Expression of LL-37 by human gastric epithelial cells as a potential host defense mechanism against Helicobacter pylori

GASTROENTEROLOGY 2003;125:1613–1625

Expression of LL-37 by Human Gastric Epithelial Cells as a Potential Host Defense Mechanism Against Helicobacter pylori KOJI HASE,* MASAMOTO MURAKAMI,* MITSUTOSHI IIMURA,* SHERI P. COLE,* YOSHIMUNE HORIBE,‡ TAKAAKI OHTAKE,* MARYGORRET OBONYO,* RICHARD L. GALLO,* LARS ECKMANN,* and MARTIN F. KAGNOFF* *Department of Medicine, University of California at San Diego, La Jolla, California; and ‡Department of Surgical Pathology, 2nd Teaching Hospital, Fujita Health University School of Medicine, Nagoya, Japan

Background & Aims: LL-37/human cationic antimicrobial peptide 18 (hCAP18) is a human cathelicidin with broad-spectrum antimicrobial, lipopolysaccharide binding, and chemotactic activities. This study examined the role of LL-37/hCAP18 in gastric innate immune defense by characterizing its constitutive and regulated expression by human gastric mucosa and its bactericidal activity against the gastric pathogen Helicobacter pylori. Methods: LL-37/hCAP18 messenger RNA expression in normal and H. pylori–infected gastric mucosa and gastric epithelial cells was determined by in situ hybridization, real-time polymerase chain reaction, immunostaining, and immunoblot analysis. Bactericidal activity was measured by using a colony-forming unit assay. Results: LL-37/hCAP18 messenger RNA and protein were expressed in a distinct distribution by surface epithelial cells as well as chief and parietal cells in the fundic glands of normal gastric mucosa. LL-37/hCAP18 was significantly increased in the epithelium and gastric secretions of H. pylori–infected patients, but not in individuals with non–H. pylori–induced gastric inflammation. Infection of cultured gastric epithelial cells with a wildtype but not an isogenic ⌬cagE mutant strain of H. pylori increased LL-37/hCAP18 expression, indicating that H. pylori–induced regulation of LL-37/hCAP18 production required an intact type IV secretion system. LL-37, the C-terminal peptide of LL-37/hCAP18, alone or in synergy with human ␤-defensin 1, was bactericidal for several H. pylori strains. Conclusions: These data indicate that H. pylori up-regulates production of LL-37/hCAP18 by gastric epithelium and suggest this cathelicidin contributes to determining the balance between host mucosal defense and H. pylori survival mechanisms that govern chronic infection with this gastric pathogen.

ngestion of contaminated food or water exposes the gastric mucosa to a wide variety of microbial pathogens. Gastric acid and proteolytic enzymes decrease the numbers of pathogenic bacteria in the stomach and their subsequent access to the small intestine, whereas gastric epithelial cells and the mucous layer overlying those cells

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contribute to the protective physical barrier that prevents mucosal invasion. The production of proteins and peptides with antimicrobial activity by gastric epithelial cells (e.g., ␤-defensins, lysozyme)1–3 provides a further layer of host innate defense. Antimicrobial peptides in the gastrointestinal tract function within a specialized microenvironment that includes the mucous layer overlying the surface epithelium. One family of antimicrobial peptides, the defensins, is produced by epithelial cells at mucosal surfaces and by neutrophils.4 –7 ␣-Defensins are produced by Paneth cells in the crypts of the small intestine or by neutrophils that infiltrate the small and large intestine during inflammatory reactions, whereas ␤-defensins are expressed by columnar epithelial cells throughout the colon.8 In the stomach, gastric epithelial cells constitutively express human ␤-defensin (hBD)-1, whereas hBD-2 is induced in response to proinflammatory cytokines or microbial infection.1,2,9 The cathelicidins comprise another group of mammalian antimicrobial proteins.10 –12 The precursor form of the cathelicidins is characterized by a conserved N-terminal signal sequence and a cathelin domain, followed by a C-terminal peptide domain that is proteolytically cleaved to release a small molecular weight antimicrobial peptide. The C-terminal peptide domains of cathelicidins vary markedly among different species.12 The single known cathelicidin in man is human cationic antibacterial protein of 18 kDa (hCAP18), which is an 18-kDa protein whose C-terminal 37 amino acid peptide is termed LL-37. LL-37/hCAP18 has a distinct distribution Abbreviations used in this paper: ATCC, American Type Culture Collection; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hBD, human ␤-defensin; hCAP18, human cationic antibacterial protein of 18 kDa; LPS, lipopolysaccharide; NF-␬B, nuclear factor-␬B. © 2003 by the American Gastroenterological Association 0016-5085/03/$30.00 doi:10.1053/j.gastro.2003.08.028

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in the intestinal tract, with its expression being greatest in the surface and upper crypt epithelium in the colon and sparse to absent in small intestinal epithelium, with the exception of Brunner’s glands.13 LL-37 has been shown to have bactericidal activity against Gram-positive and Gram-negative bacteria14,15 as well as lipopolysaccharide (LPS)-binding and neutralizing activities16 –18; histamine-releasing activity on mast cells19; and chemotactic activity for monocytes, neutrophils, and CD4⫹ T cells.20 Several studies have suggested the expression of LL37/hCAP18 correlates with human disease and point to the putative clinical importance of this molecule. For example, the bronchial secretions of cystic fibrosis patients inactivate LL-37 antimicrobial activity against Pseudomonas aeruginosa,21,22 the major airway pathogen in that disease. Shigella infection is accompanied by decreased LL-37/hCAP18 messenger RNA (mRNA) and protein expression in human colon epithelial cells,23 possibly contributing to impaired host innate defense and increased tissue-destructive inflammation. Atopic dermatitis patients have decreased LL-37/hCAP18 and hBD-2 mRNA and protein expression, directly correlating with their relative susceptibility to skin infections.24 In further support of the likely physiologic importance of LL-37 in antimicrobial defense, mice that lack the murine counterpart of LL-37/hCAP18 (termed cathelicidinrelated antimicrobial peptide or CRAMP) manifest defective resistance to cutaneous infection with Group A Streptococci and develop more severe necrotizing cutaneous infection than wild-type controls.25 Helicobacter pylori can chronically colonize the human gastric mucosa, where it is found in the mucus layer and adhering to epithelial cells.26,27 H. pylori adherence to gastric epithelium is associated with effacement of the microvilli and actin rearrangements, alterations in protein tyrosine phosphorylation, and activation of mitogenactivated protein kinases and the transcription factor nuclear factor-␬B (NF-␬B).28,29 Activation of these latter signaling pathways results in the up-regulated expression of multiple target genes, including those that code for neutrophil chemoattractants (e.g., IL-8)30 –32 and the antimicrobial peptide hBD-2.1,8,33 Moreover, this requires the presence of the H. pylori cag pathogenicity island31,34 –36 that encodes a type IV secretion system.37,38 Little is known about the role of antimicrobial peptides produced by gastric epithelial cells in innate host defense against H. pylori. To better define the role LL37/hCAP18 might play in gastric host defense against H. pylori, we have characterized the expression of this protein in gastric mucosa under normal conditions and in

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patients with gastritis caused by H. pylori infection. We show that surface gastric epithelial cells and epithelial cells in the fundic glands constitutively express LL-37/ hCAP18 and that the peptide is released into gastric secretions. Moreover, epithelial LL-37/hCAP18 production is up-regulated by H. pylori infection but not by proinflammatory cytokines. Furthermore, LL-37 either alone, or in synergy with hBD-1, has significant antimicrobial activity against several strains of H. pylori.

Materials and Methods Antibodies, Cytokines, and Antimicrobial Peptides Preimmune chicken IgY, chicken IgY anti-LL-37 that recognizes the LL-37 peptide domain, and chicken IgY antihCAP18 antibody that recognizes the cathelin domain were as described earlier.39 Rabbit IgG anti-LL-37 antibody was a gift of B. Agerberth. Rabbit anti-H⫹, K⫹-ATPase (␣-subunit) was from Calbiochem (San Diego, CA). Control rabbit serum and IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). Synthetic LL-37 peptide was from SynPep Corp. (Dublin, CA).40 Synthetic hBD-1 and recombinant hBD-2 were from Alpha-diagnostic Inc. (San Antonio, TX) and Peprotec Inc. (Rocky Hill, NJ), respectively. Recombinant human (rh) IL-1␣, IL-6, IFN-␥, and TNF␣ were from Peprotech. Bacterial LPS (Escherichia coli O111:B4) was from Sigma (St. Louis, MO).

Gastric Mucosal Biopsy Specimens Mucosal biopsy specimens were obtained at the time of upper gastrointestinal endoscopy from the gastric antrum and body. Samples were from 5 individuals with endoscopically and histologically normal gastric mucosa; 5 individuals whose gastric mucosa appeared inflamed at endoscopy and on hematoxylin and eosin stained sections and who were shown to be infected with H. pylori by urease production (Hpfast, GI Supply, Camp Hill, PA) and histological examination after Giemsa staining; and 3 individuals with endoscopically inflamed appearing mucosa, gastritis characterized by a neutrophil infiltrate on histology, a history of nonsteroidal antiinflammatory drug (NSAID) usage and no evidence of H. pylori infection by histological examination or urease production. None of the patients had gastric or duodenal ulcers. Gastric tubular adenoma, gastric hyperplastic polyp, and gastric adenocarcinoma specimens were obtained from patients undergoing endoscopy or gastric surgery. Biopsy and tissue specimens were flash frozen in liquid nitrogen for later RNA and protein analysis, frozen in isopentane/dry ice for immunohistochemistry or fixed in 10% formalin or 1% zinc sulfate/10% formalin, and embedded in paraffin. Histological sections were evaluated in a blinded fashion by 2 observers. These studies were approved by the University of California at San Diego (UCSD) Committee on Human Subjects.

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In Situ Hybridization Digoxigenin-labeled riboprobes for in situ hybridization to the cathelin domain sequence and LL-37 sequence were as described earlier.39 Fresh frozen sections of normal gastric mucosa were fixed in 4% paraformaldehyde for 10 minutes at room temperature and pretreated with 1 ␮g/mL proteinase K in PBS for 15 minutes at 37°C, after which they were incubated with a 1 mol/L triethanolamine solution (pH 8.0) containing 0.25% acetic anhydride for 15 minutes at 37°C, and prehybridized with 50% formamide in 2⫻ sodium saline citrate for 30 minutes at 45°C. Sections were hybridized for 16 hours at 45°C with sense or antisense digoxigenin-labeled complementary RNA probes (1␮g/mL) in hybridization solution (1 mg/mL yeast tRNA, 20 mmol/L Tris-HCl buffer [pH 8.0], 2.5 mmol/L EDTA, 1⫻ Denhart’s solution, 0.3 mol/L NaCl, 50% deionized formamide, 50% dextran sulfate). After hybridization, sections were treated with RNase-A (Roche Molecular Biochemicals, Mannheim, Germany) to digest nonhybridized probe. Specific hybridization of the probe was detected with alkaline phosphate-labeled Fab fragments of anti-digoxigenin antibody (1:500) (Roche Molecular Biochemicals) and visualized with 5-bromo-4-chloro-3-indolyl phosphate (X-phosphate) and nitroblue tetrazolium with addition of levamisole solution (DAKO, Carpinteria, CA). Nuclei were counterstained with methyl green.

RNA Extraction and RT-PCR Analysis Total cellular RNA was extracted from cells and mucosal biopsies using a commercial kit (RNeasy, QIAGEN, Valencia, CA). Five micrograms of total cellular RNA were reverse-transcribed and 2 ␮L of each cDNA sample from the reverse transcription reactions were amplified by polymerase chain reaction (PCR) using primers and amplification conditions described earlier.8,13 Real-time PCR to quantify the mRNA expression level of LL-37/hCAP18, hBD-1, and hBD-2 was done as described previously using an ABI Prism 7700 Sequence Detection System and software from the manufacturer (PE Applied Biosystems, Foster City, CA).13 Amplification of the expected single products was confirmed using 1% agarose gels and ethidium bromide staining.

Immunohistochemistry For LL-37/hCAP18 staining, sections of gastric mucosa (5 ␮m) were deparaffinized, rehydrated, and treated with 0.3% H2O2 in phosphate-buffered saline (PBS) for 20 minutes at room temperature to block endogenous peroxidase activity. Sections were incubated with 20% BlokHen II (Aves Labs, Tigard, OR) in PBS for 30 minutes at room temperature and then incubated overnight at 4°C with chicken IgY anti-LL-37 (0.16 ␮g/mL), chicken IgY to the cathelin domain of hCAP18 (0.11 ␮g/mL), or identical concentrations of preimmune chicken IgY. Binding of primary antibody was detected with biotin-labeled goat antichicken Ig (1:300 dilution; Vector Laboratories, Burlingame, CA) followed by streptavidin-horseradish peroxidase (DAKO), visualized with 3,3⬘-diaminobenzidine (Sigma), and counterstained with hematoxylin. For H⫹,

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K⫹-adenosine triphosphatase (ATPase) staining, sections were treated with 10 mmol/L sodium citrate buffer (pH 6.0), heated to 90°C in a microwave oven for antigen retrieval, and then incubated overnight at 4°C with rabbit anti-H⫹, K⫹-ATPase (1:7000) or an identical concentration of normal rabbit serum. After addition of goat anti-rabbit IgG (9 ␮g/mL; Jackson ImmunoResearch Laboratories) as secondary antibody, immunostaining was visualized using an APAAP kit (DAKO). The immunostaining data shown in each case is representative of the immunostaining in 6 or more fields from 4 – 8 sections of 2 or more biopsies from each H. pylori–infected or control subject.

Immunoblot Analysis Human gastric mucosal biopsy specimens were homogenized in lysis buffer (150 mmol/L NaCl, 20 mmol/L Tris [pH 7.5], 1 mmol/L EDTA, 0.1% Triton X-100) containing 0.5% protease inhibitor cocktail III (Calbiochem). Homogenates were sonicated and centrifuged at 12,000 rpm at 4°C for 20 minutes after which supernatants were concentrated (Centricon YM-3; Millipore, Bedford, MA) and protein content was measured by Lowry assay (Bio-Rad, Hercules, CA). Human gastric juice obtained at the time of endoscopy was centrifuged before use to remove any debris. Aliquots (10 or 25 ␮g protein) were mixed with tricine sample buffer (Bio-Rad) containing 2% mercaptoethanol, boiled for 3 minutes, and electrophoresed on 16.5 % tris-tricine gels. After transfer to nitrocellulose membranes (Hybond ECL; Amersham Pharmacia, Piscataway, NJ), membranes were blocked with PBS, 0.1% Tween-20 containing 10% (w/v) dry milk and 1% bovine serum albumin for 2 hours at room temperature. Membranes were probed with rabbit anti-LL-37/hCAP18 antibody (1.5 ␮g/mL) or control rabbit IgG, followed by donkey antirabbit IgG coupled to horseradish peroxidase (1:1000 dilution; Amersham Pharmacia), developed using an ECL detection kit (Amersham Pharmacia) and exposed to x-ray film (XAR5, Kodak, Rochester, NY). LL-37 concentrations in cell culture supernatants and gastric juice were determined by a dot-blot assay. Two microliters of supernatant from each culture well; 1 ␮L of gastric juice (1:5–1:25 dilution in PBS) or 1 ␮L of titrated concentrations of LL-37 peptide were applied to a nitrocellulose filter (Hybond ECL, Amersham Pharmacia). After blocking with 10% (w/v) dry milk and 1% BSA in PBS, 0.1% Tween-20, membranes were probed with rabbit anti-LL-37 antibody (1.5 ␮g/mL) or control rabbit IgG followed by horseradish peroxidase-conjugated donkey antirabbit IgG, and developed by ECL (Amersham Pharmacia). X-ray films were analyzed by imaging densitometry. LL-37 content in supernatants was determined by using a standard curve derived from synthetic LL-37 peptide.

Bacterial Strains and Gastric Epithelial Cell Lines Enteroinvasive E. coli serotype O29:NM (ATCC 43892) was from the American Type Culture Collection (Ma-

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nassas, VA). Salmonella dublin phoP was provided by D. Guiney, UCSD. H. pylori strains SD4 and SD14 were originally isolated from gastric biopsy specimens of duodenal ulcer patients.1,30 Both strains are cagA⫹ and vacA⫹ and induce IL-8 secretion by human gastric epithelial cell lines.30 SD4 with a cagE mutation (⌬cagE) was constructed by insertion of a chloramphenicol resistance gene (a gift of D. Smith) into an EcoRI site located 756 bp from the 5⬘ end of the cagE gene sequence37 of strain NCTC11638. cagE mutants are unable to assemble the type IV secretion system,41 and consistent with this, IL-8 production by AGS cells stimulated with this isogenic mutant was decreased by more than 90% compared with cells stimulated with wild-type SD4. H. pylori strain SS1 (Sydney strain)42 was a gift of D. Berg, and H. pylori strain 26695, whose complete genome has been sequenced,43 was from the American Type Culture Collection (ATCC). Bacterial cultures were maintained on Columbia agar plates containing 5% laked sheep or horse blood and 1% fungizone under microaerophilic conditions. The human gastric adenocarcinoma cell line AGS (ATCC CRL 1739) was grown in RPMI medium 1640 supplemented with 10% heat-inactivated fetal bovine serum and 2 mmol/L L-glutamine. Cells were maintained in 95% air/5% CO2 at 37°C. Cells grown in 6-well culture plates were left uninfected or were infected with H. pylori strains at a multiplicity of infection of 1:100 as described earlier.1,30

Bactericidal Assays Aliquots of H. pylori (SD4, SD14, SS1, or 26695) (2.5 ⫻ 107 bacteria/mL) were suspended in brain heart infusion broth (⬃120 mmol/L NaCl) supplemented with 0.1% cyclodextrin, in the presence or absence of titrated concentrations of LL-37, hBD-1, or hBD-2. After incubation for 6 hours at 37°C in an atmosphere of 5% O2 , 10% CO2 , and 85% N2 , bacterial suspensions were plated on Columbia agar containing 5% laked sheep or horse blood and 1% fungizone under microaerophilic conditions, and incubated at 37°C for 5 days, after which colony-forming units (CFU) were counted. The bactericidal assay for S. dublin phoP was performed by incubating bacteria (5.0 ⫻ 104/mL) for 2 hours at 37°C with concentrated gastric secretions or synthetic LL-37, after which bacterial suspensions were plated and CFU were counted. The bactericidal assay for E. coli O29:NM was as described earlier.13

Immunodepletion of LL-37 From Gastric Juice Gastric secretions were centrifuged to remove debris and dialyzed against PBS. Samples containing 300 ␮g protein were incubated with 10 ␮g of rabbit IgG anti-LL-37 or preimmune rabbit IgG for 1 hour, after which protein G (Sigma) was added and samples were incubated overnight. After centrifugation, supernatants were dialyzed against H2O, lyophilized, and resuspended at 1/5 volume of bactericidal assay buffer containing the bacteria. Bactericidal activity against S. dublin phoP was decreased by 60%– 80% when synthetic LL-37 in PBS was immunodepleted with rabbit IgG

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anti-LL37 compared with treatment with preimmune rabbit IgG.

Statistical Analysis Statistical analysis of differences between groups was performed by using an unpaired t test or 1-way analysis of variance followed by Scheffe´ ’s post test.

Results LL-37/hCAP18 mRNA and Protein Expression by Normal Human Gastric Epithelium To determine if LL-37/hCAP18 was expressed by human gastric epithelium in vivo, mucosal biopsies of normal human antrum were analyzed for LL-37/hCAP18 mRNA expression by in situ hybridization. LL-37/ hCAP18 mRNA was expressed by the luminal surface and adjacent gastric pit epithelium and by epithelium in the deeper gastric glands (Figure 1A and C). Similar results were obtained using gastric tissues from 3 additional individuals using probes directed to either the LL-37 or cathelin domains (data not shown). Consistent with the previously described data, immunohistochemical analysis of gastric biopsy specimens with anti-LL-37 showed that LL-37 is expressed in a distinct pattern by normal gastric epithelium in the body and antrum of the stomach (Figure 2). In the body, LL-37 immunostaining was present mainly in mucous producing surface epithelial cells and epithelial cells in the upper gastric pits, and in fundic glands located in the basal region (Figure 2A and E). In contrast, epithelial LL-37 immunostaining was not seen in the generative zone containing isthmus epithelium (Figure 2A). In the antrum, LL-37 immunostaining was most marked in the surface cells (Figure 2C) and in the deeper glands (not shown). To determine which cells in the fundic glands of the fundus and body produce LL-37, adjacent sections were stained with either anti-LL-37 or anti-H⫹, K⫹-ATPase, a specific marker of parietal cells. Staining was observed in both chief and parietal cells (Figure 2E–G). The same staining pattern was seen with an anti-hCAP18 antibody that recognizes an epitope of the cathelin domain of hCAP18, showing both the specificity of the immunostaining and the presence in gastric epithelial cells of both the cathelin and LL-37 domains (data not shown). Decreased Expression of Epithelial LL-37/ hCAP18 in Gastric Hyperplastic Polyps, Tubular Adenomas, and Adenocarcinomas Immunostaining of normal gastric mucosa suggested a possible correlation between LL-37/hCAP18 expression and cell differentiation, a correlation we had

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from different patients, surface epithelium lacked LL-37, whereas LL-37 was expressed in the gastric glands (Figure 3A). Similarly, LL-37 immunostaining was not detected in the surface epithelium of the 3 gastric tubular adenomas examined (Figure 3B), although it was present in the deeper fundic glands (not shown). In contrast, the gastric adenocarcinomas examined lacked both surface and glandular LL-37 (Figure 3C). Increased Gastric LL-37/hCAP18 Expression in H. pylori Infection

Figure 1. In situ hybridization of LL-37/hCAP18 mRNA in normal human stomach. (A and C) Antisense cathelin probe; (B and D) sense probe. Panels A and B, original magnification 100⫻. Panels C and D show gastric gland areas indicated by the insets in panels A and B, respectively, with original magnification 400⫻.

also noted for colon epithelium.13 To test this possibility further, gastric mucosa from gastric neoplasms that are characterized by abnormal epithelial cell differentiation were immunostained for LL-37. In 3 hyperplastic polyps

Figure 2. LL-37 expression in normal human gastric mucosa. Mucosal biopsies from the gastric body (A, B, E–H) and gastric antrum (C and D) were immunostained for LL-37 expression using chicken anti-LL-37 antibody (A, C, and E) or control chicken IgY (B, D, G). (F and H ) Sections were stained with rabbit anti-H⫹, K⫹-ATPase or control rabbit IgG, respectively. (E) LL-37 staining shown is more prominent in chief cells, whereas in some of the sections, staining of chief and parietal cell staining was of similar intensity. Original magnification 100⫻ (A and B), 200⫻ (C and D), and 400⫻ (E–H ). Similar results were obtained when sections were stained with chicken anticathelin antibody (not shown).

H. pylori infection up-regulates gastric hBD-2 but not hBD-1 expression.1,2 To determine if H. pylori infection altered LL-37/hCAP18 expression by human gastric epithelium, gastric mucosal biopsies from patients infected with H. pylori were immunostained for LL-37. As shown in Figure 4, LL-37 staining of the epithelium in biopsies from H. pylori infected individuals was more widespread compared with normal controls, with staining throughout the gastric tubular units, including isthmus cells in the generative zone (compare Figure 2C and Figure 4B). The distribution of LL-37 immunostaining in the epithelium of patients with gastritis associated with NSAID usage was similar to that of healthy controls, with LL-37 immunostaining predominantly in the mucous producing surface epithelial cells and fundic glands (Figure 4F, compare with Figure 2C). Consistent

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Figure 3. LL-37 immunostaining of gastric neoplasms. (A) Hyperplastic polyp. (B) Tubular adenoma. (C) Poorly differentiated adenocarcinoma. Sections were stained using chicken anti-LL-37. Original magnification, 100⫻ (A and C) and 400⫻ (B). Similar results were obtained when sections were stained with chicken anticathelin antibody (not shown).

with prior reports of LL-37 production by neutrophils,44,45 neutrophils in the gastric mucosa of patients with gastritis, irrespective of the cause, but not other infiltrating leukocytes stained for LL-37 (Figure 4B and D). LL-37 Peptide Is Secreted Into Gastric Juice To determine if LL-37/hCAP18 produced by gastric epithelial cells was released into gastric secretions,

Figure 4. LL-37 expression in chronic gastritis associated with H. pylori infection or NSAID use. (A–E) Gastritis associated with H. pylori infection. (A) Giemsa stain showing H. pylori (arrows). (B and C) Adjacent sections of gastric antrum immunostained with chicken LL37/hCAP18 specific antibody (B) or control chicken IgY (C). (D and E) Adjacent sections of fundic glands immunostained with chicken LL-37/hCAP18 specific antibody (D) or control chicken IgY (E). Arrows in D indicate neutrophils. (F and G) NSAID-associated gastritis. Adjacent sections of gastric antrum were immunostained with chicken LL-37/hCAP18 specific antibody (F ) or control chicken IgY (G). Original magnification: (A) 1000⫻; (B–E) 400⫻; (F and G) 200⫻. Similar results were obtained when sections were stained with chicken anticathelin antibody (not shown).

we analyzed gastric secretions from healthy and H. pylori– infected subjects for LL-37/hCAP18. Gastric secretions from control subjects with histologically normal gastric mucosa and no evidence for H. pylori infection contained both the mature 18-kDa form of LL-37/hCAP18 and the 4.5 kDa LL-37 peptide (Figure 5). In addition, gastric secretions contained a 6 kDa form of the molecule that was not seen in neutrophil lysates used as a control and

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H. pylori Infection Up-regulates LL-37/hCAP18 but not hBD-1 mRNA Levels in Human Gastric Tissue

Figure 5. LL-37/hCAP18 in human gastric secretions. Gastric secretions from healthy controls or H. pylori–infected subjects were analyzed for LL-37/hCAP18 by immunoblot analysis using rabbit anti-LL37/hCAP18. Human neutrophil homogenate and LL-37 synthetic peptide were loaded as positive controls. No bands were seen using control rabbit IgG (not shown). Data are from a control subject without gastric inflammation and an individual with chronic H. pylori gastritis. Similar data was obtained from 2 additional control subjects without gastric inflammation and 2 additional individuals with chronic H. pylori infection.

likely represents an additional cleavage product. In contrast to gastric secretions, homogenates of gastric mucosa from H. pylori–infected patients and healthy subjects contained predominately the 18 kDa form of LL-37/hCAP18, with little or no smaller molecular weight (4.5 kDa and 6 kDa) forms (data not shown). LL-37 concentrations in gastric secretions from H. pylori–infected patients were significantly greater than those in healthy subjects (H. pylori–infected subjects, mean 7.3 ⫾ 3.3 ␮g/mL, n ⫽ 4; healthy subjects mean 1.0 ⫾ 0.6 ␮g/mL, n ⫽ 5; P ⬍ 0.05). The H. pylori– infected subjects with the 2 highest concentrations of LL-37 in their gastric secretions carried CagA⫹ strains as determined by PCR analysis. Notably, these patients had the least infiltration of inflammatory cells in the gastric mucosa. To determine if LL-37/hCAP18 in gastric secretions from H. pylori–infected individuals was bioactive, we compared the bactericidal activity of gastric secretions from 2 H. pylori–infected and 2 healthy individuals against an attenuated strain of Salmonella dublin before and after immunodepletion of LL-37. Specific depletion of LL-37 from the gastric secretions of the H. pylori– infected subjects with anti–LL-37 antibody decreased bactericidal activity by 28% ⫾ 13% (n ⫽ 4, P ⬍ 0.05) relative to treatment with preimmune IgG. In contrast, the identical treatment had no effect on the bactericidal activity of gastric secretions of the healthy control subjects (n ⫽ 4).

To assay LL-37/hCAP18 mRNA expression in H. pylori–infected compared with normal gastric mucosa, LL-37/hCAP18 mRNA levels were determined by realtime PCR in histologically normal gastric mucosal biopsies (n ⫽ 4) and in biopsies from individuals infected with H. pylori (n ⫽ 4) (Figure 6). Further, to determine whether or not possible changes in LL-37/hCAP18 mRNA were simply a feature of chronic gastritis unrelated to H. pylori infection, LL37/hCAP mRNA levels were assessed in 3 additional subjects with chronic gastritis associated with NSAID usage who were not infected with H. pylori. As controls, we also assessed mRNA levels for hBD-1 and hBD-2, as hBD-1 was previously shown to be unaffected by gastric mucosal inflammation, irrespective of H. pylori infection, whereas hBD-2 was up-regulated by mucosal inflammation, whether or not caused by H. pylori infection.1 As shown in Figure 6, LL-37/hCAP18 mRNA levels were significantly increased by approximately 11-fold in the H. pylori–infected compared with healthy subjects but were not increased in chronic gastritis unrelated to H. pylori infection. hBD-1 mRNA levels were not increased by chronic mucosal inflammation, whether or not it was because of H. pylori infection, whereas hBD-2 mRNA levels were significantly increased by 5- to 76-fold in H. pylori gastritis and 3- to 9-fold in gastritis associated with NSAID use, compared with healthy subjects (Figure 6).

Figure 6. Expression of LL-37/hCAP18, hBD-1, and hBD-2 mRNA in human gastric biopsies. Gastric biopsies from 4 control subjects without gastric inflammation (open bars), 4 H. pylori–infected patients with gastritis (cross-hatched bars), and 3 patients with gastritis associated with NSAID use (solid bars) were analyzed by real-time PCR for LL-37/hCAP18, hBD-1, and hBD-2 mRNA expression. Levels are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are expressed as fold-change in mRNA transcript levels relative to control subjects without gastric inflammation. Values are mean ⫾ SEM of 3 repeated experiments. *P ⬍ 0.05 compared with normal controls and patients with gastritis associated with NSAID use.

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subsequent decrease by 24 hours after infection (Figure 7A). As assayed by real-time PCR (Figure 7B), LL-37/ hCAP18 mRNA expression in response to infection with wild-type H. pylori SD4 began to increase by 20 hours and was significantly increased at 32 hours after infection. Moreover, this response required an intact cag type IV secretion system because there was little increase in LL-37/hCAP18 mRNA expression in cells infected with an isogenic cagE mutant of H. pylori SD4. Supernatants of H. pylori SD4 wild-type–infected cultures contained significantly greater quantities of LL-37 than those infected with the H. pylori SD4 ⌬cagE mutant (wild-type SD4infected 28.8 ⫾ 0.9 ng/mL, n ⫽ 3; ⌬cage-infected 14.7 ⫾ 5.0 ng/mL, n ⫽ 3; uninfected 8.4 ⫾ 2.0 ng/mL, n ⫽ 3; P ⬍ 0.05 wild-type vs. ⌬cage at 32 hours after infection). Because LL-37/hCAP18 mRNA was not increased in mucosal biopsies from patients with gastric mucosal inflammation unrelated to H. pylori, we asked if LL-37 mRNA levels in AGS cells were up-regulated by proinflammatory stimuli (IL-1␣, IL-6, TNF␣, IFN-␥, or E. coli LPS) after 6 or 24 hours stimulation. None of these agents up-regulated LL-37/hCAP18 mRNA levels whereas hBD-2 mRNA, measured as a positive control,1 was up-regulated by 6 hours after stimulation with IL-1␣ or TNF␣ (data not shown). Figure 7. LL37/hCAP18 mRNA expression by human gastric epithelial cells infected with H. pylori. (A) AGS cells were left uninfected (control) or were infected with H. pylori SD4 strain. Expression of LL-37/hCAP18, hBD-2, and GAPDH mRNA was assessed by qualitative RT-PCR at 8 and 24 hours after infection. (B) LL-37/hCAP mRNA levels were analyzed by real-time PCR. Cells were infected with wildtype H. pylori SD4 (●), an isogenic ⌬CagE mutant of SD4 (‚), or left uninfected (E). LL-37/hCAP18 mRNA levels, normalized to those of GAPDH, are expressed as fold increase over uninfected controls at time 0. Data are from a representative experiment. Values are mean ⫾ SD, n ⫽ 3; *P ⬍ 0.05. Similar results were obtained in 2 repeated experiments.

H. pylori Up-regulates LL-37/hCAP18 Expression in Cultured Human Gastric Epithelial Cells To further characterize the regulated expression of LL-37/hCAP18 in response to H. pylori infection, we used the human gastric epithelial cell line, AGS. As shown in Figure 7A, AGS cells constitutively expressed LL-37/hCAP18 mRNA, and incubation of those cells with H. pylori SD4, a cagA⫹, vacA⫹ strain, resulted in the increased expression of LL-37/hCAP18 mRNA by 24 hours after infection. In contrast, induction of hBD-2 mRNA expression, used as a positive control, was more rapid, with increased levels noted by 8 hours, and a

LL-37 Has Antimicrobial Activity Against H. pylori Because H. pylori infection up-regulated LL-37/ hCAP18 production, we investigated whether LL-37 has antimicrobial activity against H. pylori. As shown in Figure 8, LL-37 killed H. pylori SD14 in a concentrationand time-dependent manner. The EC50 of LL-37 for H. pylori SD14 was 1.4 ␮mol/L, and significant antimicrobial activity was seen by 3 hours. Similar results were obtained for H. pylori SD4 (not shown). To determine if LL-37 is active against a broader range of H. pylori strains, we examined its antimicrobial activity against the widely studied H. pylori strains SS1 and 26695.42,43 Incubation of H. pylori SS1 with LL-37 (16 ␮mol/L) resulted in a ⬎4 log decrease in bacterial viability within 6 hours, whereas LL-37 alone was less effective against H. pylori strain 26695 (Figure 9B). The ␤-defensins are bactericidal for a broad array of Gram-positive and Gram-negative bacteria, but optimal killing requires incubation in solutions containing low concentration of NaCl. This also was reported to be the case for killing of H. pylori by hBD-2.2 In further studies, we compared the antimicrobial activity of LL-37 for H. pylori with that of hBD-1 and hBD-2. As shown in

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Discussion Gastric epithelial cells express LL-37/hCAP18 mRNA and protein and release this cathelicidin into gastric secretions. LL-37/hCAP18 was produced by those epithelial cells that line the luminal surface and upper gastric pits and by cells deeper in gastric glands but was absent from cells in the regenerative mucus neck zone. In addition, there was little, if any, LL-37/hCAP18 expression in regions of increased epithelial cell proliferation in hyperplastic polyps, by the surface epithelium in tubular adenomas, or by epithelial cells in gastric adenocarcinomas. This distinct distribution of LL-37/hCAP18 suggests that cell differentiation is a critical determinant of its expression in gastric epithelial cells because cells from the generative mucous neck zone differentiate as they

Figure 8. Antimicrobial activity of LL-37. (A) H. pylori SD14 were incubated in the presence of the indicated concentrations of LL-37 for 6 hours, after which CFU were determined. (B) H. pylori SD14 was cultured for the indicated times in the absence (E) or presence (F) of LL-37 (16 ␮mol/L). Values both panels are mean ⫾ SD, n ⫽ 4. Results in both panels are from single representative experiments. Similar results were obtained in 2 or more repeated experiments.

Figure 9A, LL-37 killed H. pylori SD4 and enteroinvasive E. coli O29:NM. In contrast, hBD-1 at the same concentration killed H. pylori SD4 but had markedly less activity against E. coli O29:NM, whereas hBD-2 killed E. coli more effectively than H. pylori SD4. Similar results were obtained also for killing of wild-type H. pylori SD14 by each of these peptides (data not shown). Because different antimicrobial innate defense molecules in combination can have synergistic activities in killing bacteria,14 we assessed the bactericidal effect of LL-37 in combination with hBD1 against H. pylori 26695. As shown in Figure 9B, hBD-1 potentiated LL-37 killing of strain 26695 as hBD-1 in combination with LL-37 decreased H. pylori viability by an average of 50-fold compared with LL-37 alone.

Figure 9. Antimicrobial activity of LL-37, hBD-1, and hBD-2. (A) H. pylori SD4 (solid bars) or E. coli O29:NM (open bars) were incubated with or without LL-37, hBD-1, or hBD-2 (each at 16 ␮mol/L) for 6 hours, after which CFU were determined. Values are mean ⫾ SD, n ⫽ 4; P ⬍ 0.05 compared to control. Results are from a single experiment. Similar results were obtained in an additional repeated experiment. (B) H. pylori strain 26695 was left untreated or cultured with LL-37 (16 ␮mol/L), hBD-1 (16 ␮mol/L), or LL-37 in combination with hBD-1 for 6 hours. Values are mean ⫾ SD, n ⫽ 4; *P ⬍ 0.05 compared with control and hBD-1 alone; **P ⬍ 0.05 compared to LL-37 alone. Results are from a single experiment. Similar results were obtained in an additional repeated experiment.

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either migrate to areas deeper in the gastric mucosa as chief, parietal, or endocrine cells or upward to the gastric pits and luminal surface as mucin-producing cells.46 – 48 The distribution of LL-37/hCAP18 in the more differentiated epithelial cell populations of the gastric mucosa differs from that reported for lysozyme3 but resembles its distribution in the colon, where the greatest expression of LL-37/hCAP18 is also seen in the more differentiated surface epithelial cells.13 Taken together with the known differential distribution of the ␣- and ␤-defensins among different epithelial cell populations, these data support an emerging concept whereby arrays of antimicrobial peptides and proteins have distinct patterns of expression within the same region of the intestinal tract and between different regions that encompass the proximal and more distal intestinal tract. Gastric mucosa from H. pylori—infected patients expressed higher levels of LL-37/hCAP18 mRNA and their gastric secretions contained bioactive LL-37 in significantly greater amounts than uninfected controls. In the infected patients, LL-37/hCAP18 was not restricted to surface cells or gastric glands but was produced throughout the length of the gastric tubules. Nonetheless, increased LL-37 production was not simply because of the presence of inflammatory mediators in H. pylori–infected patients because increased LL-37/hCAP18 production was not seen in noninfected patients with gastritis associated with NSAID use. Consistent with this, LL-37/ hCAP18 expression was not increased in cultured gastric epithelial cells stimulated with proinflammatory mediators but was increased when those cells were infected with wild-type H. pylori. These data indicate that increased LL-37/hCAP18 production by gastric epithelial cells is not solely because of the host inflammatory response and suggest that its increased expression in H. pylori–infected patients results more directly from the epithelial cell response to this pathogen. H. pylori infections characterized by gastritis, gastric ulcers, duodenal ulcers, and gastric cancer are more common with H. pylori having the cag pathogenicity island.35,37,49 –51 The cag pathogenicity island encodes a type IV secretion system that is important for the injection of bacterial components into host cells. cagE encodes a protein that is necessary for the assembly of the type IV secretion system.37,38,41,52 As shown herein, an isogenic ⌬cagE mutant of H. pylori strain SD4 did not up-regulate LL-37 production by AGS cells, suggesting an H. pylori product that is secreted into epithelial cells is important in the regulation of epithelial cell LL-37/hCAP18 production. Products of the cag pathogenicity island are known to be essential for the activation of the transcrip-

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tion factor NF-␬B by H. pylori and the promoter region of LL-37/hCAP18 contains a putative NF-␬B– binding site. Nonetheless, LL-37 was not up-regulated in AGS cells in response to activators of NF-␬B that were shown in positive controls to activate known NF-␬B target genes (e.g., hBD2), indicating either that LL-37/ hCAP18 does not function as a NF-␬B target gene or, alternatively, that NF-␬B is not the sole transcription factor required for LL-37/hCAP18 gene transcription. The in vivo microenvironment in which cationic antimicrobial peptides interact with bacteria has a key role in determining their antimicrobial activity. The activity of LL-37 is affected both by anion concentration and pH, with LL-37 preferentially having an ␣-helical structure needed for antimicrobial activity at pH ⬎ 5.53,54 However, in the stomach, a large pH gradient exists across the mucous layer that is adjacent to the epithelium (e.g., pH can vary from approximately 2 in the gastric lumen to approximately 7 in the mucus layer adherent to epithelium).46,55 This suggests LL-37 produced by surface epithelial cells has its greatest activity in close proximity to the epithelial surface (i.e., the site at which H. pylori preferentially colonizes). LL-37 as well as hBD-1, but not hBD-2, killed several strains of H. pylori in vitro, whereas those peptides manifested different antimicrobial activity against enteroinvasive E. coli O29:NM. Antimicrobial peptides vary in their bactericidal activity at physiologic salt concentrations, which in part reflects their cationic surface charge.56 The defensins, for example, are most active in hypotonic NaCl environments. However, the different bactericidal activity of LL-37 and hBD-2 for H. pylori does not appear to simply reflect their different cationic charge as LL-37/hCAP18 and hBD-2 are both strongly cationic. This suggests that other factors (e.g., differences in the membrane composition or structure of H. pylori compared with E. coli) also may be important in determining the bioactivity of these antimicrobial peptides. LL-37 alone was bactericidal for H. pylori strains SD4, SD14, and SS1 but not strain 26695 indicating heterogeneity among H. pylori strains in their susceptibility to LL-37. Furthermore, hBD-1 alone did not kill H. pylori 26695 but significantly potentiated the antimicrobial activity of LL-37 against this strain. The potentiation of LL-37 antimicrobial activity by hBD-1 is consistent with the different mechanisms these molecules use to mediate microbial killing. Whereas LL-37 forms an amphipathic ␣-helical structure that is thought to integrate within or deform the membrane resulting in its disruption,54 defensins are thought to aggregate and form pores in the bacterial membrane.57,58 In other studies, LL-37 has been

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shown to increase the antimicrobial effect of lysozyme and lactoferrin.14 Although individual antimicrobial peptides may have little antimicrobial activity, combinations of those peptides can work synergistically to kill their microbial targets. This indicates the importance of understanding the interactions between multiple antimicrobial agents present in the complex microenvironment at mucosal surfaces to define their relevant physiologic functions. H. pylori, once established in its ecological niche in the stomach, can persist in the stomach for the host’s lifetime without directly invading or killing the host.59 Failure of the host to clear H. pylori variably has been attributed to the relatively low stimulatory activity of its LPS for macrophages,60 the expression of Lewis blood group antigens,61,62 and its resistance to phagocytosis by neutrophils.63 Nonetheless, LL-37 alone or together with hBD-1 effectively killed all of the strains of H. pylori tested herein, suggesting they could play a role in innate defense to this pathogen and consistent with this, infected patients with the highest levels of LL-37 in gastric secretions had the least mucosal inflammation despite carrying CagA⫹ strains. On the other hand, to successfully colonize and infect the host, various bacteria have developed strategies to prevent their destruction by antimicrobial peptides,64,65 and this may be the case also for some strains of H. pylori. Such mechanisms include, for example, the degradation of antimicrobial peptides by bacterial proteases, expelling cationic peptides through an energy-dependent efflux system, or decreasing the anionic charge at the bacterial membrane (e.g., NH4⫹ generated by bacterial urease).27,64,66 In addition, bacteria may use the production of antimicrobial peptides to their own advantage to avoid host defenses. In this regard, LL-37 has been noted to inhibit macrophage stimulation by bacterial components (e.g., LPS, lipoteichoic acid) and to alter macrophage cytokine and chemokine expression.67 This may also explain why H. pylori–infected patients with the highest levels of LL-37 in their gastric secretions had the least mucosal inflammatory infiltrate. Finally, we note that one of the most striking features of H. pylori infection is its persistence as a relatively lowgrade chronic infection. Given the data herein, we suggest that antimicrobial peptides like LL-37 in the gastric mucosa may contribute to maintaining a critical balance between the host and H. pylori, which allows the persistence of this “controlled infection.”

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Received March 11, 2003. Accepted August 21, 2003. Address requests for reprints to: Martin F. Kagnoff, M.D., Laboratory of Mucosal Immunology, Department of Medicine (0623D), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0623. e-mail: [email protected]; fax: (858) 534-5691. Supported by NIH grants DK58960 and DK35108 (to M.F.K.), DK53649 (to S.P.C.), AI052453 (to R.L.G.), and a VA Merit Award (to R.L.G.). We thank John Leopard and Jennifer Smith for technical assistance and Dr. Nissi Varki for pathological consultation.