala S, Ogura Y, Osborne C, Hor SY, Bromfield A, Davies S, Ogunbiyi O, Nuñez G, Keshav S. Crohn's disease and the NOD2 gene: a role for paneth cells. Gastroenterology 2003; 125:47-57. [PMID: 12851870 DOI: 10.1016/s0016-5085(03)00661-9] [Citation(s) in RCA: 360] [Impact Index Per Article: 16.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
BACKGROUND & AIMS The NOD2 gene, which is strongly associated with susceptibility to Crohn's disease (CD) of the terminal ileum, interacts with bacterial lipopolysaccharide (LPS), inducing cellular activation. However, the mechanisms by which NOD2 mutations cause terminal ileitis are unknown, and NOD2 is expressed most highly by peripheral blood monocytes, which are distributed ubiquitously and readily respond to LPS via cell-surface receptors. Paneth cells on the other hand, are most numerous in the terminal ileum, are critically important in enteric antibacterial defense, and respond to LPS through as yet undefined pathways. We therefore determined if these specialized intestinal epithelial cells also expressed the NOD2 gene. METHODS In situ hybridization, immunohistochemistry, and laser-capture microdissection were used to determine RNA and protein expression in tissue sections, and real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantitate gene expression in intestinal epithelial cells and peripheral blood mononuclear cells. RESULTS NOD2 was detected readily in monocytes, but not in mature macrophages in the lamina propria or within granulomas, and levels declined as monocytes differentiated into macrophages in vitro, so that Caco-2 cells expressed more NOD2 mRNA than macrophages. NOD2 mRNA was enriched in crypts compared with villi, and in situ, Paneth cells were the most prominent cells expressing NOD2 in normal and CD-affected intestinal tissue, where they also strongly expressed tumor necrosis factor alpha (TNFalpha) RNA. CONCLUSIONS The NOD2 gene product is most abundant in Paneth cells in the terminal ileum, which could therefore play a critical and hitherto unrecognized role in the pathogenesis of NOD2-associated CD.
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Affiliation(s)
- Sanjay Lala
- Department of Medicine, Royal Free & University College Medical School, London, United Kingdom
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28
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Hsueh W, Caplan MS, Qu XW, Tan XD, De Plaen IG, Gonzalez-Crussi F. Neonatal necrotizing enterocolitis: clinical considerations and pathogenetic concepts. Pediatr Dev Pathol 2003; 6:6-23. [PMID: 12424605 PMCID: PMC7098425 DOI: 10.1007/s10024-002-0602-z] [Citation(s) in RCA: 275] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/12/2002] [Accepted: 08/21/2002] [Indexed: 12/13/2022]
Abstract
Necrotizing enterocolitis (NEC), a disease affecting predominantly premature infants, is a leading cause of morbidity and mortality in neonatal intensive care units. Although several predisposing factors have been identified, such as prematurity, enteral feeding, and infection, its pathogenesis remains elusive. In the past 20 years, we have established several animal models of NEC in rats and found several endogenous mediators, especially platelet-activating factor (PAF), which may play a pivotal role in NEC. Injection of PAF induces intestinal necrosis, and PAF antagonists prevent the bowel injury induced by bacterial endotoxin, hypoxia, or challenge with tumor necrosis factor-a (TNF) plus endotoxin in adult rats. The same is true for lesions induced by hypoxia and enteral feeding in neonatal animals. Human patients with NEC show high levels of PAF and decreased plasma PAF-acetylhydrolase, the enzyme degrading PAF. The initial event in our experimental models of NEC is probably polymorphonuclear leukocyte (PMN) activation and adhesion to venules in the intestine, which initiates a local inflammatory reaction involving proinflammatory mediators including TNF, complement, prostaglandins, and leukotriene C4. Subsequent norepinephrine release and mesenteric vasoconstriction result in splanchnic ischemia and reperfusion. Bacterial products (e.g., endotoxin) enter the intestinal tissue during local mucosal barrier breakdown, and endotoxin synergizes with PAF to amplify the inflammation. Reactive oxygen species produced by the activated leukocytes and by intestinal epithelial xanthine oxidase may be the final pathway for tissue injury. Protective mechanisms include nitric oxide produced by the constitutive (mainly neuronal) nitric oxide synthase, and indigenous probiotics such as Bifidobacteria infantis. The former maintains intestinal perfusion and the integrity of the mucosal barrier, and the latter keep virulent bacteria in check. The development of tissue injury depends on the balance between injurious and protective mechanisms.
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MESH Headings
- Animals
- Animals, Newborn
- Disease Models, Animal
- Enterocolitis, Necrotizing/blood
- Enterocolitis, Necrotizing/etiology
- Enterocolitis, Necrotizing/pathology
- Humans
- Infant, Newborn
- Infant, Newborn, Diseases/blood
- Infant, Newborn, Diseases/etiology
- Infant, Newborn, Diseases/pathology
- Platelet Activating Factor/analysis
- Species Specificity
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Affiliation(s)
- Wei Hsueh
- Department of Pathology, Children's Memorial Hospital, Northwestern University Medical School, 2300 Children's Plaza, Chicago, IL 60614, USA.
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29
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Savard CE, Blinman TA, Choi HS, Lee SK, Pandol SJ, Lee SP. Expression of cytokine and chemokine mRNA and secretion of tumor necrosis factor-alpha by gallbladder epithelial cells: response to bacterial lipopolysaccharides. BMC Gastroenterol 2002; 2:23. [PMID: 12377103 PMCID: PMC130965 DOI: 10.1186/1471-230x-2-23] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/13/2002] [Accepted: 10/11/2002] [Indexed: 01/30/2023] Open
Abstract
BACKGROUND In addition to immune cells, many other cell types are known to produce cytokines. Cultured normal mouse gallbladder epithelial cells, used as a model system for gallbladder epithelium, were examined for their ability to express the mRNA of various cytokines and chemokines in response to bacterial lipopolysaccharide. The synthesis and secretion of the tumor necrosis factor-alpha (TNF-alpha) protein by these cells was also measured. RESULTS Untreated mouse gallbladder cells expressed mRNA for TNF-alpha, RANTES, and macrophage inflammatory protein-2 (MIP-2). Upon treatment with lipopolysaccharide, these cells now produced mRNA for Interleukin-1beta (IL-1beta), IL-6, monocyte chemoattractant protein-1 (MCP-1), and showed increased expression of TNF-alpha and MIP-2 mRNA. Untreated mouse gallbladder cells did not synthesize TNF-alpha protein; however, they did synthesize and secrete TNF-alpha upon treatment with lipopolysaccharide. METHODS Cells were treated with lipopolysaccharides from 3 strains of bacteria. Qualitative and semi-quantitative RT-PCR, using cytokine or chemokine-specific primers, was used to measure mRNA levels of TNFalpha, IL-1beta, IL-6, IL-10, KC, RANTES, MCP-1, and MIP-2. TNF-alpha protein was measured by immunoassays. CONCLUSION This research demonstrates that gallbladder epithelial cells in response to lipopolysaccharide exposure can alter their cytokine and chemokine RNA expression pattern and can synthesize and secrete TNFalpha protein. This suggests a mechanism whereby gallbladder epithelial cells in vivo may mediate gallbladder secretory function, inflammation and diseases in an autocrine/paracrine fashion by producing and secreting cytokines and/or chemokines during sepsis.
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Affiliation(s)
- Christopher E Savard
- Department of Medicine, University of Washington and VA Puget Sound Health Care System, Seattle, Washington USA
| | - Thane A Blinman
- Department of Medicine, University of California at Los Angeles and West Los Angeles VA Medical Center, Los Angeles, California USA
| | - Ho-Soon Choi
- Department of Medicine, University of Washington and VA Puget Sound Health Care System, Seattle, Washington USA
| | - Sung-Koo Lee
- Department of Medicine, University of Washington and VA Puget Sound Health Care System, Seattle, Washington USA
| | - Stephen J Pandol
- Department of Medicine, University of California at Los Angeles and West Los Angeles VA Medical Center, Los Angeles, California USA
| | - Sum P Lee
- Department of Medicine, University of Washington and VA Puget Sound Health Care System, Seattle, Washington USA
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Ogle CK, Noel JG, Guo X, Wells DA, Valente JF, Ogle JD, Alexander JW. The ability of endotoxin-stimulated enterocytes to produce bactericidal factors. Crit Care Med 2002; 30:428-34. [PMID: 11889324 DOI: 10.1097/00003246-200202000-00027] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
OBJECTIVE Bactericidal peptides, specifically defensins, are produced by polymorphonuclear cells. Intestinal epithelial cells also produce bactericidal peptides, perhaps as part of their barrier function, to the greatest load of endogenous bacteria present in the body. We sought to determine whether and under what conditions intestinal cell lines could produce bactericidal compounds. DESIGN Laboratory investigation. SETTING Children's burn hospital. SUBJECTS Caco-2, IEC-6, and HT-29 cell lines. INTERVENTIONS Three different enterocyte lines were cultured for 1 day +/- lipopolysaccharide (1 or 10 microg/mL), and their supernatants were tested for bactericidal activity. Also, reverse transcription-polymerase chain reaction of Caco-2 cells was performed to assess the expression of defensin-6 mRNA. MEASUREMENTS AND MAIN RESULTS After culture, enterocytes all were found to release one or more soluble factors with bactericidal activity (as measured fluorometrically by using a metabolizable dye) when stimulated by lipopolysaccharide (1 microg/mL). The bactericidal activity of these culture supernatants was saturated by increased bacterial load, additive to the effects of normal human peripheral blood polymorphonuclear cells, and was reduced by serial supernatant dilution. Enterocyte stimulation with larger amounts of lipopolysaccharide (10 microg/mL) resulted in greater bactericidal activity. After supernatant fractionation based on molecular weight, the bactericidal effect was best retained in the <10-kDa fraction. In addition, the expression of mRNA for defensin-6, a bactericidal peptide produced by neutrophils, was seen in Caco-2 cells. CONCLUSION Enterocytes are shown to produce a soluble, low molecular weight, bactericidal compound in response to endotoxin stimulation. The expression of defensin-6 mRNA in Caco-2 cells suggests that intestinal cells may release defensins as bactericidal peptides. This experimental system provides an in vitro model to study the activity and production of bactericidal factors by enterocytes.
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Affiliation(s)
- Cora K Ogle
- Shriners Burns Hospital for Children and the Department of Surgery, University of Cincinnati, Cincinnati, OH 45229-3095, USA.
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31
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Fontan E, Saklani-Jusforgues H, Goossens PL. Early translocation of acid-adapted Listeria monocytogenes during enteric infection in TNF/LTalpha-/- mice. FEMS Microbiol Lett 2001; 205:179-83. [PMID: 11750799 DOI: 10.1111/j.1574-6968.2001.tb10944.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
TNF/LTalpha deficient mice are devoid of Peyer's patches and lack mesenteric lymph nodes. Translocation, especially in the early steps after intragastric delivery of Listeria monocytogenes, has been explored in this study, and the role of TNFalpha has been addressed. We showed that L. monocytogenes translocation occurred at least as efficiently in TNF/LTalpha-/- mice as in TNF/LTalpha+/+ littermates. Even very low inocula (2.7x10(4) cfu) could initiate infection in the TNF/LTalpha deficient mice. Early kinetics of dissemination to the spleen and liver were similar, L. monocytogenes reaching these organs at 8 h post inoculation. However, a 10-fold higher bacterial load was observed at this early time point in the TNF/LTalpha deficient mice. rTNF pretreatment (4 h before intragastric inoculation) had no effect on the L. monocytogenes associated with the caecum-colon walls at 10 h after inoculation, although bacterial levels in the caecum-colon lumen and in spleen and liver were already controlled.
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Affiliation(s)
- E Fontan
- Unité d'Immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, 25 rue du Docteur Roux, 75724 Cedex 15, Paris, France.
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Kamal M, Wakelin D, Ouellette AJ, Smith A, Podolsky DK, Mahida YR. Mucosal T cells regulate Paneth and intermediate cell numbers in the small intestine of T. spiralis-infected mice. Clin Exp Immunol 2001; 126:117-25. [PMID: 11678907 PMCID: PMC1906161 DOI: 10.1046/j.1365-2249.2001.01589.x] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/16/2001] [Indexed: 12/24/2022] Open
Abstract
Secretions of Paneth, intermediate and goblet cells have been implicated in innate intestinal host defense. We have investigated the role of T cells in effecting alterations in small intestinal epithelial cell populations induced by infection with the nematode Trichinella spiralis. Small intestinal tissue sections from euthymic and athymic (nude) mice, and mice with combined deficiency in T-cell receptor beta and delta genes [TCR(beta/delta)-/-] infected orally with T. spiralis larvae, were examined by electron microscopy and after histochemical and lineage-specific immunohistochemical staining. Compared with uninfected controls, Paneth and intermediate cell numbers increased significantly in infected euthymic and nude mice but not infected TCR(beta/delta)-/- mice. Transfer of mesenteric lymph node cells before infection led to an increase in Paneth and intermediate cells in TCR(beta/delta)-/- mice. In infected euthymic mice, Paneth cells and intermediate cells expressed cryptdins (alpha-defensins) but not intestinal trefoil factor (ITF), and goblet cells expressed ITF but not cryptdins. In conclusion, a unique, likely thymic-independent population of mucosal T cells modulates innate small intestinal host defense in mice by increasing the number of Paneth and intermediate cells in response to T. spiralis infection.
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Affiliation(s)
- M Kamal
- Division of Gastroenterology, University of NottinghamUK
- School of Biological Sciences, University of NottinghamUK
| | - D Wakelin
- School of Biological Sciences, University of NottinghamUK
| | - A J Ouellette
- Department of Pathology, University of CaliforniaIrvine, USA
| | - A Smith
- Institute of Animal HealthCompton, UK
| | - D K Podolsky
- Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical SchoolBoston, USA
| | - Y R Mahida
- Division of Gastroenterology, University of NottinghamUK
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Morita Y, Sawada M, Seno H, Takaishi S, Fukuzawa H, Miyake N, Hiai H, Chiba T. Identification of xanthine dehydrogenase/xanthine oxidase as a rat Paneth cell zinc-binding protein. BIOCHIMICA ET BIOPHYSICA ACTA 2001; 1540:43-9. [PMID: 11476893 DOI: 10.1016/s0167-4889(01)00118-5] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Paneth cells are zinc-containing cells localized in small intestinal crypts, but their function has not been fully elucidated. Previously, we showed that an intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induced selective killing of Paneth cells, and purified a zinc-binding protein in Paneth cells. In the present study, we further characterized one of these proteins, named zinc-binding protein of Paneth cells (ZBPP)-1. Partial amino acid sequences of ZBPP-1 showed identity with rat xanthine dehydrogenase (XD)/xanthine oxidase (XO). Anti-rat XD antibody (Ab) recognized ZBPP-1, and conversely anti ZBPP-1 Ab recognized 85 kDa fragment of rat XD in Western blotting. Messenger RNA and protein levels of XD were consistent with our previous data on the fluctuation of Paneth cell population after dithizone injection. Thus, ZBPP-1 is an 85 kDa fragment of XD/XO in Paneth cells. XD/XO in Paneth cells may play important roles in intestinal function.
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Affiliation(s)
- Y Morita
- Department of Internal Medicine, Graduate School of Medicine, Kyoto University, Japan
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Yamamoto S, Tanabe M, Wakabayashi G, Shimazu M, Matsumoto K, Kitajima M. The role of tumor necrosis factor-alpha and interleukin-1beta in ischemia-reperfusion injury of the rat small intestine. J Surg Res 2001; 99:134-41. [PMID: 11421615 DOI: 10.1006/jsre.2001.6106] [Citation(s) in RCA: 102] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
BACKGROUND To determine the role of tumor necrosis factor (TNF) and interleukin (IL)-1 in small-intestinal ischemia-reperfusion (I-R) injury, we investigated the effect of FR 167653, a specific IL-1 and TNF inhibitor, on warm I-R injury of the rat small intestine. MATERIALS AND METHODS Male rats treated with either saline (NS group) or FR 167653 (FR group) underwent 150 min of warm small-intestinal ischemia by applying a vascular clip at the origin of the superior mesenteric artery. In addition to the survival analyses, we investigated plasma TNF-alpha and endotoxin levels, intestinal tissue TNF-alpha and IL-1beta levels, hematocrit values and the amount of exudates in the intestinal lumen, glutamic aspartate aminotransferase (AST), and histological findings up to 120 min after reperfusion. RESULTS TNF-alpha and IL-1beta levels in the intestinal tissue, and plasma TNF-alpha and endotoxin levels, were significantly (P < 0.05) reduced in the FR group. Severe mucosal damage on histological findings (120 min after reperfusion) and a large amount of intraluminal exudates (60 min after reperfusion) were shown in the NS group, but these findings were significantly (P < 0.05) ameliorated in the FR group. Serum AST levels in the NS group increased 120 min after reperfusion, but this change was significantly (P<0.05) reduced in the FR group. The 30-day survival rate was 80% in the FR group and 30% in the NS group (P<0.05). CONCLUSIONS Dual inhibition of TNF and IL-1 effectively alleviated intestinal I-R injury, suggesting the key role of TNF and IL-1 in this pathophysiology.
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Affiliation(s)
- S Yamamoto
- Department of Surgery, Keio University School of Medicine, Shinanomachi 35, Shinjuku-ku, Tokyo 160-8582, Japan
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Schaeffer C, Diab-Assef M, Plateroti M, Laurent-Huck F, Reimund JM, Kedinger M, Foltzer-Jourdainne C. Cytokine gene expression during postnatal small intestinal development: regulation by glucocorticoids. Gut 2000; 47:192-8. [PMID: 10896909 PMCID: PMC1728015 DOI: 10.1136/gut.47.2.192] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/08/2022]
Abstract
BACKGROUND In the intestinal mucosa, numerous cytokines produced by the epithelium, fibroblasts, and immune cells were shown to affect epithelial differentiation and proliferation through epithelial-mesenchymal and epithelial-immune cell interactions. To date, the importance of cytokines in postnatal development of the rat small intestine has not been established. AIM To investigate the developmental changes in expression of mucosal cytokines in the postnatal maturation of the rat small intestinal epithelium and their regulation by glucocorticoids (GC). METHODS Mucosal maturation was assessed by the onset of sucrase-isomaltase (SI) mRNA, analysed by in situ hybridisation. The amount of transforming growth factor beta1 (TGF-beta1), beta2 (TGF-beta2), tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and TGF-alpha was analysed by reverse transcription-polymerase chain reaction (RT-PCR) in mucosal extracts from weaning (14-21 days old) and adult rats, or one day after an injection of hydrocortisone (HC) in 11 day old rats. Similarly, expression of cytokines and the regulatory effect of GC were studied on cultured subepithelial myofibroblasts cloned from postnatal jejunum and ileum cultured in the absence or presence of dexamethasone (DX). RESULTS TGF-beta1, TGF-beta2, and IL-1beta decreased during the third week of life while levels of TNF-alpha increased and TGF-alpha remained constant. In parallel, SI transcripts increased and showed a progressive accumulation in the apical part of the enterocytes first localised at the base of the villi from 18 days onwards. Interestingly, precocious induction of SI mRNA by HC paralleled the decrease in expression of TGF-beta isoforms and of IL-1beta. All cytokines were expressed in the myofibroblast cell lines. In addition, the results showed that TNF-alpha was differentially expressed in basal culture conditions and after DX stimulation in jejunal and ileal myofibroblasts. DX decreased IL-1beta but not the TGF-beta isoforms, similar to that in vivo. CONCLUSIONS This study shows that mucosal cytokines are developmentally regulated and that GC are potentially involved in this regulation in parallel with maturation of the gut mucosa at weaning.
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Affiliation(s)
- C D Johnson
- The University of Tennessee, Memphis, TN 38163, USA
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Condon MR, Viera A, D'Alessio M, Diamond G. Induction of a rat enteric defensin gene by hemorrhagic shock. Infect Immun 1999; 67:4787-93. [PMID: 10456932 PMCID: PMC96810 DOI: 10.1128/iai.67.9.4787-4793.1999] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Multicellular organisms utilize a battery of extracellular and cellular mechanisms to defend against microbial infiltration. Among the armamentarium used by the small intestine to defend against microbial invasion are antimicrobial peptides called defensins. We previously have shown that gut barrier function is impaired following hemorrhagic shock, resulting in translocation of bacteria or endotoxin. Using a rat model, we examined the effect of hemorrhagic shock on alpha-defensin expression. We utilized the anchored reverse transcriptase PCR strategy to isolate a rat enteric defensin cDNA. The cDNA is 406 bases in length and encodes a putative prepro-enteric defensin that we have named rat defensin 5 (RD-5). RD-5 expression is restricted to the small intestine and is specifically localized by in situ hybridization to the Paneth cells. A 10-fold increase in its steady state levels was observed in the distal intestine immediately after the termination of shock. This is the first study to show that enteric defensins are inducible following injury. We suggest that enteric defensins may contribute to the complex and integrated barrier function of the intestinal mucosal surface.
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Affiliation(s)
- M R Condon
- Department of Veterans Affairs Medical Center, East Orange, New Jersey, USA.
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Stüber E, Büschenfeld A, von Freier A, Arendt T, Fölsch UR. Intestinal crypt cell apoptosis in murine acute graft versus host disease is mediated by tumour necrosis factor alpha and not by the FasL-Fas interaction: effect of pentoxifylline on the development of mucosal atrophy. Gut 1999; 45:229-35. [PMID: 10403735 PMCID: PMC1727618 DOI: 10.1136/gut.45.2.229] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/08/2022]
Abstract
BACKGROUND Murine T cell mediated acute semiallogeneic graft versus host disease (GVHD) is characterised by lymphocytic infiltrates, crypt hyperplasia, and villous atrophy. It has been shown that programmed cell death (apoptosis) of the crypt epithelium takes place during the intestinal manifestation of acute GVHD. AIMS To investigate which of the two most investigated inductors of apoptosis (Fas ligand (FasL) and tumour necrosis factor alpha (TNF-alpha)) is responsible for the induction of apoptosis in this animal model. METHODS Animals undergoing acute semiallogeneic GvH reaction were treated with neutralising anti-TNF-alpha, two different anti-FasL antibodies, or pentoxifylline. RESULTS Anti-TNF-alpha application inhibited the appearance of apoptotic cells in the intestinal mucosa, whereas anti-FasL antibodies had no influence on mucosal apoptosis. In addition, the transfer of FasL deficient (gld) donor lymphocytes still induced crypt cell apoptosis, villous atrophy, and crypt hyperplasia. Furthermore, when the animals were treated with pentoxifylline, a known inhibitor of TNF-alpha secretion in vitro and in vivo, there was significant normalisation of the intestinal morphology accompanied by inhibition of epithelial apoptosis. CONCLUSIONS The FasL-Fas interaction is not involved in the induction of apoptosis during acute GVHD. However, neutralisation of TNF-alpha by an antibody or by pentoxifylline inhibits the occurrence of apoptosis and of mucosal atrophy in this animal model. These results have implications for the treatment of immunologically mediated human atrophic gut diseases-for example, diet refractory cases of coeliac disease.
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Affiliation(s)
- E Stüber
- I. Medizinische Universitätsklinik, Department of Internal Medicine, Christian-Albechts-Universität, Kiel, Germany
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Qu XW, Rozenfeld RA, Huang W, Sun X, Tan XD, Hsueh W. Roles of nitric oxide synthases in platelet-activating factor-induced intestinal necrosis in rats. Crit Care Med 1999; 27:356-64. [PMID: 10075061 DOI: 10.1097/00003246-199902000-00043] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
OBJECTIVE To examine the role of constitutive and inducible nitric oxide synthases (cNOS and iNOS) in platelet-activating factor (PAF)-induced shock and intestinal injury. DESIGN Prospective, randomized, controlled experimental study. SETTING Hospital research laboratory. SUBJECTS Young adult male Sprague-Dawley rats were anesthetized and studied. INTERVENTIONS Rats were injected with PAF, either alone or after the following pretreatments: a) selective iNOS inhibitors aminoguanidine or S-methylisothiourea; b) 3-morpholinosydnonimine, a NO donor; c) S-methylisothiourea + 3-morpholinosydnonimine; and d) antineutrophil antibody (to deplete neutrophils). MEASUREMENTS AND MAIN RESULTS Blood pressure, hematocrit, white blood cell counts, intestinal injury, and intestinal cNOS and iNOS activities were assessed. We found that: a) cNOS is the predominant NOS in the intestine and its activity is inversely correlated to the level of tissue injury; b) there is a time-dependent increase in cNOS activity in sham-operated animals, which was abolished by PAF; c) Western blotting and immunohistochemistry showed iNOS present in the normal intestine, localizing mainly in crypt cells; d) iNOS inhibitors attenuated PAF-induced injury in animals with high cNOS activity, but had no protective effect in animals with low cNOS activity; e) 3-morpholinosydnonimine, alone or together with S-methylisothiourea, alleviated PAF-induced injury; and f) neutrophil depletion blocked the suppressive effect of PAF on cNOS and prevented injury. CONCLUSIONS We conclude that cNOS and iNOS play different roles in PAF-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of iNOS inhibitors.
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Affiliation(s)
- X W Qu
- Department of Pathology, Children's Memorial Hospital, Northwestern University Medical School, Chicago, IL 60614, USA
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Vitkus SJ, Hanifin SA, McGee DW. Factors affecting Caco-2 intestinal epithelial cell interleukin-6 secretion. In Vitro Cell Dev Biol Anim 1998; 34:660-4. [PMID: 9769153 DOI: 10.1007/s11626-996-0017-7] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Intestinal epithelial cells (IEC) have previously been shown to produce several cytokines including interleukin-6 (IL-6). However, many factors which may regulate IL-6 secretion by human IEC still remain a mystery due in part to the lack of appropriate model cell lines and the difficulty of culturing human IEC over long periods of time. We have determined that the human colonic carcinoma cell line Caco-2 is capable of secreting IL-6 when stimulated by the inflammatory cytokines IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and stimulation of these cells with IL-1beta plus TNF-alpha induced a synergistic enhancement of IL-6 secretion. The inflammatory cytokine-induced enhancement in IL-6 secretion was greatest when the cells were cultured in a 10% CO2 atmosphere as compared to cells grown in 5% CO2, suggesting that environmental CO2 levels may affect IEC cytokine secretion. Finally, long-term culture of the Caco-2 cells to induce cellular differentiation had no effect on the capacity of these cells to produce IL-6, indicating that the regulation of IL-6 secretion was not affected by differentiation. Taken together, these studies provide important information on the factors which regulate IL-6 secretion by human IEC as they may contribute to the cytokine network during a mucosal inflammation. The results also suggest that the Caco-2 cell line is an appropriate model for further studies on the regulation of cytokine secretion by human IEC.
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Affiliation(s)
- S J Vitkus
- Department of Biological Sciences, Binghamton University (SUNY), New York 13902-6000, USA
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41
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Krammer PH, Galle PR, Möller P, Debatin KM. CD95(APO-1/Fas)-mediated apoptosis in normal and malignant liver, colon, and hematopoietic cells. Adv Cancer Res 1998; 75:251-73. [PMID: 9709812 DOI: 10.1016/s0065-230x(08)60744-7] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Affiliation(s)
- P H Krammer
- Tumorimmunology Program, German Cancer Research Center, Heidelberg, Germany
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42
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Hsueh W, Caplan MS, Tan X, MacKendrick W, Gonzalez-Crussi F. Necrotizing enterocolitis of the newborn: pathogenetic concepts in perspective. Pediatr Dev Pathol 1998; 1:2-16. [PMID: 10463267 PMCID: PMC7088176 DOI: 10.1007/s100249900002] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Affiliation(s)
- W Hsueh
- Department of Pathology, Children's Memorial Hospital, Northwestern University Medical School, Chicago, IL 60614, USA
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Autenrieth IB, Bucheler N, Bohn E, Heinze G, Horak I. Cytokine mRNA expression in intestinal tissue of interleukin-2 deficient mice with bowel inflammation. Gut 1997; 41:793-800. [PMID: 9462212 PMCID: PMC1891612 DOI: 10.1136/gut.41.6.793] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND Mice deficient in interleukin-2 (IL-2) develop inflammatory bowel disease resembling ulcerative colitis in humans. Recent studies provided evidence that alpha beta T cells, particularly CD4 T cells, rather than B cells, are involved in the pathogenesis of bowel inflammation of IL-2 deficient mice. AIM To analyse the pattern of expression of cytokine mRNA in intestinal tissue of normal and IL-2 deficient mice. METHODS Expression of beta-actin, IL-1 alpha, IL-1 beta, IL-6, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and transforming growth factor beta 1 (TGF-beta 1) mRNA was analysed in colon and small intestinal tissue of both IL-2 deficient (IL-2-/-) mice and normal (wild type) litter mates (IL-2+/+) at different ages by using qualitative, as well as semiquantitative, competitive reverse transcription polymerase chain reaction (RT-PCR). Results were correlated with the phase of progression of the disease, as determined by histology. RESULTS IL-2-/- mice had expressed low levels of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, and IFN-gamma mRNA in the colon by 1.5 weeks of age. In advance of the development of histologically and clinically detectable bowel inflammation, expression of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, IFN-gamma, and IL-10, but not TGF-beta 1, mRNA increased in the colon of IL-2 deficient mice. In contrast, IL-2+/+ mice expressed TGF-beta 1 mRNA in colon tissue at 13 and 23 weeks of age, but not IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, IL-10, or IFN-gamma mRNA. Levels of expression of cytokine mRNA in tissue from the small intestine were comparable in IL-2-/- and IL-2+/+ mice. CONCLUSIONS Bowel inflammation in IL-2 deficient mice is preceded by an increase in IL-1 alpha, IL-1 beta, TNF-alpha, and IFN-gamma mRNA expression in colon tissue. Low levels of TGF-beta 1, but high levels of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, IFN-gamma, and IL-10 mRNA expression correlate with the manifestation of severe colitis, and suggest that T cells and macrophages are involved in bowel inflammation of IL-2 deficient mice.
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Affiliation(s)
- I B Autenrieth
- Max v. Pettenkofer Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität, München, Germany
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Amiot F, Boussadia O, Cases S, Fitting C, Lebastard M, Cavaillon JM, Milon G, Dautry F. Mice heterozygous for a deletion of the tumor necrosis factor-alpha and lymphotoxin-alpha genes: biological importance of a nonlinear response of tumor necrosis factor-alpha to gene dosage. Eur J Immunol 1997; 27:1035-42. [PMID: 9130661 DOI: 10.1002/eji.1830270434] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The tumor necrosis factors (TNF-alpha and lymphotoxin, or LT-alpha) are important mediators of the immune and inflammatory responses, and it has been proposed that a positive feedback loop could boost the expression of the TNF to sufficiently high levels to fend off infections. To investigate this phenomenon and its biological consequences, we have generated LT-alpha/TNF-alpha knockout mice and compared mice having one or two functional LT-alpha/TNF-alpha alleles. In response to lipopolysaccharide (LPS) stimulation, TNF-alpha levels in the circulation or in the supernatant of macrophage cultures were 20- to 100-fold lower in heterozygous samples than in their wild-type counterparts. This differential increased with the intensity of stimulation and throughout the response, supporting the involvement of a positive feedback loop. Moreover, the heterozygous mice had an increased bacterial load following Listeria monocytogenes infection and exhibited a bimodal response to the association of D-galactosamine and LPS which was similar to that of wild-type mice at low doses of LPS and more like that of homozygous mutants at high doses. These results therefore establish the biological importance of the nonlinear response of TNF-alpha levels to gene dosage, and these mice provide a unique tool to study how the propensity to produce TNF can determine the immunological fitness of individuals.
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Affiliation(s)
- F Amiot
- Génétique Moléculaire et Intégration des Fonctions Cellulaires, CNRS UPR 9044, Institut de Recherches sur le Cancer, Villejuif, France
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45
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Affiliation(s)
- Y R Mahida
- Division of Gastroenterology, University Hospital, Queen's Medical Centre, Nottingham
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Qu XD, Lloyd KC, Walsh JH, Lehrer RI. Secretion of type II phospholipase A2 and cryptdin by rat small intestinal Paneth cells. Infect Immun 1996; 64:5161-5. [PMID: 8945560 PMCID: PMC174502 DOI: 10.1128/iai.64.12.5161-5165.1996] [Citation(s) in RCA: 86] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
We examined the secretion of antimicrobial proteins and peptides into surgically isolated and continuously perfused segments of rat small intestine. Up to nine discrete antimicrobial molecules appeared in the intestinal perfusates following intravenous administration of bethanechol, a cholinergic agonist, or intralumenal instillation of lipopolysaccharide (LPS). Among them were three markers of Paneth cell secretion: lysozyme; type II (secretory) phospholipase A2; and at least one intestinal defensin, RIP-3, that appeared to be an alternatively processed variant of the rat neutrophil defensin RatNP-3. Both bethanechol- and LPS-stimulated intestinal lumenal perfusates (washings) contained molecules that killed Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes in vitro. These molecules were more active against the avirulent S. typhimurium strain 7953S (phoP) than against its virulent parent, S. typhimurium 14028S. These data demonstrate that small intestinal Paneth cells secrete antimicrobial peptides in vivo, that this secretion is regulated by the autonomic (parasympathetic) cholinergic nervous system, and that the release of antimicrobial molecules can be triggered by the presence of bacterial LPS in the intestinal lumen.
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Affiliation(s)
- X D Qu
- Department of Medicine, UCLA School of Medicine, Los Angeles, California 90095-1690, USA
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47
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Stadnyk AW, Kearsey JA. Pattern of proinflammatory cytokine mRNA expression during Trichinella spiralis infection of the rat. Infect Immun 1996; 64:5138-43. [PMID: 8945557 PMCID: PMC174499 DOI: 10.1128/iai.64.12.5138-5143.1996] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Trichinella spiralis occupies an intramulticellular niche in the small intestinal epithelium, and thus we examined the intestine and gut-associated tissues for proinflammatory cytokines during the infection. We document the patterns of interleukin-1 (IL-1), IL-6, gamma interferon, and tumor necrosis factor alpha mRNA expression in the duodenum, jejunum, Peyer's patches, mesenteric lymph node, spleen, and liver in T. spiralis-infected rats. By reverse transcription-PCR detection of mRNAs, IL-1beta was found increased in the jejunum but only on day 2. The jejunal IL-1beta increase was attributed to the epithelium by isolating epithelial cells and then depleting them of intraepithelial lymphocytes prior to analysis. The only cytokine for which mRNA was substantially increased in tissues later in infection was tumor necrosis factor alpha in the spleen and, to a lesser extent, in the mesenteric lymph node. In fact mRNA levels for some cytokines declined below uninfected levels in some organs during the infection. IL-1 may be important in the initiation of the intestinal inflammatory response to this infection.
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Affiliation(s)
- A W Stadnyk
- Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
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48
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Möller P, Walczak H, Reidl S, Sträter J, Krammer PH. Paneth cells express high levels of CD95 ligand transcripts: a unique property among gastrointestinal epithelia. THE AMERICAN JOURNAL OF PATHOLOGY 1996; 149:9-13. [PMID: 8686766 PMCID: PMC1865214] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
CD95 (APO-1/Fas), a cell surface receptor and member of the tumor necrosis factor receptor superfamily, induces apoptosis upon oligomerization. CD95 is broadly expressed in normal tissues. The CD95 ligand (CD95L) is a member of the tumor necrosis factor family of cytokines and exists in a membrane-bound and in a soluble form. In vitro, CD95L is expressed and released by activated T lymphocytes. The range of cell types capable of expressing CD95L in vivo is unknown so far. Using a specific probe for human CD95L and sensitive in situ hybridization, we examined CD95L mRNA expression along the gastrointestinal tract. The scarce lymphohistiocytic infiltrate within the lamina propria contained small subsets of medium-sized labeled cells, some of which bad short cytoplasmic protrusions and others of which were lymphoid in morphology. Autochthonous cells of the gastrointestinal tract did not contain any detectable transcripts except for Paneth cells that expressed CD95L mRNA at high levels. In ulcerative colitis, CD95L mRNA-positive inflammatory cells were increased in number, and metaplastic Paneth cells were the only epithelial cells expressing CD95L. Paneth cells are CD95 negative. Therefore, these cells may not commit CD95-mediated autocrine suicide. By secreting soluble CD95L, however, the Paneth cells might contribute to mucosal integrity.
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Affiliation(s)
- P Möller
- Institute of Pathology of the University of Ulm, Germany
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Wilson CL, Heppner KJ, Rudolph LA, Matrisian LM. The metalloproteinase matrilysin is preferentially expressed by epithelial cells in a tissue-restricted pattern in the mouse. Mol Biol Cell 1995; 6:851-69. [PMID: 7579699 PMCID: PMC301245 DOI: 10.1091/mbc.6.7.851] [Citation(s) in RCA: 106] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
To explore the role of the matrix metalloproteinase matrilysin (MAT) in normal tissue remodeling, we cloned the murine homologue of MAT from postpartum uterus using RACE polymerase chain reaction and examined its pattern of expression in embryonic, neonatal, and adult mice. The murine coding sequence and the corresponding predicted protein sequence were found to be 75% and 70% identical to the human sequences, respectively, and organization of the six exons comprising the gene is similar to the human gene. Northern analysis and in situ hybridization revealed that MAT is expressed in the normal cycling, pregnant, and postpartum uterus, with levels of expression highest in the involuting uterus at early time points (6 h to 1.5 days postpartum). The mRNA was confined to epithelial cells lining the lumen and some glandular structures. High constitutive levels of MAT transcripts were also detected in the small intestine, where expression was localized to the epithelial Paneth cells at the base of the crypts. Similarly, MAT expression was found in epithelial cells of the efferent ducts, in the initial segment and cauda of the epididymis, and in an extra-hepatic branch of the bile duct. MAT transcripts were detectable only by reverse transcription-polymerase chain reaction in the colon, kidney, lung, skeletal muscle, skin, stomach, juvenile uterus, and normal, lactating, and involuting mammary gland, as was expression primarily late in embryogenesis. Analysis of MAT expression during postnatal development indicated that although MAT is expressed in the juvenile small intestine and reproductive organs, the accumulation of significant levels of MAT mRNA appears to correlate with organ maturation. These results show that MAT expression is restricted to specific organs in the mouse, where the mRNA is produced exclusively by epithelial cells, and suggest that in addition to matrix degradation and remodeling, MAT may play an important role in the differentiated function of these organs.
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Affiliation(s)
- C L Wilson
- Department of Cell Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA
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Shmakov AN, Panteleeva NG, Fedjanov AV, Trufakin VA. The role of lympho-epithelial interactions in the regulation of small intestinal epithelium proliferation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 1995; 371A:265-9. [PMID: 8525921 DOI: 10.1007/978-1-4615-1941-6_55] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Affiliation(s)
- A N Shmakov
- Siberian Branch, Russian Academy of Medical Sciences, Institute of Clinical and Experimental Lymphology, Novosibirsk
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