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And Its Natural Deletion in C57BL/10 Mice of a Novel Goblet Cell Lectin

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And Its Natural Deletion in C57BL/10 Mice of a Novel Goblet Cell Lectin Innate BALB/c Enteric Epithelial Responses to Trichinella spiralis: Inducible Expression of a Novel Goblet Cell Lectin, Intelectin-2, and Its Natural Deletion in C57BL/10 Mice This information is current as of September 23, 2021. Alan D. Pemberton, Pamela A. Knight, John Gamble, William H. Colledge, Jin-Kyu Lee, Michael Pierce and Hugh R. P. Miller J Immunol 2004; 173:1894-1901; ; doi: 10.4049/jimmunol.173.3.1894 Downloaded from http://www.jimmunol.org/content/173/3/1894 References This article cites 26 articles, 6 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/173/3/1894.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 23, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Innate BALB/c Enteric Epithelial Responses to Trichinella spiralis: Inducible Expression of a Novel Goblet Cell Lectin, Intelectin-2, and Its Natural Deletion in C57BL/10 Mice1 Alan D. Pemberton,2* Pamela A. Knight,* John Gamble,† William H. Colledge,† Jin-Kyu Lee,‡ Michael Pierce,‡ and Hugh R. P. Miller* Infection of mice with the nematode parasite Trichinella spiralis induces changes in the proteome of the jejunal epithelium, including substantial up-regulation of a novel variant of interlectin. In this study we sequence this novel lectin, termed intelectin-2, and compare expression levels during T. spiralis infection of resistant (BALB/c) with susceptible (C57BL/10) mouse strains. Intelectin-2 was cloned and sequenced from BALB/c mRNA extracted on day 14 of infection, and was found to have 91% amino acid identity with intelectin (within our study termed intelectin-1). Intelectin-2 transcripts were up-regulated early (day 3) during Downloaded from infection with T. spiralis in BALB/c mice, suggesting an innate response, and levels remained high through to day 14 (time of parasite rejection). Immunohistochemistry of jejunal sections with a rabbit polyclonal Ab to Xenopus laevis 35-kDa cortical granule lectin (XL35; 68% identity with intelectin-2) followed a similar pattern, with intense labeling of goblet and Paneth cells at day 14. However, intelectin-2 transcripts and protein were absent, and immunohistochemistry negative when C57BL/10 mice were infected with T. spiralis. Genomic PCR and Southern blotting confirmed that the intelectin-2 gene is absent from the C57BL/10 genome. The presence of intelectin-2 in resistant BALB/c mice, its absence from the susceptible C57BL/10 strain and http://www.jimmunol.org/ the kinetics of its up-regulation during T. spiralis infection suggest that this novel lectin may serve a protective role in the innate immune response to parasite infection. The Journal of Immunology, 2004, 173: 1894–1901. he mucus layer that coats and protects the simple epithelia tein, which was absent in uninfected mice, appeared to be a closely of the gut and lung is composed of mucin glycoproteins related variant of mouse intelectin, which is expressed by small T and additional components that help to protect the epithe- intestinal Paneth cells (8). Mouse intelectin belongs to a small lium against invasion by pathogens. In the gastrointestinal tract, family of related lectins, with members also described in Xenopus such components include antibacterial defensins (1), lysozyme (2), laevis (9–11) and in human (12, 13). Various roles have been trefoil factors (3), and members of the galactose-binding lectin postulated for the Xenopus lectins in fertility and development (10, by guest on September 23, 2021 family (galectins) (4). The mucus layer plays a significant role in 11). Human intelectin (12) (also known as HL-1 (13) and lacto- the response to intestinal nematodes in which the worms become ferrin receptor (14)) was expressed as a recombinant protein, and physically entrapped in mucus and are expelled (reviewed in Ref. found to bind oligosaccharides, including galactofuranose moeties 5). Mucus trapping is associated with changes in the physical prop- and to galactofuranose-containing bacterial cell wall preparations erties of mucus, and worm expulsion in primary infection is asso- (12). Hence, a role in recognition of bacteria was proposed. This ciated with goblet cell hyperplasia and augmented mucus synthesis lectin is expressed in the small intestine and colon, but is also (5). Certain lectins, such as the sheep galectin, ovgal11, also ap- highly expressed in heart tissue (12, 13), and has been immuno- pear to be up-regulated in response to parasite infection (6). Pro- localized to endothelium (13). The second human intelectin vari- posed roles for such lectins in the lumenal environment include ant, HL-2, was found to be expressed specifically in the small opsonization of bacteria and binding to mucins, to alter the vis- intestine (13). coelastic properties of mucus itself (4, 6). The aim of this study was to define the site of expression of the We recently described (7) the appearance of a novel lectin in novel mouse intelectin (7) and its relationship to the development extracts of exfoliated jejunal epithelium from BALB/c mice in- of resistance against the intestinal nematode T. spiralis. We have fected with the nematode parasite Trichinella spiralis. This pro- sequenced this intelectin variant and compared its expression with that of the previously described mouse intelectin (8). Our results show that the expression of the intelectin variant is confined to the *Division of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Vet- ileum in normal BALB/c mice, but that during infection there is † erinary Centre, Roslin, Midlothian, United Kingdom; Department of Physiology, strong jejunal expression in goblet and Paneth cells that peaks at University of Cambridge, Cambridge, United Kingdom; and ‡Department of Bio- chemistry, University of Georgia, Athens, GA 30602-7229 the time of worm expulsion. In contrast, the genome of the Received for publication January 22, 2004. Accepted for publication May 13, 2004. C57BL/10 strain, in which expulsion of T. spiralis is delayed (15), The costs of publication of this article were defrayed in part by the payment of page lacks this gene and there is no detectable expression in the gut. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 This work was supported by a Grant from the Wellcome Trust (Award No. 060312) Mouse strains and from the Cystic Fibrosis Trust. 2 Address correspondence and reprint requests to Dr. Alan Pemberton, Division For all experiments, with the exception of Southern blot analysis, BALB/c of Veterinary Clinical Studies, Easter Bush Veterinary Centre, University of and C57BL/10 mice were supplied by BD Biosciences (Cowley, U.K.) and Edinburgh, Roslin, EH25 9RG Midlothian, United Kingdom. E-mail address: 129/SvEv mice were purchased from B&K Universal (Hull, U.K.). For [email protected] Southern blot analysis, BALB/c mice were supplied by Charles River Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 1895 Breeding Laboratories (Margate, U.K.); C57BL/6J and C57BL/10 mice by dard protocols and hybridized using dioxygenin-labeled gene-specific probes, Harlan Olac (Bicester, U.K.); and 129S6/SvEv mice were a gift from R. using methods previously described (20). Positive PCR products were cloned Thresher (University of Cambridge, Cambridge, U.K.). into the TA Cloning vector pCR2.1 (Invitrogen, Leek, The Netherlands). Positive clones were identified by restriction enzyme digestion and Southern Parasite infections and tissue preparation hybridizations. All nucleotide sequences were determined by dideoxy chain- termination cycle sequencing (DBS Genomics, School of Biological and Maintenance, infection, and recovery of T. spiralis larvae was based on Biomedical Sciences, University of Durham, Durham, U.K.). Sequence data standard methods (16). Sex-matched 8- to 15-wk-old BALB/c or were analyzed with the help of web-based programs: Genestream Resource C57BL/10 mice were infected by gavage with 200–300 muscle larvae per Center (http://www2.igh.cnrs.fr/bin/lalign-guess.cgi); Java Molecular Biology mouse in 0.2 ml of PBS/0.1% agar. Larvae were freshly isolated from Directory (http://members.aol.com/_ht_a/lucatoldo/myhomepage/JaMBW); muscle cysts from 30 to 90 day infected BALB/c mice. To check infections Restriction Mapper (http://www.restrictionmapper.org); National Centre for were successful, adult worms were isolated from groups of four to five of Biotechnology Information (http://www.ncbi.nlm.nih.gov); and The Sequence the mice at 6–7 days after infection as previously described (17). For Manipulation Site (http://www.ualberta.ca/ϳstothard/javascript/index.html). RT-PCR analysis, mice were killed on day 14 following infection (worm expulsion phase) and epithelium exfoliated by EDTA perfusion as previ- Detection of transcripts by semiquantitative RT-PCR ously described (18, 19). Samples were also prepared from age-matched uninfected controls. Additional infections were set up in age- and sex- Total RNA (1 ␮g) from isolated epithelium, or other tissues as already ␮ matched BALB/c mice, which were killed on days 1, 3, 7, 14, 28, and 56 described, was reverse transcribed using 2.5 M (dT)15 as previously de- postinfection (p.i.)3 for epithelium isolation.
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