IAI Accepts, published online ahead of print on 13 August...

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The role of Interleukin-32 in Helicobacter pylori–induced gastric inflammation 1

Running Title: H. pylori induces IL-32 in gastritis. 2

Key Words: Helicobacter pylori, interleukin-32, gastritis, gastric cancer 3

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Kosuke Sakitani1, Yoshihiro Hirata1, Yoku Hayakawa1, Takako Serizawa2, Wachiko Nakata1, 5

Ryota Takahashi1, Hiroto Kinoshita1, Kei Sakamoto2, Hayato Nakagawa1, Masao Akanuma2, 6

Haruhiko Yoshida1, Shin Maeda3, Kazuhiko Koike1 7

1 Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, 8

Japan 9

2 Division of Gastroenterology, Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan 10

3 Gastroenterology Division, Yokohama City University Graduate School of Medicine, Yokohama, 11

Japan 12

Correspondence: 13

Yoshihiro Hirata, Department of Gastroenterology, Graduate School of Medicine, The University of 14

Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan 15

Email: [email protected] 16

Fax: +81-3-3814-0021 17

Tel: +81-3-3815-5411 18

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00637-12 IAI Accepts, published online ahead of print on 13 August 2012

on July 13, 2018 by guesthttp://iai.asm

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ABSTRACT 19

Helicobacter pylori (H. pylori) infection is associated with gastritis and gastric cancer. An H. pylori 20

virulence factor, the cag pathogenicity island (PAI), is related to host cell cytokine induction and gastric 21

inflammation. Since elucidation of the mechanisms of inflammation is important for therapy, the 22

associations between cytokines and inflammatory diseases have been vigorously investigated. Levels of 23

interleukin (IL)-32, a recently described inflammatory cytokine, are increased in various inflammatory 24

diseases, such as rheumatoid arthritis and Crohn’s disease, and in malignancies, including gastric cancer. 25

In this report, we examined IL-32 expression in human gastric disease. We also investigated the function 26

of IL-32 in activation of the inflammatory cytokines in gastritis. IL-32 expression paralleled human 27

gastric tissue pathology, with low IL-32 expression in H. pylori–uninfected gastric mucosa, and more in 28

gastritis and gastric cancer tissue. H. pylori infection increased IL-32 expression in human gastric 29

epithelial cell lines. H. pylori–induced IL-32 expression was dependent on the bacterial cagPAI genes 30

and activation of nuclear factor-κB (NF-κB). IL-32 expression induced by H. pylori was not detected in 31

the supernatant of AGS cells, but was found in the cytosol. Expression of H. pylori–induced cytokines, 32

CXCL1, CXCL2, and IL-8, was decreased in IL-32-knockdown AGS cell lines compared to a control 33

AGS cell line. We also found that NF-κB activation was decreased in H. pylori–infected 34

IL-32-knockdown cells. These results suggest that IL-32 has important functions in the regulation of 35

cytokine expression in H. pylori–infected gastric mucosa.36


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INTRODUCTION 37

The relationship between microbial infections, inflammatory disorders, and cancer have been 38

extensively investigated and the associations are now widely accepted, partly owing to the discovery of 39

Helicobacter pylori (H. pylori) (5, 19). H. pylori was first isolated from gastritis patients in 1983 by 40

Warren and Marshall (52). Gastric inflammation caused by H. pylori infection increases the risk of 41

gastric cancer, the common cause of cancer death worldwide (8, 42, 51). One of the virulence factors 42

responsible for the progression of gastric diseases is the cag pathogenicity island (PAI) of H. pylori, a 43

cluster of approximately 30 genes. H. pylori and its cagPAI genes are associated with severe gastric 44

diseases, including gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma 45

(29, 40). 46

H. pylori cagPAI activates nuclear factor-κB (NF-κB) and mitogen-activated protein kinases 47

(MAPKs) signaling pathways in infected epithelial cells. NF-κB is a transcription factor that regulates 48

various cellular responses including inflammation, cell survival or death, and cell proliferation. NF-κB 49

activation induces production of inflammatory cytokines, such as interleukin (IL)-1β, IL-8, and TNF-α. 50

IL-8, which is induced by H. pylori infection via NF-κB activation in gastric epithelial cells, plays a 51

critical role in gastritis and gastric carcinogenesis (6, 39, 49). IL-8 causes neutrophil infiltration into 52

gastric tissue, which elicits additional inflammation. In Japanese populations, a single polymorphism in 53

the IL-8 gene is associated with up-regulation of IL-8 and is associated with an increased risk of 54


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atrophic gastritis, and gastric cancer (50). Similarly, polymorphisms in the IL-1β and TNF-α genes have 55

been associated with gastritis and gastric cancer (9, 47). 56

The importance of understanding inflammation has recently been highlighted. First, particular 57

cytokines induced in inflammatory diseases are good therapeutic targets; for example, tumor necrosis 58

factor (TNF)-α, which leads to the sequential release of cytokines and causes inflammatory reactions. 59

Antibodies used for anti-TNF therapy have been shown to control rheumatoid arthritis and Crohn’s 60

disease (34). Also, the identification and elimination of pathogens in inflammatory disease have 61

decreased the incidence of inflammation-associated cancer. Indeed, some studies on H. pylori have 62

shown that eradication therapy reduces the risk of gastric cancer (10, 38, 53). 63

IL-32, formerly called NK-4, is a newly described inflammatory cytokine, and is reported to induce 64

the production of several other cytokines, such as TNF-α and IL-1 (7, 23). IL-32 does not share 65

sequence homology with other cytokines, and no homolog has been found in rodents. Previous reports 66

showed that IL-32 expression is increased in various inflammatory diseases, and it is involved in the 67

pathogenesis of rheumatoid arthritis and Crohn’s disease (14, 44, 45). IL-32 expression is induced by 68

hepatitis B virus, hepatitis C virus, and Mycobacterium tuberculosis (3, 32, 35, 41). Furthermore, IL-32 69

expression is associated with several malignancies, including lung cancer, pancreatic cancer, and gastric 70

cancer (22, 36, 46). 71

The mechanisms underlying IL-32 expression in gastric tissues as well as the roles of IL-32 in the 72


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development of gastric disease have not been fully clarified. In this study, we investigated IL-32 73

expression in H. pylori–infected gastric epithelial cells. We further examined the association of IL-32 74

and other inflammatory cytokines induced in gastritis. 75

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MATERIALS AND METHODS 77

Human gastric tissue samples. 78

Gastric tissues from patients were obtained from the archives of the University of Tokyo Hospital and 79

Motojima General Hospital with the approval of the medical ethics committee and with informed 80

consent. H. pylori–induced gastritis was defined by positive H. pylori test results, a rapid urease test 81

(Helicocheck, Otsuka Pharmaceuticals, Tokyo, Japan), and microscopic verification. Normal gastric 82

mucosa was defined by the absence of pathological inflammation and negative results for the H. pylori 83

test. 84

85

Cell lines. 86

Three gastric cancer cell lines, AGS, TMK-1, and MKN45, were described previously (15, 30, 31). AGS 87

cells were maintained in Ham’s F12 (Sigma, St. Louis, MO), which contained 10% fetal bovine serum 88

(FBS). TMK-1 and MKN45 cells were maintained in RPMI 1640 (Sigma) contained 10% FBS. 89

90


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Helicobacter pylori strains. 91

H. pylori strain TN2, which is positive for cagPAI, was maintained under microaerobic conditions in 92

Brucella broth. TN2 isogenic cagPAI-negative and cagE-negative mutants (TN2-ΔcagPAI and 93

TN2-ΔcagE, respectively) were constructed as described previously (39). In co-culture experiments, H. 94

pylori was washed with PBS, resuspended in Ham’s F12 (Sigma), and used in the assays. The bacteria to 95

cell ratio was approximately 100:1 in all assays. 96

97

Reagents. 98

Recombinant human IL-1β, recombinant human TNF-α, and recombinant IL-32β were purchased from 99

R&D Systems (Minneapolis, MN). Chemical inhibitors for IKKβ (SC-514) and p38 inhibitor 100

(SB203580) were purchased from Merck (Nottingham, U.K). SC-514 and SB203580 were dissolved in 101

4% dimethyl sulfoxide and added to 12-well plates at concentration of 20 μM, one hour before H. pylori 102

infection. 103

104

Plasmids. 105

The luciferase reporter plasmids, -133-IL-8-Luc (a gift from Dr. K Matsushima), pNF-κB-Luc 106

(Stratagene, La Jolla, CA), and pRL-TK (Promega, Madison, WI) were described previously (1, 31). 107

pSilencer vectors (Ambion, Austin, TX) encoding siRNA for IL-32 were constructed using sequences 108


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reported previously (3). Two siRNA sequences were used to generate pSi-IL-32-6 and pSi-IL-32-7, 109

respectively. The IL-32 expression vector (pcDNA-IL32β) was constructed by cloning IL-32β cDNA 110

into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA). Full-length IL-32β was amplified by RT-PCR 111

from RNA obtained from AGS cells infected with H. pylori and sequenced. For IL-32 rescue 112

experiments, a mutant IL-32β expression vector (pcDNA-mIL32β) was generated by mutagenesis. We 113

constructed primers to insert three mutations in the target sequence of pSi-IL32-6, and mutagenesis was 114

performed on pcDNA-IL32β using QuickChange Site-Directed Mutagenesis Kit (Stratagene) according 115

to the manufacture’s protocol. 116

117

Immunoblotting and immunoprecipitation. 118

Cells were disrupted in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 119

1% Triton X (Sigma), 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM Na3VO4, 1 μg/ml 120

leupeptin). CagA immunoprecipitation was performed as described previously (16). Cell lysates were 121

separated by SDS-PAGE, transferred to a PVDF membrane (Amersham Pharmacia Biotech, UK), and 122

blocked for one hour with TBST plus 5% bovine serum albumen (BSA). The membrane was incubated 123

overnight at 4°C with the primary antibody, and subsequently washed and incubated with secondary 124

HRP-conjugated antibody. The immunocomplexes were detected with Immunostar LD (Wako, Tokyo, 125

Japan). Images were obtained using the LAS 3000 image analyzer (Fujifilm, Tokyo, Japan). The primary 126


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antibodies used for immunoblotting were: anti-human IL32 (R&D), anti-human β-actin (Sigma), 127

anti-urease (Santa Cruz, Santa Cruz, CA), anti-phospho-IκBα (Ser32, Cell Signaling, Beverly, MA), 128

anti-phospho-JNK (Cell Signaling), anti-phospho-p38 (Cell Signaling), anti-phosphotyrosine antibody 129

(PY99, Santa Cruz), and anti-CagA antibody (16). 130

131

Immunohistochemistry. 132

IL-32 expression in human gastric tissues was examined immunohistochemically. Formalin-fixed 133

paraffin-embedded gastric specimens were cut at a thickness of 3 μm, and de-paraffinized. Endogenous 134

peroxidase activity was blocked using 3% H2O2. The primary anti-IL-32 antibody (KU32-52; 135

BioLegend, San Diego, CA) was added and the tissue sections were incubated overnight. For the second 136

antibody, the tissues were exposed to Histofine Simple Stain MAX PO (Nichirei, Tokyo, Japan) for 30 137

min. Mouse IgG1 isotype antibody (R&D) was used as the negative control. 138

IL-32 expression in AGS was examined by immunofluorescence staining. AGS cells were seeded 139

into four-well chamber slides (Nalge Nunc International, Naperville, IL), and infected with H. pylori for 140

24 h. The cells were washed with PBS, fixed with 3% paraformaldehyde for 30 min, incubated with 141

0.5% Triton X (Sigma) for 10 min, and then blocked with 2% BSA for 10 min. IL-32 antibody 142

(KU32-52) was added and incubated overnight. Cells were incubated with Alexa Flour 555-conjugated 143

goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) for 30 min, and the nuclei were 144


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visualized by staining with Hoechst 33342 (Takara, Tokyo, Japan) for one min. 145

146

ELISA. 147

IL-32, IL-8, CXCL1, CXCL2, and TNF-α levels in culture supernatants or cell lysates were quantitated 148

by an enzyme-linked immunosorbent assay (ELISA). IL-32 levels were measured as described 149

previously (22), KU32-56 (BioLegend) was used as a capture antibody and biotinylated KU32-52 150

(BioLegend) as the detection antibody. IL-8, CXCL1, and TNF-α levels were measured with Quantikine 151

ELISA kits (R&D), according to the manufacturer’s protocol. CXCL2 levels were measured with a 152

human CXCL2 assay kit (IBL, Gunma, Japan). 153

154

Cloning and sequencing. 155

Cloning and sequence analysis was conducted to identify IL-32 isoforms in human gastritis tissue. Total 156

RNA was extracted from human gastric tissue of H. pylori-positive gastritis patients using NucleoSpin 157

RNAII (Takara), and cDNA was synthesized from 1 μg of RNA using the ImProm-II Reverse 158

Transcription System (Promega). The full-length coding region of IL-32 cDNA was amplified by PCR, 159

and cloned into the TOPO TA cloning vector (Invitrogen). Plasmid DNAs from 20 colonies were 160

sequenced using the ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA). 161

162


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Real-time RT-PCR. 163

Total cellular RNA samples were isolated from AGS cells using NucleoSpin RNA II (Takara). The 164

cDNAs were generated from 1 μg of total RNA by reverse transcription using the ImProm-II Reverse 165

Transcription System (Promega). The mRNA expression levels of total IL-32, IL-32β, IL-8, CXCL1, 166

CXCL2, TNF-α, IL-11, and GAPDH was determined by quantitative real-time RT-PCR using the ABI 167

PRISM 7000 Quantitative PCR System (Applied Biosystems). GAPDH mRNA was used as the internal 168

control. The primer sequences used are available on request. 169

170

RNA interference. 171

The siRNAs for IKKβ (5’-GGUGAAGAGGUGGUGGUGAGC-3’) and IL-8 (Qiagen, Chatsworth, CA) 172

were used. The siRNAs or the non-silencing control (5’-AATTCTCCGAACGTGTCACGT-3’) were 173

transfected with Lipofectamine RNAimax (Invitrogen) for 48 h, and cells were infected with H. pylori 174

for the indicated times. 175

176

Construction of stable cell lines. 177

To establish IL-32-knockdown stable cell lines and a control cell line, AGS cells were transfected with 178

pSi-IL-32-6 and pSi-IL-32-7, or a scrambled DNA sequence for control cells, using Effectene 179

Transfection Reagent (Qiagen). The transfectants were cultured with G418 (50 μg/ml) and selected. We 180


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established two independent knockdown cell lines, AGS-IL32KD1 and AGS-IL32KD2, using 181

pSi-IL-32-6 and pSi-IL-32-7, respectively. 182

To obtain IL-32 over-expressing cell lines, AGS cells were transfected with pcDNA-IL32β and 183

selected with G418. Two cell lines were established (AGS-IL-32 clone 15 and clone 19). The control 184

cell line was generated by transfection with empty pcDNA3.1 and selection with G418. 185

186

Luciferase assay. 187

AGS cells, cultured in 24-well tissue culture plates, were transfected with 100 ng/μl -133-IL-8-Luc or 188

pNF-κB-Luc and 10 ng/μl pRL-TK using the Effectene transfection reagent (Qiagen). In the rescue 189

experiment, AGS-IL32KD1 cells were transfected with 100 ng of pcDNA-mIL32β or the empty 190

pcDNA3.1 vector. After 24 h, H. pylori was added (cells: bacteria = 1: 100) and co-cultured for a further 191

24 h. Cells were washed with PBS and lysed in luciferase lysis buffer. The lysates were assayed using a 192

Pica-Gene Dual-Luciferase Reporter Assay System (Toyo ink, Tokyo, Japan) as described previously 193

(31). All assays were performed at least three times independently in triplicate. 194

195

Statistical analysis. 196

Statistical analysis was performed using Student’s t-test, two-sided or one-way ANOVA with Dunnett’s 197

multiple comparison, or Tukey’s HSD. Differences were considered statistically significant at values of 198


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p <0.05. 199

200

RESULTS 201

IL-32 expression correlates with the severity of gastric disease. 202

To investigate the involvement of IL-32 in H. pylori–induced gastric disease, we first examined the 203

expression levels of IL-32 in human gastric tissues. IL-32 levels were elevated in gastritis tissues and 204

gastric cancer tissues compared to those in H. pylori–negative normal gastric mucosa, as determined by 205

immunoblot analysis (Fig. 1A). Immunohistochemical analysis indicated that high levels of IL-32 were 206

present in the cytoplasm of gastric epithelial cells of gastritis patients (Fig. 1B, right), while the negative 207

isotype control with mouse IgG1 antibody exhibited negative staining (Fig. 1B, left). IL-32 expression 208

was weak or not detectable in normal gastric mucosa (Fig. 1C, left). IL-32 was strongly positive in the 209

cytoplasm of gastric cancer cells (Fig. 1C, right). 210

To quantitate IL-32 expression, IL-32 concentrations in gastric tissues were determined by ELISA. 211

As shown in Fig. 1D, IL-32 protein levels increased depending on the pathology, ranging from very low 212

in normal gastric mucosa to intermediate in H. pylori–infected gastric mucosa, and high in gastric cancer 213

tissue. IL-32 concentration in gastritis (643 pg/ml ± 492) was significantly higher than that of normal 214

gastric tissue (208 pg/ml ± 133). Gastric cancer tissue exhibited an even higher IL-32 expression (1651 215

pg/ml ± 488), which was statistically significant compared to that in gastritis. 216


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To determine the IL-32 isoforms that are expressed in gastric tissue, full-length IL-32 mRNA was 217

amplified from the cDNA obtained from gastritis tissue. As shown in Fig. 1E (left), human gastritis 218

tissue, as well as the AGS cells, expressed full-length IL-32. By cloning the IL-32 mRNA from gastritis 219

tissue, we found that 90% of IL-32 isoforms were IL-32β (18/20), 10% were IL-32ε (2/20); no other 220

isoforms were detected (Fig. 1E, right). 221

222

H. pylori infection induces IL-32 expression in gastric cancer cells. 223

Next, we investigated the expression of IL-32 in vitro using the gastric cancer cell lines. IL-1β and 224

TNF-α have been reported to strongly induce IL-32 in several cell lines (21, 37, 54). Twenty-four hours 225

after stimulation with IL-1β, TNF-α, and cagPAI-positive H. pylori strain TN2, IL-32 expression was 226

induced in AGS cells (Fig. 2A). Western blot analysis revealed that IL-32 was also induced by H. pylori 227

in the TMK-1 and MKN45 gastric cancer cell lines, in a time-dependent manner (Fig.2 B). IL-32 mRNA 228

levels were then determined by real-time RT-PCR. IL-32 mRNA levels in AGS cells increased after 229

infection with H. pylori for three hours, similar to the increase in IL-8 mRNA levels (Fig. 2C). These 230

results indicate that H. pylori enhances IL-32 mRNA and protein expression in infected gastric cells in 231

vitro. 232

233

IL-32 is induced by the NF-κB signaling pathway in AGS co-cultured with H. pylori. 234


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Progression of gastric disease is affected by the presence of H. pylori virulence factors, such as cagPAI. 235

We next examined the role of cagPAI on IL-32 expression using isogenic mutant strains of TN2. 236

Immunoblot analysis showed that IL-32 expression after infection with TN2-ΔcagPAI and TN2-ΔcagE 237

was significantly weaker than that induced by wild-type H. pylori (Fig. 3A), suggesting that these 238

virulence factors up-regulate IL-32 expression. 239

Since cagPAI-positive H. pylori exhibits a higher capacity to activate NF-κB and MAPK, especially 240

in epithelial cells (11, 16), we next investigated the involvement of these signaling pathways in the 241

expression of IL-32 in H. pylori–infected cells. When we examined the effects of the chemical inhibitors 242

of IKKβ (SC-514) and p38 (SB203580) on H. pylori–induced IL-32 expression, H. pylori–induced 243

IL-32 mRNA expression was significantly reduced in cells pre-incubated with SC-514, but not with 244

SB203580 (Fig. 3B). To confirm the involvement of IKKβ in H. pylori–induced IL-32 expression, 245

siRNA for IKKβ was transfected into cells. H. pylori–induced IL-32 levels were reduced by IKKβ 246

siRNA (Fig. 3C). These results indicate that IL-32 is induced via the NF-κB signaling pathway in AGS 247

cells infected with cagPAI-positive H. pylori. 248

249

IL-32 is intracellular in H. pylori–infected cells. 250

Next, we investigated IL-32 expression by gastric cells. First, we examined IL-32 levels in the 251

supernatant of H. pylori–infected AGS cells. While 24 hours infection with wild-type H. pylori typically 252


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produces 1000 pg/ml of IL-8 protein in AGS cell supernatants (data not shown), our ELISA results 253

showed that IL-32 concentrations were below the detection limit (< 16 pg/ml) in the same supernatant in 254

our ELISA. When we disrupted the cell membrane by freeze-thawing, IL-32 protein was detected in the 255

supernatant of AGS cells, suggesting an intracellular accumulation of IL-32 in H. pylori–infected cells 256

(Fig. 4A, left). We also examined intracellular protein levels in AGS cells with or without H. pylori 257

infection by ELISA. Lysates of uninfected AGS cells contained 800 pg/mg of IL-32. However, H. pylori 258

infection increased the intracellular IL-32 level significantly (Fig. 4A, right). 259

To address the localization of IL-32 in AGS cells, we performed immunofluorescence staining. As 260

shown in Fig. 4B, uninfected AGS cells contained low levels of IL-32 in the cytosol. H. pylori infection 261

increased both cytosolic and nuclear IL-32 levels, questioning whether IL-32 in gastric cells functions as 262

a typical cytokine. Thus, we examined whether extracellular IL-32 could activate intracellular signaling 263

in gastric epithelial cells, as it has been reported to do in macrophages (23). In contrast to TNF-α, 264

stimulation of AGS cells with recombinant IL-32β did not enhance IκBα, JNK or p38 phosphorylation in 265

AGS cells, suggesting that IL-32 has a specific intracellular function in gastric cells (Fig. 4C). 266

267

IL-32 expression up-regulates IL-8, CXCL1, CXCL2, and TNF-α expression. 268

Previous reports showed that IL-32 functions as a proinflammatory cytokine, which elicits production of 269

other inflammatory cytokines in diseases such as rheumatoid arthritis. Due to its intracellular 270


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localization in AGS cells, next we determined whether IL-32 regulates inflammatory cytokines. We 271

established two stable IL-32-knockdown AGS cell lines, AGS-IL32KD1 and AGS-IL32KD2, and 272

decreased IL-32 mRNA expression was confirmed by real-time RT-PCR in both (Fig. 5A, left). Next, we 273

examined whether IL-32 affects the expression of cytokines in H. pylori–infected AGS cells. As shown 274

in Fig. 5A (right), the expression of IL-8 mRNA was significantly decreased in both AGS-IL32KD1 and 275

AGS-IL32KD2 cells co-cultured with H. pylori, compared with the control cell line. We also measured 276

the expression of other cytokines that are induced by H. pylori (17, 28, 48). CXCL1, CXCL2, and 277

TNF-α mRNA levels were also decreased in the IL-32-knockdown cells (Fig. 5B). IL-11 mRNA levels 278

were not decreased in the IL-32-knockdown cells (Fig. 5B). ELISA analysis indicated that IL-8, CXCL1, 279

and CXCL2 levels in supernatants of AGS-IL32KD1 cells co-cultured with H. pylori were decreased 280

compared to the control cell line (Fig. 5C). The levels of TNF-α were below the detection limit of the 281

ELISA (< 16 pg/ml, data not shown). To confirm the role of IL-32 in regulation of the expression of 282

other cytokines, we established two stable AGS cell lines (AGS IL-32 clone15, and AGS IL-32 clone19) 283

that over-expressed IL-32. IL-8 mRNA levels were increased in these IL-32 over-expressing cells. In the 284

two IL-32 over-expressing lines, IL-8 levels were two-fold higher than that in control cells (Fig. 5D). 285

Next we asked whether the reduced IL-8 levels in IL-32-knockdown AGS cells could be rescued by 286

over-expressing IL-32 cDNA. We re-introduced a mutant IL-32 cDNA (pcDNA-mIL32β), which was 287

genetically modified to be resistant to the inhibitory effects of the siRNA, into AGS-IL32KD1 cells and 288


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examined IL-8 activity using reporter assays. Immunoblot analysis confirmed that IL-32 was suppressed 289

in AGS-IL32KD1, and could be rescued by over-expression of mIL-32 cDNA (Fig. 5E, upper right). The 290

suppression of H. pylori–induced IL-8 reporter activation in AGS-IL32KD1 was rescued by 291

over-expression of mIL-32 in AGS-IL32KD1 cells (Fig. 5E). These results suggest that IL-32 functions 292

as an intracellular regulator of cytokine expression in H. pylori–infected gastric cells. 293

294

IL-32 regulates NF-κB activation in H. pylori–infected AGS cells. 295

We examined whether IL-32 suppression impairs the type IV secretion system of H. pylori, which is 296

essential for induction of cytokine production in epithelial cells. CagA phosphorylation and the 297

subsequent hummingbird phenotype of infected AGS cells are the hallmark of the type IV secretion 298

system (43). Microscopic analyses indicated that both AGS-IL32KD1 and AGS control cells co-cultured 299

with H. pylori developed the hummingbird phenotype (Fig. 6A, upper). CagA was phosphorylated in 300

AGS-IL32KD1 and AGS control cells (Fig. 6A, lower), indicating that IL-32 expression does not affect 301

type IV secretion. 302

Next, we examined NF-κB activation in AGS IL-32 stable-knockdown cells. A luciferase reporter 303

assay indicated that NF-κB activation induced by H. pylori was decreased in AGS-IL32KD cells 304

compared with the control cell line (Fig. 6B). Immunoblot analysis showed that phosphorylation of IκBα 305

was decreased in AGS-IL32KD1 co-cultured with H. pylori, compared with the control cell line. This 306


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indicates that NF-κB signaling is defective in AGS-IL32KD1 cells (Fig. 6C). 307

Finally, we tested the contribution of down-regulation of IL-8 mRNA to the level of IL-32 mRNA. 308

The siRNA for IL-8 was transfected into AGS cell lines, and decreased IL-8 mRNA expression was 309

confirmed by real-time RT-PCR (Fig. 6D, left). H. pylori–induced IL-32 levels were not significantly 310

different in AGS transfected with scramble siRNA and AGS transfected with siRNA for IL-8 (Fig. 6D, 311

right). 312

These results indicate that intracellular IL-32 expression is associated with NF-κB activation and 313

subsequent cytokine production in gastric epithelial cells infected with H. pylori. 314

315

DISCUSSION 316

In this study we show that expression of IL-32 is elevated in human gastritis and gastric cancer tissue. 317

We also demonstrate that IL-32 is induced by H. pylori in vitro. In addition, our results indicate that 318

IL-32 expression activates NF-κB and induces cytokine production, including IL-8. These results 319

suggest that IL-32 is fundamental to the gastric inflammation caused by H. pylori. H. pylori induces 320

NF-κB activation in gastric epithelial cells in a cagPAI-dependent manner, which is required for IL-32 321

up-regulation. In turn, intracellular IL-32 amplifies NF-κB activity and cytokine production, which 322

accelerates the inflammatory responses of gastric tissue infected with H. pylori (Fig. 6D). 323

IL-32 has six described mRNA splice variants, named IL-32α, β, γ, δ, ε, and ζ (12, 23). In our 324


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analysis, IL-32β was the major isoform detected in the gastritis tissue. IL-32β is reportedly a major 325

isoform in endothelial cells, and it has a critical function in vascular inflammation and sepsis (25). The 326

longest IL-32 isoform, IL-32γ, was the most abundant in rheumatoid arthritis synovial tissue (14). 327

IL-32γ exerted the greatest induction of TNF-α in human peripheral blood mononuclear cells (4). 328

However, IL-32γ mRNA was not detected in gastritis tissue. A possible explanation for the differing 329

IL-32 isoform abundance among disease tissues may be that the splicing of IL-32 differs in different cell 330

types or due to varying inductive stimuli. The biological differences and the mechanisms of 331

tissue-specific expression of the IL-32 isoforms need to be investigated to understand the roles of this 332

cytokine in humans. 333

We also investigated the mechanisms of IL-32 induction by H. pylori in AGS cells. Previous reports 334

on IL-32 expression suggested the involvement of the JNK (33), p38 (24), ERK (37), and the NF-κB 335

(37) pathway. We found that cagPAI-positive H. pylori induced production of IL-32, in agreement with 336

the activation of NF-κB by cagPAI-positive H. pylori in the pathogenesis of gastric diseases (2, 11). As 337

expected, inhibition of the NF-κB pathway resulted in decreased IL-32 induction in the H. 338

pylori–infected gastric epithelial cell model. These results indicate that IL-32 is induced in gastric 339

epithelial cells through the activation of NF-κB by H. pylori infection. 340

To determine its role in gastritis, we examined the pattern of IL-32 expression in AGS cells, and 341

found it to be retained intracellularly. This is in contrast to most cytokines, which are generally secreted 342


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from cells. In our investigation, recombinant IL-32β, unlike TNF-α, failed to activate IκBα, JNK, and 343

p38 in AGS cells (Fig. 4C), which differed from the situation in macrophages (23). Furthermore, IL-32 344

was not detected by ELISA in supernatants of AGS cells co-cultured with H. pylori. Thus, autocrine 345

signaling activation by IL-32 in gastric cells seems unlikely. Instead, disruption of the cell membrane 346

increased the IL-32 concentration in the supernatant to detectable levels (Fig. 4A). These results are 347

consistent with previous reports that described intracellular IL-32 expression and leakage from apoptotic 348

cells (12, 13, 26). Of note, neutrophil proteinase 3, an IL-32 binding protein, that is expressed in 349

neutrophils and monocytes is highly expressed in H. pylori–related gastritis (18). IL-32 from damaged 350

epithelial cells may activate myeloid cells through an IL-32-neutrophil-proteinase 3-interaction to 351

enhance inflammation of gastric tissue. Although intracellular receptors for IL-32 has not been 352

discovered, some interleukins such as IL-1 and IL-33 is known to have intracellular targets and to play 353

roles as both intracellular and secreted mediators (27). Thus, IL-32 may act intracellularly in gastric 354

epithelial cells and act extracellularly on myeloid cells. 355

We found that IL-32 expression levels increased concurrent with disease progression in the gastric 356

mucosa. IL-32 staining was increased in inflamed lesions of chronic pancreatitis, and strongly positive 357

in pancreatic cancer cells, suggesting the involvement of this cytokine in pancreatic carcinogenesis (36). 358

However, the mechanism by which IL-32 expression modifies the inflammatory processes ultimately 359

leading to malignancy is largely unknown. Our results showing that H. pylori–induced NF-κB activation 360


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enhances IL-32 expression, and that IL-32 in turn amplifies the NF-κB signaling pathway, may 361

contribute to inflammation-induced carcinogenesis. Interestingly, when IL-32 was knocked down, 362

expression of the anti-apoptotic genes, Bcl-2 and Bcl-xL, in pancreatic cell lines was reduced (36); these 363

are regulated by the NF-κB pathway (20). Thus, it is plausible that IL-32 expression also amplifies 364

NF-κB signaling in pancreatic cancer cells. Taken together, our findings suggest that IL-32 is involved 365

in the inflammation-cancer process through amplification of NF-κB expression and up-regulation of its 366

targets, including IL-32 itself. 367

In conclusion, the inflammatory molecule IL-32 plays an important role in gastritis. H. pylori 368

infection induces IL-32 through NF-κB activation, in a cag-dependent manner. IL-32 amplifies the 369

NF-κB pathway and induces production of other inflammatory cytokines, including IL-8. IL-32 may be 370

a future target for medical treatment of gastric inflammation and carcinogenesis. 371

372

ACKNOWLEDGEMENTS 373

We thank Mitsuko Tsubouchi for technical assistance. We thank Dr. Takaaki Sano and Dr. Teiji 374

Motojima (Motojima General Hospital, Gumma, Japan) for provision of gastric carcinoma specimens. 375

This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of 376

Education, Culture, Sports, Science and Technology of Japan to Y. Hirata. 377

378


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556

FIGURE LEGENDS 557

FIG 1. Expression of IL-32 in human gastric tissues. 558


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(A) Immunoblot analysis of IL-32 protein in normal human gastric tissues, gastritis tissues, and gastric 559

cancer tissues. (B) Representative immunohistochemical analysis of IL-32 expression in human gastritis 560

tissue (right). Mouse IgG1 isotype antibody was the negative control (left). (C) Typical examples of 561

immunohistochemical analysis of IL-32 expression in normal human gastric tissue (left), gastritis tissue 562

(center), and gastric cancer tissue (right). (D) Graphic representation of IL-32 expression in human 563

gastric samples, including normal gastric mucosa, gastritis tissues and gastric cancer tissues. Expression 564

was quantified by ELISA. The box plot indicates the median (horizontal line), inter-quartile range (the 565

box itself), and the sample minimum and maximum (bars). *, p <0.05 by Tukey’s HSD. (E) The 566

full-length coding region of IL-32 cDNA, synthesized from RNA extracted from human gastritis tissue 567

and AGS cells infected with H. pylori, was amplified by PCR (left). The known structure of IL-32 568

isoforms (splice variants) and the number of each clone detected in human gastritis tissue are depicted 569

(right). The exon numbers are shown beneath the boxes. 570

FIG 2. H. pylori infection induces IL-32 expression in gastric cancer cells. 571

(A) AGS cells were infected with cagPAI-positive H. pylori strains TN2 for 24 h, or stimulated with 572

IL-1β, and TNF-α for 24 h at the indicated concentrations. IL-32 expression was detected by 573

immunoblot analysis. (B) AGS, TMK-1 and MKN45, gastric cancer cell lines, were co-cultured with H. 574

pylori for the indicated hours, and IL-32 expression was evaluated by immunoblot analysis. (C) IL-32 575


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and IL-8 mRNA levels in AGS cells after infection with H. pylori for six hours were determined by 576

real-time RT-PCR. Data are shown as means ± SEM (n=3). *, p <0.05 by Student’s t-test. 577

FIG 3. H. pylori infection induces IL-32 production in AGS cells via the NF-κB signaling pathway. 578

(A) Levels of Il-32 in AGS cells infected with wild-type H. pylori (TN2-WT), TN2-ΔcagPAI, and 579

TN2-ΔcagE for the indicated times were determined by immunoblot analysis. Anti-urease staining was 580

used to control for H. pylori strain variations. (B) H. pylori–induced IL-32 mRNA expression with or 581

without chemical inhibitors was examined by real-time RT-PCR. The IKKβ inhibitor SC-514 (SC) and 582

p38 inhibitor SB203580 (SB) were added at a concentration of 20 μM, one-hour before H. pylori 583

infection. Cells were co-cultured with H. pylori for 24 h. Data are the means ± SEM (n=3). *, p <0.05 by 584

Dunnett’s multiple comparison. NS indicates not significant. (C) H. pylori-induced IL-32 expression in 585

AGS cells treated with control siRNA and IKKβ siRNA as determined by immunoblot analysis. AGS 586

cells were transfected with the siRNAs for 48 h and then infected with H. pylori for the indicated times. 587

588

FIG 4. IL-32 is intracellular in H. pylori–infected cells. 589

(A) IL-32 concentrations in the supernatant were quantitated by ELISA. Supernatants were collected 590

from AGS cells infected with H. pylori for 24 h, and the same cells that had been freeze-thawed to 591

facilitate cell membrane destruction (Fig. 4A, left). IL-32 concentrations in AGS cell lysates with or 592


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without H. pylori infection for 24 h were determined by ELISA (Fig. 4A, right). Data are shown as 593

means ± SEM (n=3). * p <0.05 by Student’s t-test. (B) AGS cells were co-cultured with H. pylori for 24 594

h, and IL-32 expression was analyzed by immunofluorescence staining. Nuclear staining was conducted 595

using Hoechst 33342. (C) Immunoblots of the indicated proteins in AGS cells stimulated with TNF-α (1 596

ng/ml), and recombinant IL-32β (1 ng/ml) for 0, 15, 30, and 60 min. 597

FIG 5. IL-32 expression affects induction of other cytokines involved in gastritis. 598

(A) Control (Ctrl) and stable IL-32 knockdown AGS cell lines (IL32KD1 and IL32KD2) were cultured 599

with or without H. pylori for six hours. IL-32β mRNA and IL-8 mRNA levels were determined by 600

real-time RT-PCR. Data are shown as means ± SEM (n=3). *, p <0.05 by Dunnett’s multiple comparison. 601

(B) CXCL1, CXCL2, TNF-α and IL-11 mRNA levels were evaluated in AGS control cells (Ctrl) and 602

IL32KD1 cells cultured with or without H. pylori for six hours by real-time RT-PCR. Data are shown as 603

means ± SEM (n=3). *, p <0.05 by Student’s t-test. NS indicates not significant. (C) ELISA of IL-8, 604

CXCL1, and CXCL2 expression in AGS cell supernatants of control (Ctrl) and IL32KD1 cells cultured 605

with or without H. pylori for 24 h. Data are shown as means ± SEM (n=3). *, p <0.05 by Student’s t-test. 606

(D) Two stable IL-32-overexpressing AGS cell lines (AGS IL-32 clone 15, and AGS IL-32 clone 19) 607

were established. The increased IL-32 mRNA level was confirmed by real-time RT-PCR (left). IL-8 608

mRNA levels in the control (Ctrl), IL-32 clone 15, and IL-32 clone 19 cells were determined by 609


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real-time RT-PCR (right). Data are shown as means ± SEM (n=3). *, p <0.05 by Dunnett’s multiple 610

comparison. (E) IL-8 reporter activity was examined in AGS control (Ctrl), IL32KD1, and IL32KD1 611

cells transfected with IL-32β cDNA (pcDNA-mIL32β). Cells were left uninfected or infected with H. 612

pylori for 24 h. Data are shown as means ± SEM (n=3). *, p <0.05 by Student’s t-test. IL-32 levels in 613

each cell are shown in the upper right panel. 614

615

FIG 6. Role of IL-32 in H. pylori–induced cell signaling. 616

(A) Micrographs of uninfected AGS cells transfected with the non-silencing control (AGS Ctrl), AGS 617

Ctrl cells infected with H. pylori, and stable IL-32 knockdown AGS (AGS-IL32KD1) cells infected with 618

H. pylori for 24 h (Fig. 6A, upper). Cell lysates of uninfected AGS Ctrl cells, AGS Ctrl cells infected 619

with H. pylori, and AGS-IL32KD1 cells infected with H. pylori for 24 h were immunoprecipitated using 620

anti-CagA antibody and the immunoprecipitates were immunoblotted using anti-phosphotyrosine and 621

anti-CagA antibodies (Fig. 6A, lower). (B) NF-κB activation induced by 24-h H. pylori infection of AGS 622

Ctrl cells and AGS-IL32KD1 was determined using luciferase activity assays. Data are shown as means 623

± SEM (n=3). *, p <0.05 by Student’s t-test. (C) Immunoblot analysis of IL-32 and pIκBα levels in AGS 624

Ctrl cells and AGS-IL32KD1 cells infected with H. pylori for the indicated times. Anti-urease staining 625

was used to control the H. pylori-load. (D) H. pylori-induced IL-32β mRNA (Fig. 6D right) and IL-8 626

mRNA (Fig. 6D left) levels in AGS cells treated with control siRNA (AGS Ctrl) and IL-8 siRNA 627


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(AGS-siIL8) as determined by real-time RT-PCR. AGS cells were transfected with the siRNAs for 48 h 628

and then infected with H. pylori for an additional 24 h. Data are shown as means ± SEM (n=3). *, p 629

<0.05 by Student’s t-test. NS indicates not significant. (E) Summary of the role of IL-32 in gastritis. H. 630

pylori induces NF-κB activation in gastric epithelial cells in a cagPAI-dependent manner. NF-κB 631

activation is required for IL-32 expression. Intracellular IL-32 expression amplifies both NF-κB 632

activation and IL-8 expression. 633

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IAI Accepts, published online ahead of print on 13 August...

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