Volume 125, Issue 6, Pages 1613-1625 (December 2003) 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, Martin F Kagnoff  Gastroenterology  Volume 125, Issue 6, Pages 1613-1625 (December 2003) DOI: 10.1053/j.gastro.2003.08.028

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×. Gastroenterology 2003 125, 1613-1625DOI: (10.1053/j.gastro.2003.08.028)

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 anti-cathelin antibody (not shown). Gastroenterology 2003 125, 1613-1625DOI: (10.1053/j.gastro.2003.08.028)

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). Gastroenterology 2003 125, 1613-1625DOI: (10.1053/j.gastro.2003.08.028)

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 LL-37/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). Gastroenterology 2003 125, 1613-1625DOI: (10.1053/j.gastro.2003.08.028)

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-LL-37/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. Gastroenterology 2003 125, 1613-1625DOI: (10.1053/j.gastro.2003.08.028)

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. Gastroenterology 2003 125, 1613-1625DOI: (10.1053/j.gastro.2003.08.028)

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 wild-type H. pylori SD4 (•), an isogenic ΔCagE mutant of SD4 (▵), or left uninfected (○). 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. Gastroenterology 2003 125, 1613-1625DOI: (10.1053/j.gastro.2003.08.028)

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 (○) or presence (•) of LL-37 (16 μmol/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. Gastroenterology 2003 125, 1613-1625DOI: (10.1053/j.gastro.2003.08.028)

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 μmol/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 μmol/L), hBD-1 (16 μmol/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. Gastroenterology 2003 125, 1613-1625DOI: (10.1053/j.gastro.2003.08.028)