CD74 and Stimulates Procarcinogenic Events Produced by Gastric

Helicobacter pylori CagA-Dependent Macrophage Migration Inhibitory Factor Produced by Gastric Epithelial Cells Binds to CD74 and Stimulates Procarcinogenic Events This information is current as of September 28, 2021. Ellen J. Beswick, Irina V. Pinchuk, Giovanni Suarez, Johanna C. Sierra and Victor E. Reyes J Immunol 2006; 176:6794-6801; ; doi: 10.4049/jimmunol.176.11.6794 http://www.jimmunol.org/content/176/11/6794 Downloaded from

References This article cites 52 articles, 17 of which you can access for free at:

http://www.jimmunol.org/content/176/11/6794.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Helicobacter pylori CagA-Dependent Macrophage Migration Inhibitory Factor Produced by Gastric Epithelial Cells Binds to CD74 and Stimulates Procarcinogenic Events1

Ellen J. Beswick,* Irina V. Pinchuk,*† Giovanni Suarez,* Johanna C. Sierra,* and Victor E. Reyes2*‡

Macrophage migration inhibitory factor (MIF) is a proinﬂammatory cytokine that has recently been implicated in carcinogenesis. Helicobacter pylori, which is closely linked to gastric cancer, induces the gastric epithelium to produce proinﬂammatory cytokines, including MIF. MIF can bind to CD74, which we have previously shown to be highly expressed on the surface of gastric epithelial cells (GEC) during H. pylori infection. In this study, we sought to investigate the role of the H. pylori-induced MIF on epithelial

proliferation and procarcinogenic events. Upon establishing a role for the H. pylori CagA virulence factor in MIF production, MIF Downloaded from binding to CD74 on GEC was conﬁrmed. rMIF and H. pylori were shown to increase GEC proliferation, which was decreased when cagA؊ strains were used and when CD74 was blocked by mAbs. Apoptosis was also decreased by MIF, but increased by cagA؊ strains that induced much lower amounts of MIF than the wild-type bacteria. Furthermore, MIF binding to CD74 was also shown to decrease p53 phosphorylation and up-regulate Bcl-2 expression. This data describes a novel system in which an H. pylori virulence factor contributes to the production of a host factor that in turn up-regulates procarcinogenic events by the gastric

epithelium. The Journal of Immunology, 2006, 176: 6794–6801. http://www.jimmunol.org/

elicobacter pylori is a common human pathogen that tyrosine phosphorylation-dependent manner and stimulates phos- infects over 50% of the world’s population. Infection phatase activity (8, 9). This activity induces various cell responses H with this organism is a major cause of chronic gastritis, that result in cytoskeleton rearrangement leading to a cell scatter- gastric and duodenal ulcers, and gastric carcinomas throughout the ing/hummingbird phenotype, and cell-to-cell disassociation that world (1, 2). Pathogenesis of infection often includes inflamma- may result in disruption of the epithelial barrier integrity. ERK1/2 tion, mucosal damage, or gastric atrophy, and requires close asso- activation is also suggested to be induced after CagA is phosphor- ciation of the bacteria with the gastric epithelium, leading to ylated (10), and may also lead to increased inflammatory responses by guest on September 28, 2021 chronic epithelial responses. There is now considerable evidence associated with H. pylori infection (11, 12). Another important linking this pathogen to changes in the gastric mucosa that con- observation about CagAϩ H. pylori infections is an increase in tribute to gastric cancer (3, 4). Gastric cancer is the second leading gastric cell proliferation (13, 14), which has been observed both in cause of mortalities due to cancer in the world, is often undetect- vivo and in vitro. The mechanisms leading to this increase in pro- able in the early stages, and has an overall survival rate of only liferation remain unknown. 10–20% (5). Links between chronic inflammation and carcinogen- H. pylori has recently been shown to induce production of mac- esis may play an important role in gastric cancer, but are not well rophage migration inhibitory factor (MIF) by several cell types understood. (15). MIF is a versatile cytokine that mediates both innate and The main virulence factor of H. pylori that has been suggested adaptive immunity and plays a role in chronic inflammation. It has to induce procarcinogenic events in gastric epithelial cells (GECs)3 been shown to up-regulate NF-␬B, ERK1/2, AP-1, and protein is the cytotoxin-associated gene A product, CagA protein (6, 7). kinase C pathways leading to increased expression of IL-1␤ and CagA is injected into cells by H. pylori’s type IV secretion system. IL-8 mRNA (16, 17). Also, it is important to consider the role that Once CagA is injected into the cell, it specifically binds the Src MIF plays in cell growth and procarcinogenic signaling. It is homology 2-containing protein tyrosine phosphatase, SHP-2, in a thought to promote tumor growth and viability by modulating immune responses and supporting tumor-associated angiogenesis

*Department of Pediatrics, †Department of Internal Medicine, and ‡Department of (18, 19). The central mechanisms MIF affects to increase cell pro- Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX liferation and decrease apoptosis are p53 inhibition and reduction 77555 of oxidative stress-induced apoptosis (20, 21). The inhibition of Received for publication January 10, 2006. Accepted for publication March 22, 2006. p53 appears to result in a more robust inﬂammatory response, The costs of publication of this article were defrayed in part by the payment of page which may also explain its importance in chronic inﬂammation. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. CD74, or the class II MHC-associated invariant chain, can act as 1 This work was supported by the National Institutes of Health Grants DK50669 and a receptor for MIF (22). CD74 is known for its role in the regu- DK56338. E.J.B. was a recipient of a fellowship under National Institutes of Health lation of intracellular transport and Ag-presenting functions of Training Grant T32 AI007536-06. class II MHC molecules. Recent studies have shown that CD74 is 2 Address correspondence and reprint requests to Dr. Victor E. Reyes, Children’s not only expressed intracellularly, but also on a variety of cell Hospital, Room 2.300, University of Texas Medical Branch, 301 University Boule- vard, Galveston, TX 77555. E-mail address: [email protected] surfaces, including GECs (23, 24). We and others have shown that ␬ 3 Abbreviations used in this paper: GEC, gastric epithelial cell; MIF, macrophage bound CD74 can induce signaling events leading to NF- B acti- migration inhibitory factor. vation (25, 26). CD74 may also play a role in cancer. Cell surface

expression of CD74 was shown to be up-regulated during gastric cells for1hat4°Ctoprevent internalization. Some samples were incu- cancer when tumor cells were examined by immunohistochemical bated with the anti-CD74 mAb, MB-741 (BD Biosciences) for 1 h before staining (27). Patients with low CD74 expression were shown to exposure to MIF. After washing with PBS, samples were incubated with 1 ␮l of streptavidin-PE (BD Biosciences) for 30 min on ice. Samples incu- have better outcomes than those with high CD74 expression. In bated with only streptavidin-PE were used as the negative controls. Sam- more general cancer immunotherapy studies, antisense suppression ples were washed two more times and analyzed by flow cytometry on a of CD74 has been demonstrated to be an effective therapeutic treat- FACScan cytometer (BD Biosciences). ment in several studies (28, 29). Proliferation assay This study investigated GEC production of MIF in response to H. pylori. This response was shown to be CagA dependent. MIF CellTiter 96 Non-Radioactive Cell Proliferation kit (Promega) was used to measure cell proliferation. Cells were cultured in a 96-well plate overnight was shown to bind to CD74 in this system and increase cell proin serum-free medium at 1 ϫ 104 cells/well. A standard curve was estab- liferation that could be blocked by anti-CD74 mAbs. Cell prolif- lished using wells seeded with 1/2 dilutions of cells starting from 5 ϫ 103 eration was not increased by CagA knockout strains, but apoptosis to 6.4 ϫ 105. Cells were treated with 0.1, 1.0, or 10 ng/ml MIF or 1:1, 10:1, rates of GECs were increased to levels that were higher than those or 100:1 bacteria:cell ratio of wild-type and cag PAI or cagA knockout strains for 24 h. Some cells were pretreated with anti-CD74 clone MB-748 of cells treated with wild-type H. pylori. Phosphorylated p53 was (BD Biosciences) or isotype control Abs before the addition of MIF. For a increased by CagA-deficient strains, but not by wild-type strains, positive control, 1 ng/ml PMA was added to cells for 24 h. Cells were then and this increase was abrogated when rMIF was added to cell washed with medium to remove bacteria, and the dye solution added ac- cultures suggesting that MIF induced by H. pylori infection de- cording to manufacturer’s instructions for1hat37°C. After addition of creases phosphorylation of p53, and increases GEC proliferation. stop solution, OD was read at 490 nm using an ELISA plate reader. Cell numbers were calculated using the standard curve. These observations strongly suggest a link between MIF, a proin- Downloaded from flammatory cytokine, and procarcinogenic events. Apoptosis Cell Death Detection ELISA (Roche) kits were used to measure cell death. Materials and Methods Cells were treated with wild-type and cagA-deficient knockout strains us- Cell lines ing the same concentrations and time intervals that were used for proliferation assays. rMIF was added to some wells before cagA knockouts. For The GEC line N87 (CRL-5822) was obtained from the American Tissue wild-type strains, anti-MIF neutralizing Abs were added to some wells and Culture Collection (ATCC) and maintained in RPMI 1640 medium sup- isotype controls were used as well. Cells were lysed according to the man- http://www.jimmunol.org/ plemented with 10% FCS and 1 mM glutamine. The nontransformed fetal ufacturer’s instructions, lysates were used for ELISA, and OD was mea- gastric cell line HS-738 (CRL-7869) was also obtained from ATCC and sured at 405 nm. maintained in DMEM with high glucose, 10% FCS, and 1 mM glutamine. p53 phosphorylation Bacterial cultures Total and phosphorylated p53 levels were measured by intracellular bead H. pylori strains 26695, a strain isolated from a patient with gastritis in the arrays (BioSource International). Beads conjugated with mAbs for p53 United Kingdom, and 51B, a strain isolated from a patient with gastritis in were prepared according to manufacturer’s instructions and incubated with Japan, and the cagA isogenic mutants were obtained from Dr. Y. Yamaoka lysates from GECs treated with H. pylori strains 26695, 51B, or their (Baylor College of Medicine, Houston, TX). These autonomous knockouts CagA-deficient mutants for 24 h. Some samples were pretreated with rMIF

were prepared as previously described (30). All strains were grown on blood (10 ng/ml) before the addition of H. pylori. After washing, samples were by guest on September 28, 2021 agar plates (BD Biosciences) at 37°C under microaerophilic conditions, as run on a Bio-Plex System and analyzed using Bio-Plex Manager software previously described (31). Bacteria were transferred after 48 h into Brucella (Bio-Rad). broth containing 10% FBS for 24 h. After centrifugation at 2500 ϫ g for 10 Phosphorylated p53 was also detected by Western blotting. H. pylori min, bacteria were resuspended in sterile PBS. The concentration of bacteria strains or rMIF was incubated with GECs for 24 h. Cells were lysed as ϫ per milliliter was determined by measuring the OD530 using a spectrophotom- described previously (32). Lysates were centrifuged at 14,000 g for 10 eter (DU-65; BD Biosciences) and comparing the value to a standard curve min at 4°C. The resulting protein (5 ␮g) from each sample were loaded into generated by quantifying viable organisms from aliquots of bacteria at varying wells of 10% SDS gels for electrophoresis, and transferred to nitrocellulose concentrations that were also assessed by OD. membranes for Western blotting. The membranes were treated with incubated with anti-phospho (S15) p53 (R&D Systems), and anti-␤-actin MIF ELISA (Sigma-Aldrich) as a loading control. After washing four times for 15 min with TBS Tween, donkey anti-mouse HRP-conjugated secondary Ab H. pylori strains were incubated with GECs in a 100:1 bacteria:cell ratio in (Santa Cruz Biotechnology) was used diluted 1/10,000 and incubated with 24-well plates for 8-h time intervals up to 32 h. Supernatants from wells the membrane for1hatroom temperature. The membrane was again were used to quantitate the production of MIF by ELISA. The MIF ELISA washed four times for 15 min. Immunoreactive proteins were detected kit was obtained from R&D Systems, and assays were performed according using Super Signal West Pico Chemiluminescent Substrate (Pierce to the manufacturer’s instructions. Biotechnologies). MIF binding to CD74 Flow cytometry rMIF (R&D Systems), 1 ng, was added to GEC lysates, and rotated for 2 h For flow cytometry analysis of Bcl-2 and activated caspase-3, cells were ␮ at 4°C. Lysate mixtures were precleared with 10 l of protein A/G beads lysed using BD Cytofix/Cytoperm (BD Biosciences) according to the man- (Santa Cruz Biotechnology) for2hat4°C. MIF was immunoprecipitated ufacturer’s instructions. Anti-Bcl-2 (BD Biosciences) or anti-caspase-3 ac- using protein A/G beads that were preincubated with anti-MIF mAb (R&D tive form (BD Biosciences), or isotype control (BD Biosciences) PE-con- Systems) for2hatroom temperature. After washing, beads were incubated jugated Abs for1honice. Cells were washed with PBS and analyzed on with the lysate mixture of MIF and GEC lysates. Beads were then washed the FACScan Flow Cytometer (BD Biosciences) using CellQuest software. four times and the bound material was eluted, samples were run on SDS PAGE, and transferred to a nitrocellulose membrane for Western blotting. Statistical analysis The membrane was incubated with the anti-CD74 Ab clone Pin.1 (Stress- Ϯ Gen Bioreagents at a 1/3,000 dilution for2hatroom temperature. After Results are expressed as mean SEM. MIF production and cell turnover rates were compared using an ANOVA analysis of the variance and con- washing four times for 15 min with TBS Tween 20, donkey anti-mouse Ͻ HRP-conjugated secondary Ab (Santa Cruz Biotechnology) was used di- sidered significant if p 0.05. luted 1/10,000 and incubated with the membrane for1hatroom temperature. The membrane was again washed four times for 15 min. Immuno- Results reactive proteins were detected using Super Signal West Pico H. pylori induces MIF production by GECs in a Chemiluminescent Substrate (Pierce Biotechnologies). CagA-dependent manner For flow cytometry, MIF was biotinylated using the Fluoreporter Bioti- nylation kit (Molecular Probes) according to manufacturer’s instructions. MIF has recently been suggested to play an important role in both Biotinylated MIF (10 ng) was incubated with 2 ϫ 105 GECs or fibroblast innate and adaptive immunity and the dysregulation of cell cycle 6796 H. pylori-INDUCED MIF STIMULATES GEC PROLIFERATION events in carcinogenesis. Because these factors are important dur- MIF binds to CD74 on GECs ing H. pylori infection, we sought to determine MIF production by CD74 has been suggested to act as a receptor for MIF in several GECs in response to H. pylori. Furthermore, because the cag studies (22, 33). We have shown that GECs express large amounts pathogenicity island injection of CagA into cells plays such an of CD74 (24, 25), which is up-regulated during inflammatory con- important role in many GEC responses induced by H. pylori, ditions and H. pylori infection (34). Consequently, we examined cagA-deficient strains were compared with wild-type strains of H. the role of CD74 as a receptor for MIF on GECs by immunopre- pylori at a moderate infection of 10:1 bacteria:cell ratio. Fig. 1, A cipitation and Western blot analysis. rMIF was incubated with N87 and B, indicate that H. pylori strains 26695 and 51B induced MIF cell lysates. MIF was immunoprecipitated along with GEC pro- production by the HS-738 nontransformed fetal gastric cell line teins bound to it. Western blot analysis using anti-CD74 mAbs and the N87 gastric carcinoma cell line, respectively. Increases in revealed that CD74 was coprecipitated with MIF in samples and in MIF production were detected as early as 8 h postinfection and control samples immunoprecipitated with anti-CD74 as indicated continued to increase up to 32 h after infection. When the CagA- by the presence of a 33-kDa band (Fig. 2A). deficient strains were used at 24 h, the induction of MIF was just Attachment of MIF to CD74 was also analyzed by flow cytom- slightly above basal levels produced by both cell lines (Fig. 1, C ϩ etry. Biotinylated MIF was incubated with GECs and stained with and D), whereas the wild-type (CagA ) induced a robust MIF streptavidin-PE for analysis. As shown in Fig. 2B, MIF attached to response, suggesting that MIF production by GECs is dependent cells, but this attachment was dramatically decreased upon incu- on CagA injection into the cells. bating cells with anti-CD74 before the addition of MIF. These studies suggest that CD74 is the primary receptor for MIF on GEC surfaces because blocking CD74 led to very little attachment of Downloaded from MIF to the cells.

H. pylori-induced MIF increases GEC proliferation Various reports have shown that MIF increases proliferation of some cell types. In this study, we investigated the ability of MIF to induce proliferation of GECs. rMIF or H. pylori strains and their http://www.jimmunol.org/ respective CagA-deﬁcient knockout strains were incubated with cells for 24 h. Proliferation was measured by nonradioactive cell proliferation colorimetric assay, as used in multiple recent studies (14, 33). Standard curves of known amounts of cells were run with each assay to extrapolate cell number from treated samples. As seen in Fig. 3A, GEC proliferation was increased starting at 0.1 ng/ml rMIF, which was in the range of that produced by GECs in

response to H. pylori, and increased further with each 10-fold in- by guest on September 28, 2021 crease in MIF concentration. To investigate the role of CD74 in the

FIGURE 2. MIF binds to CD74 on GECs. A, rMIF was mixed with N87 cell lysates and immunoprecipitated with anti-MIF Abs with bound cell FIGURE 1. GECs produce MIF in response to H. pylori. MIF produc- proteins. Western blot analysis with anti-CD74 revealed the presence of tion by (A) HS-738 and (B) N87 GECs after exposure to H. pylori strains CD74. N87 lysates were run as a control in the center lane, and N87 cell 51B or 26695 for various time points assayed by ELISA. MIF levels were lysates immunoprecipitated with isotype control Abs were run in the left decreased when (C) HS-738 and (D) N87 cells where treated with CagA- lane. B, Biotinylated rMIF was incubated with HS-738 cells and strepta- deﬁcient mutants compared with wild-type H. pylori at 24 h. The means are vidin-PE for ﬂow cytometry. Anti-CD74 Abs blocked attachment to cells shown as the results of duplicates in four experiments, n ϭ 8. compared with the solid peak control. The Journal of Immunology 6797

observed proliferation, anti-CD74 blocking Abs were incubated with cells 30 min before addition of rMIF or H. pylori strains. CD74 blocking led to considerably decreased proliferation, at least 2-fold with high concentrations of MIF, than unblocked samples. Notably, after anti-CD74 treatment, proliferation levels were decreased to levels similar to those of untreated cells. To further investigate the role of MIF in H. pylori-induced GEC proliferation, wild-type and CagA-deficient strains were incubated with cells in ratios of 1:1, 10:1, and 100:1 bacteria:cells. Fig. 3B demonstrates that H. pylori strain 26695 increases GEC proliferation at bacterial ratios of 1:1 and 10:1, but at 100:1 the cell number is decreased compared with 10:1, suggesting the cells are over- whelmed by bacteria and began to undergo apoptosis at higher concentrations. Based on these results, wild-type strains and CagA-deficient mutants were investigated at a 10:1 bacteria:cell ratio. Because the ability of H. pylori to induce MIF appeared to depend on cagA expression, to examine the effect of CagA on cell proliferation, CagA-deficient strains were used in these assays in Downloaded from comparison to wild-type strains. As shown in Fig. 3C, CagA-deficient mutants induce proliferation to a lesser degree than wild- type strains. When anti-MIF neutralizing Abs were added to wells before the addition of wild-type H. pylori, proliferation was decreased by Ͼ50% (Fig. 3D), further suggesting the role of MIF in H. pylori-induced cell proliferation. http://www.jimmunol.org/

H. pylori-induced MIF decreases GEC apoptosis Because H. pylori is known to induce both cell proliferation and apoptosis (31, 35), apoptosis of GECs with wild-type H. pylori and CagA-deficient strains, which induce very little MIF, were investigated for their ability to induce apoptosis of GECs. Apoptosis was measured by Cell Death Detection ELISA for histone-associated DNA fragmentation (nucleosome fragment formation) at 24 by guest on September 28, 2021 and 48 h. Fig. 4A indicates the apoptosis index of HS-738 cells in the presence of H. pylori 26695 in 1:1, 10:1, and 100:1 bacteria: cell ratios. Although apoptosis rates with the 10:1 bacteria:cell ratio was only slightly increased over that of 1:1, a much larger increase in apoptosis was observed with 100:1 bacteria:cell ratio, which parallels the decrease in proliferation seen with 100:1 in Fig. 3B. When the 26695 CagA-deficient mutant strain was examined for its ability to induce GEC apoptosis, cell death was double of that induced by the wild-type, parental strain. Similar results were seen with strain 51B and its corresponding CagA-deficient mutant (data not shown here). To determine a role for MIF in the decreased apoptosis rates seen with wild-type H. pylori, rMIF (10 ng/ml) was added to cultures incubated with CagA-deficient strains in amounts equivalent to those produced during H. pylori 100:1 bacteria:cells (Fig. 1). Addition of MIF to cultures with CagA-deficient mutants resulted in a decrease in apoptosis (Fig. 4B), similar to that seen with wild-type 26695 in Fig. 4A, demon- FIGURE 3. MIF induces GEC proliferation. HS-738 and N87 cells were strating an important role for MIF in decreasing H. pylori-induced treated with (A) increasing concentrations of rMIF that led to increased pro- apoptosis. p Ͻ 0.05), which is blocked using anti-CD74 compared with To further investigate the effect of MIF on apoptosis, expression ,ء) liferation Ͻ ءء unblocked cells ( , p 0.05) (B) increasing ratios of H. pylori strain 26695 of the activated form of caspase-3 was measured in GECs after where all conditions showed significant increases in cell number (p Ͻ 0.05), exposure to 100:1 bacteria:cell ratio. Some cells were pretreated (C) H. pylori strains 26695 and 51B increase proliferation compared with p Ͻ 0.05), and their CagA-deficient knockouts led to de- with 1 ng/ml rMIF for 24 h before exposure to H. pylori strains. As ,ء) untreated cells p Ͻ 0.05) at 10 bacteria/ seen in Fig. 4C, H. pylori induced cells to produce some active ,ءء) creased cell numbers compared with wild types cell and (D) anti-CD74 blocking Abs and anti-MIF neutralizing Abs decreased caspase-3, which was produced at a higher rate with the CagA- p Ͻ 0.05). Cell proliferation deficient bacteria than the wild type. When cells were pretreated ,ء) proliferation compared with unblocked cells was measured by CellTiter 96 Non-Radioactive Cell Proliferation kit and com- with MIF before CagAϪ bacteria, the levels of active caspase-3 pared with a standard curve of known amounts of cells. The means are shown were decreased (Fig. 4D), suggesting that MIF can down-regulate ϭ as the results of duplicates in four experiments, n 8. apoptotic events. 6798 H. pylori-INDUCED MIF STIMULATES GEC PROLIFERATION

H. pylori-induced MIF decreases phosphorylation of p53 MIF has been shown to decrease p53 expression and phosphorylation in several studies (36, 37). Because CagA has been shown here to induce MIF production by GECs, the role of rMIF and CagA-induced MIF in the expression and phosphorylation of p53 was investigated. The levels of total p53 and phosphorylated p53 (S15) were also measured by intracellular bead arrays. The units of phosphorylation per nanogram of total p53 were calculated as rec- ommended by the manufacturer. Fig. 5A illustrates that the level of phosphorylated p53 detected in cell treated with the CagA-deficient bacteria was more than double that of cells treated with the wild type, which was consistent with both strains tested. When rMIF was added to cells in similar amounts to that produced when cells were incubated with wild-type H. pylori before CagAϪ strains, the level of phospho-p53 was decreased to similar levels seen in cells treated with wild-type bacteria. To further verify these findings, Western blot analysis was performed with cell samples treated under the same conditions. Similarly, treating cells with CagA-deficient 26695 resulted in higher levels of phospho-p53 Downloaded from than those induced in cells treated with wild-type 26695 (Fig. 5B). Similar results were seen with N87 cells and H. pylori strain 51B, but are not shown here. When rMIF was added, the levels of phosphorylated p53 decreased. These results, along with other studies suggesting a role for CagA in gastric cancer, and studies suggesting that MIF inhibits p53 function (36), indicate that MIF may play http://www.jimmunol.org/ an important role in regulating p53 during H. pylori infections.

H. pylori-induced MIF induces Bcl-2 expression Because Bcl-2 expression is up-regulated during H. pylori infection and gastric atrophy and cancer (38, 39), and down-regulated in by guest on September 28, 2021

FIGURE 4. MIF decreases GEC apoptosis. HS-738 cells incubated with H. pylori 24 and 48 h were examined for apoptosis by histone-associated DNA fragmentation where (A) strain 26695 incubated with cells resulted in less DNA fragmentation than the CagA-deficient knockout (p Ͻ 0.05) and (B) CagA knockout DNA fragmentation was decreased with addition of rMIF (p Ͻ 0.05). The means are shown as the results of duplicates in four experiments, FIGURE 5. p53 phosphorylation induction by H. pylori. Phosphoryla- n ϭ 8. Active caspase-3 was measured by flow cytometry as shown in example tion of p53 is shown with HS-738 cells by (A) intracellular bead array and figures where (C) H. pylori strain 26695 induces some expression, which is (B) Western blotting where p53 phosphorylation is increased by H. pylori p Ͻ 0.05), but ,ء) increased with the CagA- strain and (D) reduced when rMIF is incubated with strain 26695 and 51B compared with untreated control p Ͻ ,ءء) cells before infection in example figures compared with the isotype control increased more with the CagAϪ strain compared with 26695 .(p Ͻ 0.05 ,ءءء) shown with a solid peak when compared with solid peak isotype control. 0.05), but decreased in the presence of rMIF The Journal of Immunology 6799

conditions, and during H. pylori infection (34). This is especially significant in this system because H. pylori can also use CD74 as a point of attachment to GECs (25). The dramatic increase in CD74 expression during infection and the high turnover rate of CD74 (22) suggests that both MIF and H. pylori can use CD74 as a receptor in this system. MIF binding to CD74 has been shown to increase proliferation of different cells types (22, 45, 46). Simi- larly, rMIF induced proliferation of GECs here in a dose-dependent fashion, and at comparable amounts to what is produced during H. pylori infection. Concurrently, H. pylori infection also led to increased proliferation, which was decreased when CagA knockout strains were used. Proliferation was also decreased when CD74 was blocked with Abs or MIF was neutralized by Abs in the medium. Under the same conditions, apoptosis of cells was decreased in the presence of MIF, and cagA knockout strains, where MIF production is much lower, induced greater levels of apoptosis than wild-type strains. rMIF added to cultures with the cagA knockout strains brought apoptosis levels back down suggesting that the effects MIF has on the cells may down-regulate apoptosis. Downloaded from The results of our studies agree with several other independent studies, including those by Peek et al. and Rokkas et al. (13, 14) using biopsies which showed that cagϩ strains increase GEC proliferation without a parallel increase in apoptosis. However, a sep- arate study by Moss et al. (47) disagreed with these and suggested that there is not a deficiency in apoptosis with cagAϩ strains, but http://www.jimmunol.org/ FIGURE 6. Bcl-2 expression by GECs. Bcl-2 staining in GECs is ex- Ϫ the authors of this study also proposed that host factors may be pressed (A) at higher levels with 26695 infection than with CagA infec- more important than bacterial factors in determining the outcome tion, but (B) increased when rMIF is incubated with cells in an example of infection. Our studies agree that a host factor, MIF, may be figure of HS-738 cells compared with the solid peak isotype control. crucial in shifting the balance of proliferation/apoptosis to one that is more positive for proliferation. Concurrent with the decrease in apoptosis attributed to MIF, apoptotic cells (40), the expression of Bcl-2 was investigated during infection with H. pylori and upon pretreatment with rMIF. MIF may inhibit p53 function (36, 37), which is considered an Flow cytometric analysis revealed that Bcl-2 expression is induced important tumor suppressor because it is an important factor in by guest on September 28, 2021 in GECs upon exposure to H. pylori, but is decreased when cells apoptotic pathways. As for the effect of H. pylori on p53, there is were exposed to CagA-deficient strains in comparison to wild-type conflicting information available. Several studies suggest there are strains (Fig. 6A). Incubation of samples with rMIF alone also in- no changes in p53 levels of the gastric mucosa during infection duced expression of Bcl-2 (Fig. 6B). These results indicate that (48, 49), while others suggest that p53 levels are increased during MIF may also play an important role in the induction of Bcl-2 infection (50, 51). However, these studies only looked at total p53 expression seen in GECs during H. pylori infection. present, but failed to investigate levels of phosphorylated p53. In this study, we measured the amount of phosphorylated p53 per Discussion nanogram of total p53. We found that the total amount of p53 did Proinflammatory cytokines such as MIF and IL-8 have been at- not fluctuate much during infection, but there were changes in tributed to increased proliferative signaling in a variety of systems phosphorylated p53. Although wild-type H. pylori did induce (20, 33, 41, 42). MIF has been shown to play an important role in some phosphorylation of p53, CagA-deficient strains induced more the regulation of proinflammatory immune responses in inflamma- than double the levels. The addition of rMIF decreased these levels tory disorders, and proliferative responses in multiple cancer types. to levels similar to those induced by the wild-type bacteria. Based MIF has recently been shown to be produced in response to H. on these studies, MIF appears to have the ability to decrease p53 pylori by multiple cell types (15). Because CagA has been shown phosphorylation, which may elucidate how it decreases apoptosis. to induce inflammatory responses (11, 12), and it has been asso- There are certainly bacterial factors present that induce some p53 ciated with gastric cancer (43, 44), the role of CagA in MIF pro- phosphorylation, as seen with wild-type H. pylori, but MIF appears duction by GECs was investigated in this study. By using CagA- to decrease the overall amount of phosphorylation, and apoptosis deficient knockout strains, we established that CagA is responsible seen. This is especially evident with cells infected with CagA- for MIF production by GECs because while wild-type strains in- deficient strains along with rMIF. Because H. pylori has also been duced MIF production, very little MIF was present in cultures proposed to mutate p53, the possibility that MIF may be involved treated with CagA knockout strains. should also be investigated. GECs express CD74 and this expression increases during infec- To further examine the role for MIF in the regulation of apo- tion with H. pylori both in vivo and in vitro (24, 34). CD74 was ptosis and increased proliferation, Bcl-2 expression by infected recently shown to be used by MIF as a receptor (22). Conse- GECs was investigated. Bcl-2 regulates apoptotic processes, and quently, MIF binding to CD74 on GECs was examined here, and can lead to apoptotic resistance when highly expressed. This pro- found to be a major receptor for MIF in this system as well. Similar tein has been shown to be up-regulated during H. pylori infection to MIF, CD74 has been shown to be up-regulated in cancer studies, with cagϩ strains (52) and H. pylori-associated gastric cancers (38, including gastric cancer (27). In a previous study, we also found 39). In this study, Bcl-2 levels were expressed at much higher that CD74 surface expression is increased during inflammatory levels in GECs with wild-type strains than with CagA-deficient 6800 H. pylori-INDUCED MIF STIMULATES GEC PROLIFERATION strains. rMIF was also able to increase expression further suggest- 19. Nishihira, J., T. Ishibashi, T. Fukushima, B. Sun, Y. Sato, and S. Todo. 2003. ing the role for MIF in driving cells toward a carcinogenic Macrophage migration inhibitory factor (MIF): its potential role in tumor growth and tumor-associated angiogenesis. Ann. NY Acad. Sci. 995: 171–182. phenotype. 20. Mitchell, R. A., C. N. Metz, T. Peng, and R. Bucala. 1999. Sustained mitogen- The data presented here are the first to suggest a crucial role for activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF): regulatory role in cell prolif- MIF in the procarcinogenic signaling induced by H. pylori infec- eration and glucocorticoid action. J. Biol. Chem. 274: 18100–18106. tion of GECs. Production of MIF by these cells in response to H. 21. Nguyen, M. T., H. Lue, R. Kleemann, M. Thiele, G. Tolle, D. Finkelmeier, pylori depends upon the presence of CagA, which is also the bac- E. Wagner, A. Braun, and J. Bernhagen. 2003. The cytokine macrophage migration inhibitory factor reduces pro-oxidative stress-induced apoptosis. J. Immunol. terial factor suggested to play a role in procarcinogenic epithelial 170: 3337–3347. responses. rMIF induced the same proliferative responses that 22. Leng, L., C. N. Metz, Y. Fang, J. Xu, S. Donnelly, J. Baugh, T. Delohery, CagAϩ H. pylori strains, and could therefore be an important piece Y. Chen, R. A. Mitchell, and R. Bucala. 2003. MIF signal transduction initiated by binding to CD74. J. Exp. Med. 197: 1467–1476. to the gastric cancer puzzle. 23. Ong, G. L., D. M. Goldenberg, H. J. Hansen, and M. J. Mattes. 1999. Cell surface expression and metabolism of major histocompatibility complex class II invariant Acknowledgments chain (CD74) by diverse cell lines. Immunology 98: 296–302. We thank Dr. Yoshio Yamaoka for providing the H. pylori strains used in 24. Barrera, C. A., E. J. Beswick, J. C. Sierra, D. Bland, R. Espejo, R. Mifflin, P. Adegboyega, S. E. Crowe, P. B. Ernst, and V. E. Reyes. 2005. Polarized these studies. expression of CD74 by gastric epithelial cells. J. Histochem. Cytochem. 53: 1481–1489. Disclosures 25. Beswick, E. J., D. A. Bland, G. Suarez, C. A. Barrera, X. Fan, and V. E. Reyes. 2005. Helicobacter pylori binds to CD74 on gastric epithelial cells and stimulates The authors have no financial conflict of interest. interleukin-8 production. Infect. Immun. 73: 2736–2743.

26. Matza, D., O. Wolstein, R. Dikstein, and I. Shachar. 2001. Invariant chain in- Downloaded from References duces B cell maturation by activating a TAF(II)105-NF-␬B-dependent transcrip- 1. Dunn, B. E., H. Cohen, and M. J. Blaser. 1997. Helicobacter pylori. Clin. Mi- tion program. J. Biol. Chem. 276: 27203–27206. crobiol. Rev. 10: 720–741. 27. Ishigami, S., S. Natsugoe, K. Tokuda, A. Nakajo, H. Iwashige, K. Aridome, 2. Kuipers, E. J. 1997. Helicobacter pylori and the risk and management of asso- S. Hokita, and T. Aikou. 2001. Invariant chain expression in gastric cancer. ciated diseases: gastritis, ulcer disease, atrophic gastritis and gastric cancer. Al- Cancer Lett. 168: 87–91. iment. Pharmacol. Ther. 11(Suppl. 1): 71–88. 28. Lu, X., N. L. Kallinteris, J. Li, S. Wu, Y. Li, Z. Jiang, G. G. Hillman, J. V. Gulfo, 3. Bjorkholm, B., P. Falk, L. Engstrand, and O. Nyren. 2003. Helicobacter pylori: R. E. Humphreys, and M. Xu. 2003. Tumor immunotherapy by converting tumor resurrection of the cancer link. J. Intern. Med. 253: 102–119. cells to MHC class II-positive, Ii protein-negative phenotype. Cancer Immunol. http://www.jimmunol.org/ 4. Uemura, N., S. Okamoto, and S. Yamamoto. 2002. H. pylori infection and the Immunother. 52: 592–598. development of gastric cancer. Keio. J. Med. 51(Suppl. 2): 63–68. 29. Xu, M., X. Lu, N. L. Kallinteris, Y. Wang, S. Wu, E. von Hofe, J. V. Gulfo, 5. Msika, S., M. A. Tazi, A. M. Benhamiche, C. Couillault, M. Harb, and J. Faivre. R. E. Humphreys, and G. G. Hillman. 2004. Immunotherapy of cancer by anti- 1997. Population-based study of diagnosis, treatment and prognosis of gastric sense inhibition of Ii protein, an immunoregulator of antigen selection by MHC cancer. Br. J. Surg. 84: 1474–1478. class II molecules. Curr. Opin. Mol. Ther. 6: 160–165.

6. Deguchi, R., A. Takagi, H. Kawata, H. Inoko, and T. Miwa. 2001. Association 30. Yamaoka, Y., D. H. Kwon, and D. Y. Graham. 2000. A Mr 34,000 proinﬂam- between CagAϩ Helicobacter pylori infection and p53, bax and transforming matory outer membrane protein (oipA)ofHelicobacter pylori. Proc. Natl. Acad. growth factor-␤-RII gene mutations in gastric cancer patients. Int. J. Cancer 91: Sci. USA 97: 7533–7538. 481–485. 31. Fan, X., S. E. Crowe, S. Behar, H. Gunasena, G. Ye, H. Haeberle, N. Van Houten, 7. Farinati, F., R. Cardin, V. M. Russo, G. Busatto, M. Franco, and M. Rugge. 2003. W. K. Gourley, P. B. Ernst, and V. E. Reyes. 1998. The effect of class II major Helicobacter pylori CagA status, mucosal oxidative damage and gastritis pheno- histocompatibility complex expression on adherence of Helicobacter pylori and

type: a potential pathway to cancer? Helicobacter 8: 227–234. induction of apoptosis in gastric epithelial cells: a mechanism for T helper cell by guest on September 28, 2021 8. Higashi, H., R. Tsutsumi, S. Muto, T. Sugiyama, T. Azuma, M. Asaka, and type 1-mediated damage. J. Exp. Med. 187: 1659–1669. M. Hatakeyama. 2002. SHP-2 tyrosine phosphatase as an intracellular target of 32. Fan, X. G., D. Kelleher, X. J. Fan, H. X. Xia, and P. W. Keeling. 1996. Heli- Helicobacter pylori CagA protein. Science 295: 683–686. cobacter pylori increases proliferation of gastric epithelial cells. Gut 38: 19–22. 9. Yamazaki, S., A. Yamakawa, Y. Ito, M. Ohtani, H. Higashi, M. Hatakeyama, and 33. Meyer-Siegler, K. L., E. C. Leifheit, and P. L. Vera. 2004. Inhibition of macro- T. Azuma. 2003. The CagA protein of Helicobacter pylori is translocated into phage migration inhibitory factor decreases proliferation and cytokine expression epithelial cells and binds to SHP-2 in human gastric mucosa. J. Infect. Dis. 187: in bladder cancer cells. BMC Cancer 4: 34. 334–337. 34. Beswick, E. J., S. Das, I. V. Pinchuk, P. Adegboyega, G. Suarez, Y. Yamaoka, 10. Higashi, H., A. Nakaya, R. Tsutsumi, K. Yokoyama, Y. Fujii, S. Ishikawa, and V. E. Reyes. 2005. Helicobacter pylori-induced IL-8 production by gastric M. Higuchi, A. Takahashi, Y. Kurashima, Y. Teishikata, et al. 2004. Helicobacter epithelial cells up-regulates CD74 expression. J. Immunol. 175: 171–176. pylori CagA induces Ras-independent morphogenetic response through SHP-2 35. Peek, R. M., Jr. 2002. Helicobacter pylori strain-specific modulation of gastric recruitment and activation. J. Biol. Chem. 279: 17205–17216. mucosal cellular turnover: implications for carcinogenesis. J. Gastroenterol. 11. Crabtree, J. E., and S. M. Farmery. 1995. Helicobacter pylori and gastric mucosal 37(Suppl. 13): 10–16. cytokines: evidence that CagA-positive strains are more virulent. Lab. Invest. 73: 36. Mitchell, R. A., H. Liao, J. Chesney, G. Fingerle-Rowson, J. Baugh, J. David, and 742–745. R. Bucala. 2002. Macrophage migration inhibitory factor (MIF) sustains macro- 12. Peek, R. M., Jr., G. G. Miller, K. T. Tham, G. I. Perez-Perez, X. Zhao, phage proinflammatory function by inhibiting p53: regulatory role in the innate J. C. Atherton, and M. J. Blaser. 1995. Heightened inflammatory response and ϩ immune response. Proc. Natl. Acad. Sci. USA 99: 345–350. cytokine expression in vivo to cagA Helicobacter pylori strains. Lab. Invest. 73: 37. Fingerle-Rowson, G., O. Petrenko, C. N. Metz, T. G. Forsthuber, R. Mitchell, 760–770. R. Huss, U. Moll, W. Muller, and R. Bucala. 2003. The p53-dependent effects of 13. Peek, R. M., Jr., S. F. Moss, K. T. Tham, G. I. Perez-Perez, S. Wang, macrophage migration inhibitory factor revealed by gene targeting. Proc. Natl. G. G. Miller, J. C. Atherton, P. R. Holt, and M. J. Blaser. 1997. Helicobacter ϩ Acad. Sci. USA 100: 9354–9359. pylori cagA strains and dissociation of gastric epithelial cell proliferation from apoptosis. J. Natl. Cancer Inst. 89: 863–868. 38. Topal, D., V. Goral, F. Yilmaz, and I. H. Kara. 2004. The relation of Helicobacter 14. Rokkas, T., S. Ladas, C. Liatsos, E. Petridou, G. Papatheodorou, S. Theocharis, pylori with intestinal metaplasia, gastric atrophy and BCL-2. Turk. A. Karameris, and S. Raptis. 1999. Relationship of Helicobacter pylori CagA J. Gastroenterol. 15: 149–155. status to gastric cell proliferation and apoptosis. Dig. Dis. Sci. 44: 487–493. 39. Zhang, H., D. C. Fang, R. Q. Wang, S. M. Yang, H. F. Liu, and Y. H. Luo. 2004. 15. Xia, H. H., S. K. Lam, A. O. Chan, M. C. Lin, H. F. Kung, K. Ogura, D. E. Berg, Effect of Helicobacter pylori infection on expression of Bcl-2 family members in and B. C. Wong. 2005. Macrophage migration inhibitory factor stimulated by gastric adenocarcinoma. World J. Gastroenterol. 10: 227–230. Helicobacter pylori increases proliferation of gastric epithelial cells. World 40. Chu, S. H., J. W. Lim, K. H. Kim, and H. Kim. 2003. NF-␬B and Bcl-2 in J. Gastroenterol. 11: 1946–1950. Helicobacter pylori-induced apoptosis in gastric epithelial cells. Ann. NY Acad. 16. Onodera, S., J. Nishihira, Y. Koyama, T. Majima, Y. Aoki, H. Ichiyama, Sci. 1010: 568–572. T. Ishibashi, and A. Minami. 2004. Macrophage migration inhibitory factor up- 41. Li, A., M. L. Varney, J. Valasek, M. Godfrey, B. J. Dave, and R. K. Singh. 2005. regulates the expression of interleukin-8 messenger RNA in synovial fibroblasts Autocrine role of interleukin-8 in induction of endothelial cell proliferation, sur- of rheumatoid arthritis patients: common transcriptional regulatory mechanism vival, migration and MMP-2 production and angiogenesis. Angiogenesis 8: between interleukin-8 and interleukin-1␤. Arthritis Rheum. 50: 1437–1447. 63–71. 17. Ren, Y., H. M. Chan, Z. Li, C. Lin, J. Nicholls, C. F. Chen, P. Y. Lee, V. Lui, 42. Joh, T., H. Kataoka, S. Tanida, K. Watanabe, T. Ohshima, M. Sasaki, H. Nakao, M. Bacher, and P. K. Tam. 2004. Upregulation of macrophage migration inhib- H. Ohhara, S. Higashiyama, and M. Itoh. 2005. Helicobacter pylori-stimulated itory factor contributes to induced N-Myc expression by the activation of ERK interleukin-8 (IL-8) promotes cell proliferation through transactivation of epider- signaling pathway and increased expression of interleukin-8 and VEGF in neu- mal growth factor receptor (EGFR) by disintegrin and metalloproteinase roblastoma. Oncogene 23: 4146–4154. (ADAM) activation. Dig. Dis. Sci. 50: 2081–2089. 18. Chesney, J., C. Metz, M. Bacher, T. Peng, A. Meinhardt, and R. Bucala. 1999. An 43. Al Marhoon, M. S., S. Nunn, and R. W. Soames. 2004. The association between essential role for macrophage migration inhibitory factor (MIF) in angiogenesis cagAϩ H. pylori infection and distal gastric cancer: a proposed model. Dig. Dis. and the growth of a murine lymphoma. Mol. Med. 5: 181–191. Sci. 49: 1116–1122. The Journal of Immunology 6801

44. Azuma, T., S. Yamazaki, A. Yamakawa, M. Ohtani, A. Muramatsu, H. Suto, types and modulation of the gastric epithelial cell cycle. Cancer Res. 59: Y. Ito, M. Dojo, Y. Yamazaki, M. Kuriyama, et al. 2004. Association between 6124–6131. diversity in the Src homology 2 domain-containing tyrosine phosphatase binding 49. Berloco, P., F. Russo, F. Cariola, M. Gentile, P. Giorgio, M. L. Caruso, site of Helicobacter pylori CagA protein and gastric atrophy and cancer. J. Infect. A. M. Valentini, G. Di Matteo, and A. Di Leo. 2003. Low presence of p53 Dis. 189: 820–827. abnormalities in H. pylori-infected gastric mucosa and in gastric adenocarcinoma. 45. Meyer-Siegler, K. L., R. C. Ordorica, and P. L. Vera. 2004. Macrophage migra- J. Gastroenterol. 38: 28–36. tion inhibitory factor is upregulated in an endotoxin-induced model of bladder 50. Hibi, K., H. Mitomi, W. Koizumi, S. Tanabe, K. Saigenji, and I. Okayasu. 1997. inﬂammation in rats. J. Interferon Cytokine Res. 24: 55–63. Enhanced cellular proliferation and p53 accumulation in gastric mucosa chroni- 46. Meyer-Siegler, K. L., K. A. Iczkowski, and P. L. Vera. 2005. Further evidence for cally infected with Helicobacter pylori. Am. J. Clin. Pathol. 108: 26–34. increased macrophage migration inhibitory factor expression in prostate cancer. 51. Kodama, M., T. Fujioka, K. Murakami, T. Okimoto, R. Sato, K. Watanabe, and BMC Cancer 5: 73. M. Nasu. 2005. Eradication of Helicobacter pylori reduced the immunohisto- 47. Moss, S. F., E. M. Sordillo, A. M. Abdalla, V. Makarov, Z. Hanzely, chemical detection of p53 and MDM2 in gastric mucosa. J. Gastroenterol. Hepa- G. I. Perez-Perez, M. J. Blaser, and P. R. Holt. 2001. Increased gastric epithelial tol. 20: 941–946. cell apoptosis associated with colonization with cagAϩ Helicobacter pylori 52. Choi, I. J., J. S. Kim, J. M. Kim, H. C. Jung, and I. S. Song. 2003. Effect of strains. Cancer Res. 61: 1406–1411. inhibition of extracellular signal-regulated kinase 1 and 2 pathway on apoptosis 48. Peek, R. M., Jr., M. J. Blaser, D. J. Mays, M. H. Forsyth, T. L. Cover, S. Y. Song, and bcl-2 expression in Helicobacter pylori-infected AGS cells. Infect. Immun. U. Krishna, and J. A. Pietenpol. 1999. Helicobacter pylori strain-speciﬁc geno- 71: 830–837. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021