Impaired Luminal Processing of Human Defensin-5 in Crohn's Disease
2008; Elsevier BV; Volume: 172; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2008.070755
ISSN1525-2191
AutoresDavid Elphick, Susan Liddell, Yashwant R. Mahida,
Tópico(s)Immune Response and Inflammation
ResumoHuman defensin (HD)-5 is an antimicrobial peptide expressed in small intestinal Paneth cells, and alterations in HD-5 expression may be important in Crohn's disease (CD) pathogenesis. Levels of HD-5 in Paneth cells and ileostomy fluid from control and CD patients were studied by quantitative immunodot analysis, immunohistochemistry, acid urea-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blotting, reverse phase-high performance liquid chromatography, N-terminal amino acid sequencing, and ES-QToF mass spectrometry. In both control and CD patients, HD-5 in Paneth cell extracts was present almost exclusively in the precursor form. HD-5 levels in ileostomy fluid were lower in CD patients (n = 51) than in controls (n = 20): median (range), 7.9 (5.5 to 35.0) μg/ml versus 10.5 (6.0 to 30.4) μg/ml; P = 0.05; this difference was most marked in CD patients with homozygous/compound heterozygous mutations in NOD2 (P = 0.03). In control ileostomy fluid, HD-5 was present in the mature form only. In contrast, CD patient ileostomy fluid contained both precursor and mature forms of HD-5, with the majority present in a complex with trypsin, chymotrypsinogen/chymotrypsin, and α1-anti-trypsin. Pro-HD-5 was not associated with trypsin or chymotrypsinogen in Paneth cell extracts. In conclusion, pro-HD-5 in the intestinal lumen is processed by trypsin in a complex in which chymotrypsinogen is also cleaved for activation. The persistence of this complex in CD may be attributable to increased luminal levels of proteinase inhibitors such as α1-anti-trypsin. Human defensin (HD)-5 is an antimicrobial peptide expressed in small intestinal Paneth cells, and alterations in HD-5 expression may be important in Crohn's disease (CD) pathogenesis. Levels of HD-5 in Paneth cells and ileostomy fluid from control and CD patients were studied by quantitative immunodot analysis, immunohistochemistry, acid urea-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blotting, reverse phase-high performance liquid chromatography, N-terminal amino acid sequencing, and ES-QToF mass spectrometry. In both control and CD patients, HD-5 in Paneth cell extracts was present almost exclusively in the precursor form. HD-5 levels in ileostomy fluid were lower in CD patients (n = 51) than in controls (n = 20): median (range), 7.9 (5.5 to 35.0) μg/ml versus 10.5 (6.0 to 30.4) μg/ml; P = 0.05; this difference was most marked in CD patients with homozygous/compound heterozygous mutations in NOD2 (P = 0.03). In control ileostomy fluid, HD-5 was present in the mature form only. In contrast, CD patient ileostomy fluid contained both precursor and mature forms of HD-5, with the majority present in a complex with trypsin, chymotrypsinogen/chymotrypsin, and α1-anti-trypsin. Pro-HD-5 was not associated with trypsin or chymotrypsinogen in Paneth cell extracts. In conclusion, pro-HD-5 in the intestinal lumen is processed by trypsin in a complex in which chymotrypsinogen is also cleaved for activation. The persistence of this complex in CD may be attributable to increased luminal levels of proteinase inhibitors such as α1-anti-trypsin. Crohn's disease (CD) is a chronic inflammatory condition of the gastrointestinal tract that is believed to arise from an abnormality that results in the loss of the normal immunological unresponsiveness to the resident luminal microflora.1Podolsky DK Inflammatory bowel disease.N Engl J Med. 2002; 347: 417-429Crossref PubMed Scopus (3233) Google Scholar, 2Strober W Murray PJ Kitani A Watanabe T Signalling pathways and molecular interactions of NOD1 and NOD2.Nat Rev Immunol. 2006; 6: 9-20Crossref PubMed Scopus (676) Google Scholar The primary abnormality may affect the surface epithelium and/or the mucosal immune system. The identification of homozygous/compound heterozygous mutations in the CARD15/NOD2 gene in a proportion of patients with CD3Hugot JP Chamaillard M Zouali H Lesage S Cezard JP Belaiche J Almer S Tysk C O'Morain CA Gassull M Binder V Finkel Y Cortot A Modigliani R Laurent-Puig P Gower-Rousseau C Macry J Colombel JF Sahbatou M Thomas G Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease.Nature. 2001; 411: 599-603Crossref PubMed Scopus (4796) Google Scholar, 4Ogura Y Bonen DK Inohara N Nicolae DL Chen FF Ramos R Britton H Moran T Karaliuskas R Duerr RH Achkar JP Brant SR Bayless TM Kirschner BS Hanauer SB Nunez G Cho JH A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease.Nature. 2001; 411: 603-606Crossref PubMed Scopus (4271) Google Scholar, 5Hampe J Cuthbert A Croucher PJ Mirza MM Mascheretti S Fisher S Frenzel H King K Hasselmeyer A MacPherson AJ Bridger S van Deventer S Forbes A Nikolaus S Lennard-Jones JE Foelsch UR Krawczak M Lewis C Schreiber S Mathew CG Association between insertion mutation in NOD2 gene and Crohn's disease in German and Br populations.Lancet. 2001; 357: 1925-1928Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar has led to significant interest in the role of innate immunity in the etiopathogenesis of CD. Studies have shown that NOD2 is a cytosolic protein that binds muramyl dipeptide (derived from peptidoglycan of both Gram-positive and Gram-negative bacteria), leading to activation of nuclear factor (NF)-κB and subsequent expression of proinflammatory mediators such as interleukin-1 and tumor necrosis factor-α.6Inohara N Chamaillard M McDonald C Nunez G NOD-LRR proteins: role in host-microbial interactions and inflammatory disease.Annu Rev Biochem. 2005; 74: 355-383Crossref PubMed Scopus (826) Google Scholar In addition to monocytes, NOD2 has recently been reported to be expressed in Paneth cells,7Lala S Ogura Y Osborne C Hor SY Bromfield A Davies S Ogunbiyi O Nunez G Keshav S Crohn's disease and the NOD2 gene: a role for Paneth cells.Gastroenterology. 2003; 125: 47-57Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 8Ogura Y Lala S Xin W Smith E Dowds TA Chen FF Zimmermann E Tretiakova M Cho JH Hart J Greenson JK Keshav S Nunez G Expression of NOD2 in Paneth cells: a possible link to Crohn's ileitis.Gut. 2003; 52: 1591-1597Crossref PubMed Scopus (378) Google Scholar which are a subtype of epithelial cells normally present in the base of small intestinal crypts. Paneth cells are normally absent in the colon but are frequently seen in colonic samples of patients with inflammatory bowel disease.9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar, 10Paterson JC Watson SH Paneth cell metaplasia in ulcerative colitis.Am J Pathol. 1961; 38: 243-249PubMed Google Scholar Because they express antimicrobial peptides of the α-defensin family, antimicrobial proteins (lysozyme, secretory phospholipase A2, angiogenin 4), and an intestinal bactericidal lectin,11Cash HL Whitham CV Behrendt CL Hooper LV Symbiotic bacteria direct expression of an intestinal bactericidal lectin.Science. 2006; 313: 1126-1130Crossref PubMed Scopus (1106) Google Scholar Paneth cells are believed to be important in innate immunity in the intestine.12Elphick DA Mahida YR Paneth cells: their role in innate immunity and inflammatory disease.Gut. 2005; 54: 1802-1809Crossref PubMed Scopus (140) Google Scholar, 13Selsted ME Ouellette AJ Mammalian defensins in the antimicrobial immune response.Nat Immunol. 2005; 6: 551-557Crossref PubMed Scopus (988) Google Scholar Dysregulation of Paneth cell function leading to impaired expression or activities of antimicrobial peptides and proteins may adversely affect intestinal protection against luminal microorganisms. There is a vast and complex population of microorganisms in the colon and terminal ileum, with 30 to 40 species making up the majority of the total population.14Hooper LV Midtvedt T Gordon JI How host-microbial interactions shape the nutrient environment of the mammalian intestine.Annu Rev Nutr. 2002; 22: 283-307Crossref PubMed Scopus (1250) Google Scholar, 15Simon GL Gorbach SL Intestinal flora in health and disease.Gastroenterology. 1984; 86: 174-193PubMed Scopus (615) Google Scholar CD frequently occurs in the terminal ileum, and those with mutations in the NOD2 gene frequently develop disease in this region.16Ahmad T Armuzzi A Bunce M Mulcahy-Hawes K Marshall SE Orchard TR Crawshaw J Large O de Silva A Cook JT Barnardo M Cullen S Welsh KI Jewell DP The molecular classification of the clinical manifestations of Crohn's disease.Gastroenterology. 2002; 122: 854-866Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar Two members of the α-defensin family, human defensin(HD)-5 and HD-6, have been identified in humans and shown to be expressed by Paneth cells17Jones DE Bevins CL Paneth cells of the human small intestine express an antimicrobial peptide gene.J Biol Chem. 1992; 267: 23216-23225Abstract Full Text PDF PubMed Google Scholar and also infrequent cells designated intermediate cells.9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar HD-5 is synthesized in its precursor form (Figure 1) and studies have shown that it is stored in this form in Paneth cells.9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar, 18Ghosh D Porter E Shen B Lee SK Wilk D Drazba J Yadav SP Crabb JW Ganz T Bevins CL Paneth cell trypsin is the processing enzyme for human defensin-5.Nat Immunol. 2002; 3: 583-590Crossref PubMed Scopus (367) Google Scholar Recombinant mature HD-5 is active against many Gram-negative and Gram-positive bacteria and the predicted mature form of HD-5 has been identified in samples from the small intestinal lumen.18Ghosh D Porter E Shen B Lee SK Wilk D Drazba J Yadav SP Crabb JW Ganz T Bevins CL Paneth cell trypsin is the processing enzyme for human defensin-5.Nat Immunol. 2002; 3: 583-590Crossref PubMed Scopus (367) Google Scholar, 19Porter EM van Dam E Valore EV Ganz T Broad-spectrum antimicrobial activity of human intestinal defensin 5.Infect Immun. 1997; 65: 2396-2401Crossref PubMed Google Scholar In vitro, trypsin has been shown to be capable of processing pro-HD-5 to the mature form and this enzyme is present in Paneth cells.18Ghosh D Porter E Shen B Lee SK Wilk D Drazba J Yadav SP Crabb JW Ganz T Bevins CL Paneth cell trypsin is the processing enzyme for human defensin-5.Nat Immunol. 2002; 3: 583-590Crossref PubMed Scopus (367) Google Scholar, 20Bohe M Borgstrom A Lindstrom C Ohlsson K Pancreatic endoproteases and pancreatic secretory trypsin inhibitor immunoreactivity in human Paneth cells.J Clin Pathol. 1986; 39: 786-793Crossref PubMed Scopus (49) Google Scholar, 21Senegas-Balas FO Figarella CG Amouric MA Guy-Crotte OM Bertrand CA Balas DC Immunocytochemical demonstration of a pancreatic secretory protein of unknown function in human duodenum.J Histochem Cytochem. 1991; 39: 915-919Crossref PubMed Scopus (25) Google Scholar When Paneth cell-containing isolated terminal ileal crypts were stimulated in vitro with carbamyl choline or lipopolysaccharide, a truncated form of pro-HD-5 (but not mature form) was released.9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar Truncated forms of pro-HD-5 have also been identified in ileal neobladder urine.22Porter EM Poles MA Lee JS Naitoh J Bevins CL Ganz T Isolation of human intestinal defensins from ileal neobladder urine.FEBS Lett. 1998; 434: 272-276Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Thus, the in vivo site (inside or on the surface of Paneth cells or in the lumen) and mechanism(s) of processing of pro-HD-5 remain to be determined. This is in contrast to α-defensins (designated cryptdins) in murine Paneth cells, which are stored in mature form, after intracellular processing by matrilysin [also known as matrix metalloproteinase-7 (MMP-7)].23Wilson CL Ouellette AJ Satchell DP Ayabe T Lopez-Boado YS Stratman JL Hultgren SJ Matrisian LM Parks WC Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense.Science. 1999; 286: 113-117Crossref PubMed Scopus (928) Google Scholar The importance of cryptdin processing has been shown by increased susceptibility to orally administered Salmonella typhimurium in mice lacking MMP-7.23Wilson CL Ouellette AJ Satchell DP Ayabe T Lopez-Boado YS Stratman JL Hultgren SJ Matrisian LM Parks WC Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense.Science. 1999; 286: 113-117Crossref PubMed Scopus (928) Google Scholar In Trichinella spiralis-induced inflammation in murine small intestine, we have previously shown active Paneth cell degranulation,24Kamal 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: 117-125Crossref PubMed Scopus (78) Google Scholar which would be expected to release antimicrobial peptides and proteins into the lumen. In humans, it is not known whether the precursor form of HD-5 may be processed intracellularly in Paneth cells (eg, via an induced or activated processing enzyme) during small intestinal inflammation, as in CD. Such intracellular processing would enable the enteric defensin to be active on release by the Paneth cells, as is the case in mice. To address the above issues, we have investigated the form of HD-5 present in small intestinal Paneth cells of patients with CD. Ileostomy fluid of controls and patients with CD was used to characterize luminal expression of HD-5. Human intestinal mucosal samples were obtained from operation resection specimens, after informed consent and with approval of the Nottingham Research Ethics Committee. Histologically normal terminal ileal mucosa was obtained from right hemi-colectomy specimens (n = 8) resected for colon carcinoma affecting the cecum or ascending colon. Small intestinal (seven terminal ileum, three jejunum) mucosal samples affected by CD were also obtained from operation resection specimens, in which there was active inflammation. CD was diagnosed in these patients using standard criteria (clinical, endoscopic, radiological, and histological features). Ileostomy fluid was collected, in the presence of protease inhibitors (104 mmol/L AEBSF, 80 μmol/L aprotinin, 2 mmol/L leupeptin, 4 mmol/L bestatin, 1.5 mmol/L pepstatin A, 1.4 mmol/L E-64; Sigma, Poole, UK), from patients with an ileostomy. It was studied after centrifugation, either immediately or after storage at −80°C. Patients included 51 with CD and 20 with normal small intestine (seven ulcerative colitis, seven colonic carcinoma, two mesenteric ischemia, one intractable constipation, one familial adenomatous polyposis, one appendiceal abscess, and one ruptured ectopic pregnancy). For the ileostomy patients with CD, clinical disease activity was assessed using the Harvey-Bradshaw index,25Harvey RF Bradshaw JM A simple index of Crohn's-disease activity.Lancet. 1980; 1: 514Abstract PubMed Scopus (2292) Google Scholar with the modification that the score for number of liquid stools per day was replaced with the number of stoma bags filled per day minus the usual number of stoma bags filled when in remission. Active disease was taken as a modified Harvey-Bradshaw index of 8 or more, and inactive as 3 or less, in keeping with previous reports.26Best WR Predicting the Crohn's disease activity index from the Harvey-Bradshaw Index.Inflamm Bowel Dis. 2006; 12: 304-310Crossref PubMed Scopus (224) Google Scholar Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) was used for the resolution of cationic peptides and proteins as previously described.9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar Precast 18% Tris-tricine sodium dodecyl sulfate (SDS)-PAGE ready gels (Bio-Rad, Hemel Hempstead, UK) were also used for the resolution of peptides and small proteins. The presence of immunoreactive HD-5 in crypt cell extracts, ileostomy fluid samples, and purified fractions, was determined by dot blot and Western blot analyses using polyclonal anti-HD-5 sera.9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar This anti-sera was raised against synthetic mature HD-5 (amino acids 63 to 94 in Figure 1). It will therefore recognize both mature and pro-HD-5. Sections of formalin-fixed, paraffin-embedded intestinal tissue samples were used for immunohistochemistry, using rabbit anti-HD-5 and anti-chymotrypsin (Biogenesis, Kingston, NH) antibodies, as previously described.9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar Intestinal mucosal crypt epithelial cells were isolated from fresh small bowel tissue, lysed, and cationic peptides and proteins were extracted as previously described.9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar, 27Mahida YR Galvin AM Gray T Makh S McAlindon ME Sewell HF Podolsky DK Migration of human intestinal lamina propria lymphocytes, macrophages and eosinophils following the loss of surface epithelial cells.Clin Exp Immunol. 1997; 109: 377-386Crossref PubMed Scopus (72) Google Scholar HD-5 was purified from crypt acid extracts using cation exchange chromatography and reverse phase-high performance liquid chromatography (RP-HPLC), as previously described.9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar Ileostomy fluid was collected as described above. After addition of acetic acid (to make a 10% v/v solution), protease inhibitors and centrifugation (150,000 × g), the supernatant was diluted 15:1 with 5 mmol/L ammonium acetate (pH 6.0) buffer. Cationic peptides and proteins were obtained using cation exchange beads (Macro-Prep CM Support, Bio-Rad) and concentrated (Vivaspin 2-kDa MWCO filters). After application to RP-HPLC (as previously described9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar), HD-5-containing fractions were characterized by AU-PAGE, 18% Tris-tricine SDS-PAGE, Western blot analysis, N-terminal amino acid sequencing, and mass spectrometry. Intact protein mass determination was performed using an electrospray Q-ToF2 tandem mass spectrometer (Waters Co., Milford, MA). Spectra were processed between 3000 and 10,000 Da, at a resolution of 1 Da and a peak width of 0.75 Da. After MaxEnt1 processing, the spectra were centered with the minimum peak width at half height set at 1 and using the centroid top method at 80%. N-terminal amino acid sequencing of purified peptides was performed by Edman degradation chemistry using an automated amino acid sequencer (model 474A; Perkin Elmer Biosystems, Shelton, CT). Deduced N-terminal sequences were searched against the SwissProt protein sequence database, using the FASTA algorithm. Polymerase chain reaction (PCR) primer pairs were used to amplify regions of 2 to 400 bases around SNP 8 (sense: 5′-TTTGCTCAGACACCTCTTCAATTGTG-3′, anti-sense: 5′-AAAATGTCAACTTGAGGTGCCCAAC-3′,probe extension: 5′-TGAGTGCCAGACATCTGAGAAGGCCCTGCTC-3′), SNP 12 (sense: 5′-AGTGAGGCCACTCTGGGATTGAGT-3′, anti-sense: 5′-AGCTCCTCCCTCTTCACCTGATCTC-3′, probe extension: 5′-CCCCCTCGTCACCCACTCTGTTGC-3′) and SNP 13 (sense: 5′-GAATCTCAGACATGAGCAGGATGTGT-3′, anti-sense: 5′-CCGTCACCCCATTTTACAGATAGAAA-3′, probe extension: 5′-TTACCAGACTTCAGGATGGTGTCATTCCTTTCAAGGG-3′) of NOD2 gene. NOD2 genotyping was performed on the amplified DNA regions using the ABI Prism SNaPshot Multiplex Kit (Applied Biosystems, Foster City, CA) to perform a probe extension assay. To verify the probe extension assay, DNA samples representative of different NOD2 genotypes were sequenced using the Big Dye terminator cycle sequencing kit (Applied Biosciences, Foster City, CA) according to the manufacturer's instructions. Comparative studies of HD-5 levels in ileostomy fluid samples were performed in a blinded manner. Thus, the samples were coded before study by others not involved in this research and the code broken after the results were available. Data are expressed as median (range) and were analyzed by Kruskal-Wallis and Mann-Whitney U-tests. Immunohistochemistry showed HD-5 expression predominantly in Paneth cells in sections of both normal and Crohn's ileum (Figure 2). In Crohn's small intestine, in addition to predominant expression in Paneth cells, HD-5 immunoreactivity was also often seen in enterocytes and goblet-like intermediate cells further up the crypt, and within the crypt lumen (Figure 2), features only occasionally seen in normal small bowel. Crypts were isolated from small intestinal mucosal samples of 10 CD (seven terminal ileum, six NOD2 wild type, two NOD2 SNP12 heterozygotes, one NOD2 SNP13 heterozygote, and one NOD2 SNP13 homozygote) and 8 normal terminal ileal resection specimens, and proteins extracted in acetic acid. Figure 3 demonstrates that the electrophoretic mobility on AU-PAGE of HD-5 derived from crypt epithelial cells from Crohn's small intestine is broadly similar to that of HD-5 derived from normal small intestine. Multiple bands on AU-PAGE for control (normal) samples are similar to those previously reported9Cunliffe RN Rose FR Keyte J Abberley L Chan WC Mahida YR Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease.Gut. 2001; 48: 176-185Crossref PubMed Scopus (190) Google Scholar, 18Ghosh D Porter E Shen B Lee SK Wilk D Drazba J Yadav SP Crabb JW Ganz T Bevins CL Paneth cell trypsin is the processing enzyme for human defensin-5.Nat Immunol. 2002; 3: 583-590Crossref PubMed Scopus (367) Google Scholar for pro-HD-5 purified from normal small intestinal Paneth cells. These bands disappear on neutralization of anti-HD-5 antiserum using purified HD-5 (not shown). In contrast, mature HD-5 derived from the ileostomy fluid migrates significantly further on AU-PAGE as a single band. Eleven of the small intestinal crypt epithelial cell acid extracts [six normal and five Crohn's (two NOD2 wild type, two NOD2 SNP12 heterozygotes, and one NOD2 SNP13 heterozygote)] were fractionated using cation exchange chromatography. In all cases, HD-5 eluted at 0.63 to 0.83 mol/L NaCl, and these fractions were used for subsequent purification by RP-HPLC, in which only the fraction eluting at 39% acetonitrile contained HD-5 (Figure 4). ES-Q-ToF MS of RP-HPLC-purified HD-5 peptide from six normal ileal and three Crohn's small intestinal resection specimens showed the presence of a single mass of 8102 Da, which is identical to the predicted mass of pro-HD-5. In two samples of HD-5 purified from two CD crypt extracts (one NOD2 wild type and one NOD2 SNP12 heterozygote), a smaller peptide with mass 7360 Da was seen in addition to the mass for pro-HD-5 (8102 Da, Figure 5). This mass difference would occur if a 6-amino acid truncation (ESLQER) had occurred from the N terminus of the HD-5 pro-peptide (expected mass of truncated peptide is 7360 Da, Figure 1). These findings were confirmed by N-terminal amino acid sequencing with the sequence ESLQE for all seven samples (two normal ileum, five Crohn's small bowel) studied and also a secondary sequence, ADEAT, for the two CD samples with a smaller HD-5 mass (7360 Da, Figure 1). HD-5 concentration in ileostomy fluid was extrapolated from a standard curve obtained by densitometric analysis of dot blots of known concentrations of purified pro-HD-5 from normal small bowel. In a blinded manner, 71 samples (51 Crohn's and 20 controls) were analyzed eight times, and the mean HD-5 level, and coefficient of variation, calculated for each sample. The mean coefficient of variation over all samples was 6.6%. As shown in Table 1 and Figure 6A, luminal HD-5 levels were lower in Crohn's ileostomy fluid than control [median (range): 7.9 (5.5 to 35.0) μg/ml versus 10.5 (6.0 to 30.4) μg/ml; P = 0.05], and this was most marked in those with active disease, as defined by modified Harvey Bradshaw index ≥8 [active versus inactive CD: 6.3 (5.9 to 9.0) μg/ml versus 8.9 (5.5 to 26.2) μg/ml; P = 0.01], and in NOD2 mutant homozygotes/compound heterozygotes (P = 0.03). Two of the latter patients had jejunostomies (modified Harvey Bradshaw scores 12 and 4) and the remaining two had ileostomies (modified Harvey Bradshaw scores 4 and 13). To control for differences in intestinal fluid flux, the data were expressed as a ratio of HD-5 to total protein concentration in the ileostomy fluid, which also demonstrated lower HD-5 levels in Crohn's versus control patients; P = 0.04 (Figure 6B).Table 1Patient Data for Small Intestinal Fluid AnalysesControl patientsCrohn's patientsNumber2051Age45 (35 to 70) years39 (19 to 85) yearsGender (male/female)9/1119/32Time since ileostomy0.32 (0.08 to 34) years4 (0.17 to 28) yearsJejunostomies/ileostomies1/195/46HD-5 levels10.5 (6 to 30.4) μg/ml7.9 (5.5 to 35) μg/mlHD-5 levels according to Crohn's disease activity*Active Crohn's defined as modified Harvey Bradshaw index 8 or more. Inactive Crohn's defined as index of 3 or less. Inactive8.9 (5.5 to 26.2) μg/ml, n = 37 Active6.3 (5.9 to 9) μg/ml, n = 7HD-5 levels according to NOD2 genotype†WT indicates NOD2 wild-type genotype. Hom/Com Het indicates NOD2 mutant homozygote (SNP13, n = 3) or compound heterozygote (SNP8/SNP13, n = 1). WT10.4 (6 to 23.5) μg/ml, n = 137.9 (5.5 to 26.2) μg/ml, n = 35 Heterozygote14.2 (7.4 to 30.4), n = 78.2 (6.2 to 35) μg/ml, n = 12 Hom/Com Het5.9 (5.8 to 8.5) μg/ml, n = 4Data given as median (range).* Active Crohn's defined as modified Harvey Bradshaw index 8 or more. Inactive Crohn's defined as index of 3 or less.† WT indicates NOD2 wild-type genotype. Hom/Com Het indicates NOD2 mutant homozygote (SNP13, n = 3) or compound heterozygote (SNP8/SNP13, n = 1). Open table in a new tab Data given as median (range). Molecular forms of HD-5 present in the ileostomy fluid of control (n = 3, reasons for colonic resection: ulcerative colitis, cancer, mesenteric ischemia) and CD patients (n = 7, Table 2) were investigated. In control concentrated ileostomy fluid samples, only one HD-5-immunoreactive band with electrophoretic mobility similar to that of synthetic mature HD-5 was seen on AU-PAGE Western blot analysis (Figure 7A). By contrast, multiple HD-5-immunoreactive bands were seen on AU-PAGE Western of ileostomy fluid samples from seven patients with CD. These included bands with similar electrophoretic mobility to those seen with mature and precursor forms of HD-5. However, the most prominent HD-5-positive bands had slower electrophoretic mobility than the precursor form of HD-5 isolated from normal small intestinal crypt cell extract (Figure 7A). When analyzed by SDS-PAGE Western under reducing/denaturing conditions, the most prominent HD-5-immunoreactive bands were seen in the regions of the mature and precursor forms of HD-5. Weak HD-5-positive bands with electrophoretic mobility slower than pro-HD-5 (similar to that for the 27.5-kDa marker) were also seen (Figure 7B), which could be attributable to remnants of a complex containing mature HD-5 and other proteins responsible for delayed electrophoretic mobility of HD-5 on AU-PAGE and which becomes dissociated under the reducing/denaturing conditions of SDS-PAGE.Table 2HD5 ImmunoreactivityDetails of Crohn's disease patients and ileostomy fluid samplesFractions that were HD-5-positive on RP-HPLCHD-5-positive bands on AU-PAGE WesternHD-5-po
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