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,
,¶,*
,¶
* Department of Medicine, Division of Gastroenterology, Departments of
Surgery and
Pathology, and
Greenebaum Cancer Center, University of Maryland School of Medicine, and
¶ Veterans Affairs Maryland Health Care System, Baltimore, MD 21201;
|| Laboratoire de Chimie et Biochimie Pharmacologiques, Unité Mixte de Recherche 8601, Centre National de la Recherche Scientifique, Université René Descartes, Paris, France;
# Department of Pathology and Laboratory Medicine,
** Mental Retardation Research Center, Departments of
Psychiatry and
* Pediatrics, University of California School of Medicine, Los Angeles, CA 90024; and
* Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21231
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B-dependent induction of
arginase II, but not arginase I, was observed in RAW 264.7 macrophages
cocultured with H. pylori. The time course of apoptosis
matched those of both arginase and iNOS activities. Surprisingly,
apoptosis was blocked by the arginase inhibitors
N
-hydroxy-L-arginine
or
N-hydroxy-nor-L-arginine,
but not by the iNOS inhibitor
N-iminoethyl-L-lysine. These findings
were confirmed in peritoneal macrophages from iNOS-deficient mice and
were not dependent on bacterial-macrophage contact. Ornithine
decarboxylase (ODC), which metabolizes L-ornithine to
polyamines, was also induced in H. pylori-stimulated
macrophages. Apoptosis was abolished by inhibition of ODC and was
restored by the polyamines spermidine and spermine. We also demonstrate
that arginase II expression is up-regulated in both murine and human
H. pylori gastritis tissues, indicating the likely in
vivo relevance of our findings. Therefore, we describe arginase- and
ODC-dependent macrophage apoptosis, which implicates polyamines in the
pathophysiology of H. pylori
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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40% of the population of the U.S.
(1) and is substantially higher in underdeveloped regions.
An important hallmark of H. pylori infection is that it
induces a vigorous gastric mucosal immune response that fails to
eradicate the organism, and the infection persists for the life of the
host. The host response can result in mucosal damage that may
ultimately lead to clinical consequences of ulcer disease or cancer. In
addition to a Th1-specific lymphocyte response (2),
H. pylori infection induces activation of an innate response
program in macrophages (3), which includes increased
expression of inducible NO synthase
(iNOS)3 and
cyclooxygenase-2 (4, 5), and production of inflammatory
mediators such as reactive oxygen intermediates, TNF-
, and IL-1
(6). An important part of the induction of mucosal damage by H. pylori involves activation of apoptosis. It has been shown that H. pylori in contact with the gastric epithelium induces substantial apoptosis of these cells in vivo (7) and in vitro (8). Although H. pylori is considered a noninvasive pathogen, its products have been shown to reach the lamina propria (9), and when crypt abscesses and other forms of direct epithelial damage occur, bacteria can reach mucosal macrophages. However, the ability of H. pylori to induce macrophage apoptosis has not been examined. The death of macrophages could contribute to the mucosal inflammation by causing the release of preformed proinflammatory cytokines, such as IL-1 (10). In addition, the loss of activated macrophages is likely to decrease the effective immune response to the pathogen. This may limit the inflammation under some conditions but would contribute to the inability of the host to eliminate the infection.
Apoptosis of macrophages can be induced by numerous effector molecules, including NO (11). Because we have previously shown that iNOS expression and activity are induced by H. pylori in macrophages in vitro (4), and that mucosal macrophages are a major source of iNOS in H. pylori infection in vivo (5), we reasoned that modulation of NO production in response to H. pylori would regulate levels of macrophage apoptosis. Arginases are enzymes that can be induced in macrophages by bacterially derived products (12) and are natural competitive inhibitors of iNOS, because they metabolize the same substrate, L-arginine (13). Therefore, arginases can regulate the biological effects of NO (14, 15) and have been reported to inhibit NO-dependent apoptosis in RAW 264.7 macrophage cells (16). Importantly, arginase converts L-arginine to L-ornithine, which is then acted upon by ornithine decarboxylase (ODC) to produce polyamines. The biogenic polyamines, putrescine, spermidine, and spermine, have been shown to have numerous biological functions, including inhibition of monocyte activation (17), stimulation of cellular migration (18) and proliferation (19), and regulation of apoptosis (20).
We hypothesized that arginase is induced by H. pylori in macrophages, and that it modulates apoptosis in these cells. To determine the respective roles of the iNOS and arginase-ODC pathways in H. pylori-induced apoptosis, we have used selective inhibitors of iNOS, arginase, and ODC, and peritoneal macrophages from wild-type (WT) and iNOS-/- C57BL/6 mice. Our results show, for the first time, that H. pylori induces apoptosis in macrophages by a process that occurs independently of iNOS expression but is mediated by activation of the arginase-ODC metabolic pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All reagents for cell culture, RNA extraction, RT-PCR, and
Northern blotting were obtained from Life Technologies (Grand Island,
NY). The NF-B inhibitor, Z-Leu-Leu-Leu-CHO (MG-132), the
protein kinase A (PKA) inhibitor,
N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinoline-sulfonamide
(H89), and the protein kinase C (PKC) inhibitor,
2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)
maleimide (Gö 6983), were purchased from Calbiochem (San Diego,
CA). The iNOS-specific inhibitor,
N-iminoethyl-L-lysine
(L-NIL), and the arginase inhibitor and iNOS
substrate,
N-hydroxy-L-arginine
(NOHA), were purchased from Alexis Biochemicals (San Diego, CA). The
specific arginase inhibitor,
N-hydroxy-nor-L-arginine
(norNOHA), which is not a substrate for iNOS, was prepared as
previously described (21). -Difluoromethylornithine
(DFMO), an ODC inhibitor, was obtained from Ilex Oncology (San Antonio,
TX). All other chemicals were purchased from Sigma-Aldrich (St.
Louis, MO).
Bacteria
H. pylori SS1, a mouse-adapted human strain (22), was used. Bacteria were passaged on Brucella agar plates containing 10% sheep blood and were maintained under microaerobic conditions. For the experiments H. pylori were harvested from plates, washed twice, and suspended in PBS. Concentrations of bacteria were determined by OD at 600 nm.
Mice, cells, and culture conditions
The murine macrophage cell line RAW 264.7 was maintained in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, 1 mM sodium pyruvate, and 10 mM HEPES at 37°C in a humidified 5% CO2 atmosphere. For the coculture experiments RAW 264.7 cells were plated in the same medium without penicillin-streptomycin for 2 h, and H. pylori was then added to macrophages at a multiplicity of infection (MOI) of 10. To separate bacteria from macrophages, filter supports (0.2-µm pore size; Transwell; Nunc, Naperville, IL) were used, and under these conditions an MOI of 100 was used.
Experiments were also conducted with peritoneal macrophages isolated as previously described (23) from WT or iNOS-deficient C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME). H. pylori was added at an MOI of 10. Because serum has been shown to enhance arginase activity in resident macrophages (24), DMEM without serum, supplemented with 4 g/L BSA, 1 mM sodium pyruvate, and 10 mM HEPES, was used for these cultures.
For H. pylori mouse infection studies, C57BL/6 x Sv129
WT mice (The Jackson Laboratory) were inoculated with
109 CFU H. pylori SS1 every other day
three times by gavage, and gastric tissues were harvested 4 mo later.
Some mice were also gavaged with the same concentrations of
nonpathogenic Escherichia coli strain DH5 as a
control.
RT-PCR
RAW 264.7 macrophages were seeded at 2 x
106/well in six-well plates, and mouse peritoneal
macrophages were seeded at 5 x 105/well in
24-well plates. After stimulation, total RNA was isolated using TRIzol
reagent. RNA was also extracted from mouse and human tissues by
homogenization in TRIzol reagent. The human tissue samples were from
patients with H. pylori gastritis, as previously
described (5). Subsequently, 2 µg RNA from each sample
was reverse transcribed using 50 U Superscript II reverse
transcriptase. PCR was conducted using 2 µl cDNA and 1 U Taq DNA
polymerase. For iNOS, arginase I, and arginase II, 15 pmol each of 5'
and 3' primers were used with 3 pmol each of 
-actin primers in a
multiplex reaction (5, 25). For ODC, PCR was performed
with 6 and 18 pmol each of ODC primers and -actin primers,
respectively. One PCR cycle consisted of the following: 94°C for 1
min, 60°C for 1.5 min, and 72°C for 1.8 min. For RAW 264.7 cells,
the total cycle numbers were 35 for iNOS and arginase I, 25 for
arginase II, and 20 for ODC. For peritoneal macrophages, the total
cycle numbers were 27 for arginase I and arginase II and 37 for
-actin. For mouse tissues, the total cycle numbers were 27 for
arginase I and 28 for arginase II. For human tissues, the total cycle
numbers were 40 for arginase I and 28 for arginase II. A final
elongation step of 7 min at 72°C was used for each reaction. Sense
and antisense primer sequences and PCR product sizes were as follows:
murine iNOS, 5'-GCCTCGCTCTGGAAAGA-3' and 5'-TCCATGCAGACAACCTT-3',
499 bp; murine arginase I, 5'-AAGAAAAGGCCGATTCACCT-3' and
5'-CACCTCCTCTGCTGTCTTCC-3', 201 bp; murine arginase II,
5'-ACAGGGTTGCTGTCAGCTCT-3' and 5'-TGATCCAGACAGCCATTTCA-3', 298 bp;
human arginase I, 5'-CCCTTTGCTGACATCCCTAA-3' and
5'-GACTCCAAGATCAGGGTGGA-3', 201 bp; human arginase II,
5'-GACACTGCCCAGACCTTTGT-3' and 5'-CGTTCCATGACCTTCTGGAT, 304 bp;
murine ODC, 5'-CAGCAGGCTTCTCTTGGAAC-3' and
5'-CATGCATTTCAGGCAGGTTA-3', 602 bp; and murine/human -actin,
5'-CCAGAGCAAGAGAGGTATCC-3' and 5'-CTGTGGTGGTGAAGCTGTAG-3', 436 bp.
PCR products were run on 2% agarose gels with 0.4 µg/ml ethidium
bromide. Stained bands were visualized under UV light and photographed
with a digital gel documentation system (EDAS 290 and 1D software;
Kodak Digital Science, Rochester, NY).
Northern blot analysis
RAW 264.7 macrophages were seeded at 106/ml in 75-cm2 flasks. After incubation with H. pylori, total RNA was extracted with TRIzol, and 10 µg RNA/lane was separated on 1% agarose gels. Northern blot analysis was performed as previously described (4, 15). For iNOS mRNA detection, a 32P random primer-labeled full-length (3.9-kb) cDNA probe for murine iNOS (obtained from Q. W. Xie and C. Nathan, Cornell University Medical College, Ithaca, NY) was used. The arginase II cDNA probe (298 bp) was generated by RT-PCR amplification of arginase II derived from RAW 264.7 macrophage RNA. The concentration and loading of RNA were standardized by hybridization with a cDNA probe (1.1 kb) for GAPDH (Clontech Laboratories, Palo Alto, CA). Densitometric analysis of band intensity was determined using National Institutes of Health Image version 1.62 (http://rsb.info.nih.gov/nih-image/).
Immunoblotting for arginase II
After coculture with H. pylori, RAW 264.7 macrophages were lysed in the presence of protease inhibitors (26). Mouse tissues were homogenized in the same lysis buffer. Protein concentrations of 12,000 x g supernatants were measured using the DC Protein Assay kit (Bio-Rad, Hercules, CA), and 100 µg/lane was separated by SDS-PAGE using a 12% gel and transferred onto Immobilon-P membranes (Millipore, Bedford, MA) by electroblotting. Membranes were blocked overnight at room temperature using PBS containing 0.1% Tween and 5% nonfat dry milk. Arginase II protein was detected by incubation of blots with a polyclonal Ab to rat mammary gland arginase II (27), which does not cross-react with arginase I by immunoprecipitation or Western blotting. The primary Ab was used at a dilution of 1/500 for 2 h at room temperature, followed by a sheep anti-rabbit Ab conjugated to HRP (1/2000). Chemiluminescent detection was performed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and exposure to Hyperfilm ECL (Amersham, Little Chalfont, U.K.).
Determination of arginase activity
Colorimetric detection of macrophage arginase activity was determined as previously described (14). RAW 264.7 macrophages were lysed in 50 µl of a solution containing 0.1% Triton X-100, 0.01% antipain, 0.01% pepstatin, and 0.01% aprotinin. MnCl2 (10 mM for 10 min at 55°C) and L-arginine (0.5 M for 1 h at 37°C) were successively added, and the reaction was stopped by adding an acid solution. The concentration of urea synthesized by arginase metabolism was determined at 540 nm after addition of anti-isonitrosopropiophenone for 45 min at 100°C.
Assay for ODC activity
ODC activity was determined by a radiometric analysis in which the amount of [14C]O2 liberated from L-[14C]ornithine was estimated as previously described (28). Cells were lysed in 500 µl of a solution of 1 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.05 mM pyridoxal-5'-phosphate, and 5 mM DTT. Cells were frozen and thawed three times, then sonicated. After centrifugation at 12,000 x g for 15 min, 300 µl of the supernatant was incubated at 37°C with 10 nmol L-[14C]ornithine (sp. act., 47.7 mCi/mmol; NEN, Boston, MA) for 15 min. The [14C]O2 liberated by the activity of the ODC was trapped on filter paper impregnated with 20 µl 2 N NaOH. The reaction was stopped with 300 µl 10% TCA, and [14C]O2 present on the filter paper was measured by liquid scintillation spectroscopy. Aliquots of the 12,000 x g supernatants were assayed for protein concentration using the DC Protein Assay kit.
Measurement of NO concentration
The concentration of the oxidized product of NO, nitrite (NO2-), was assessed by the Griess reaction, as we have previously described (4, 15).
Apoptosis analysis
Macrophage apoptosis was determined by two methods.
ELISA. RAW 264.7 cells (2 x 105 cells/well) and peritoneal macrophages (104 cells/well) were cultured in 24- and 96-well plates, respectively, in the presence or the absence of H. pylori and various inhibitors. Floating and adherent cells were harvested after experiments, counted with a hemocytometer, and 104 cells were analyzed using the Cell Death Detection ELISA Plus kit from Roche Molecular Biochemicals (Indianapolis, IN), based on the determination of cytoplasmic histone-associated DNA fragments. According to the manufacturer’s instructions, in each experiment the OD of unstimulated control cells was assigned a value of 1.0, and the relative amount of apoptosis in experimental groups was determined as a ratio to control level.
DNA nick end labeling of cells. Macrophages (105/well) were cultured and stimulated in four-well plastic chamber slides (Nunc). At the end of the experiments, cells were washed with PBS and fixed with 4% formaldehyde. DNA fragmentation was analyzed by TUNEL assay using an In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD). The percentage of apoptotic cells was determined after counting 10 microscope fields at a magnification of x400.
Statistical analysis
For comparisons between multiple groups, the Student-Newman-Keuls test was used, and for single comparisons between two groups, Student’s t test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After 0–12 h of coculture with H. pylori, mRNA levels
of arginase I, arginase II, and iNOS were determined in macrophages by
RT-PCR. Arginase II was up-regulated by 8-fold after 2 h and was
decreased minimally from this level at 6 and 12 h (Fig. 1
A). The iNOS gene product was
not expressed in control macrophages or after 2 h of coculture
(Fig. 1A) but was markedly induced at 6 h. We found no
increase in the expression of type I arginase in macrophages stimulated
for 2–12 h (Fig. 1A) or after 24 h with H.
pylori (data not shown), and there was actually a decrease
from basal expression at 6 h and later time points. To confirm
that the expression of both iNOS and arginase II was increased in RAW
264.7 cells in response to H. pylori, we also assessed mRNA
levels by Northern blot analysis. As shown in Fig. 1B, both
transcripts were up-regulated in H. pylori-stimulated
macrophages compared with unstimulated cells, by 7-fold for arginase II
and 15-fold for iNOS.
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C).
The signaling mechanism responsible for the expression of arginase II
mRNA in RAW 264.7 cells in response to H. pylori was
investigated. Because the induction of arginase enzymes has been linked
to increased activity of NF-B and protein kinases, we assessed each
of these pathways. Up-regulation of arginase II mRNA expression in
response to H. pylori was completely inhibited by the
NF-B inhibitor MG-132 (Fig. 1D). In contrast, H89 and Gö 6983,
specific inhibitors of PKA and PKC, respectively, did not affect
arginase II expression.
Macrophage apoptosis correlates with increased arginase and iNOS activities
To investigate the roles of iNOS and arginase in RAW 264.7 cells
exposed to H. pylori, we assessed the activities of both
proteins and the levels of apoptosis by ELISA. In the same experiments
arginase activities and apoptosis were determined in the cell lysates,
and iNOS activity was investigated by NO2-
generation in the culture supernatants. In activated macrophages a
dramatic increase in arginase activity was observed after 12 h of
stimulation (Fig. 2A). After
24 h of stimulation the activity of macrophage arginase was
increased 7.7 ± 1.5-fold compared with that in control cells. The
production of NO2- by H.
pylori-activated macrophages was not increased before 6 h and
became significantly elevated after 12 h (Fig. 2B).
After 18 and 24 h of coculture, NO2-
levels were increased by 11.9 ± 1.7- and 27.1 ± 2.8-fold vs
the control value, respectively. Despite the significant increases in
NO production, there was no loss of viability for the RAW 264.7
macrophages (data not shown).
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C,
apoptosis was not detected between 2 and 12 h of coculture, but
was significantly increased by 2.8 ± 0.87- and 3.4 ±
0.13-fold, at 18 and 24 h, respectively. The time point of 24
h was then used for all other experiments. H. pylori-stimulated macrophage apoptosis is arginase dependent
Because both arginase and iNOS activities directly paralleled the
induction of apoptosis, we assessed the effects of the arginase
inhibitors, NOHA and norNOHA, and the selective inhibitor of iNOS,
L-NIL, on H. pylori-stimulated macrophage
apoptosis. Relative to the control level, the induction of apoptosis by
H. pylori was inhibited by 78 and 95% with NOHA and
norNOHA, respectively (Fig. 3).
Surprisingly, macrophage apoptosis was not affected by
L-NIL (Fig. 3) despite complete inhibition of NO
production. NO2- generation by RAW 264.7
macrophages was not enhanced by the arginase inhibitors, most likely
due to excess L-arginine in the medium,
as previously described (14, 15).
NO2- levels were as follows: control, 3.6
± 1.2 µM; H. pylori, 21.7 ± 5.5 µM; H.
pylori and L-NIL, 2.5 ± 1.0 µM; and
H. pylori and norNOHA, 22.9 ± 8.2 µM. In addition,
no alteration of the basal level of apoptosis was observed in control
macrophages treated with iNOS or arginase inhibitors (data not
shown).
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A), while
NO2- production was observed only with cells
from WT mice. NO2- levels were as follows:
control WT cells, 3.7 ± 1.0 µM; H. pylori WT cells,
15.6 ± 0.8 µM; control iNOS-/- cells,
2.8 ± 0.2 µM; H. pylori
iNOS-/- cells, 2.6 ± 1.5 µM. We
also found that L-NIL treatment had no effect on
H. pylori-stimulated apoptosis in WT peritoneal
macrophages; compared with unstimulated control cells there were
1.96 ± 0.26- and 2.03 ± 0.33-fold increases in apoptosis
for H. pylori alone and H. pylori plus
L-NIL, respectively, by ELISA
(p < 0.01 vs control for both). As in the RAW
264.7 cells, L-NIL completely abolished NO
production, while norNOHA had no such effect.
NO2- levels were as follows: control, 3.7
± 1.0 µM; H. pylori, 15.6 ± 0.8 µM; H.
pylori plus L-NIL, 1.9 ± 0.4 µM; and
H. pylori plus norNOHA, 13.6 ± 2.4 µM. As shown in
Fig. 4B, up-regulation of arginase II, but not arginase I,
was observed in response to H. pylori in resident
macrophages from both WT and iNOS-/-
mice.
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To further explore the involvement of the arginase-ODC metabolic
pathway in apoptosis, we assessed the expression and activity of ODC in
response to H. pylori. Kinetic analysis of ODC mRNA levels
showed that the basal expression of the gene in control macrophages was
moderately up-regulated by 1.3-, 1.7-, and 2.1-fold at 2, 6, and
12 h of coculture with H. pylori, respectively (Fig. 5A). Because ODC is regulated
mainly at the post-transcriptional level, we assessed enzymatic
activity and found that it was increased by 10.6 ± 2.5-fold in
macrophages infected with H. pylori for 18 h (Fig. 5B).
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To determine whether H. pylori-stimulated
macrophage apoptosis is mediated by ODC, we examined the effect of the
ODC inhibitor, DFMO, on apoptosis. As shown in Fig. 6A, compared with H.
pylori stimulation alone, the apoptosis observed in macrophages
cultured with the bacteria was abolished by DFMO. When DFMO was added
to unstimulated macrophages, basal apoptosis was inhibited by 22
± 4% (p < 0.01 vs control; data not shown).
Inhibition of H. pylori-stimulated apoptosis by DFMO was
significantly reversed using the polyamines spermidine and spermine
(Fig. 6A). In contrast, putrescine had no effect. In further
support of NO-independent apoptosis, we found that, despite causing
apoptosis, spermidine and spermine inhibited H.
pylori-induced NO2- generation by
5.8 ± 0.8- and 17 ± 3.7-fold, respectively
(p < 0.01 for each).
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B). Apoptosis was also enhanced with 5 µM spermidine but
was not increased further at higher concentrations (Fig. 6B). Putrescine did not stimulate apoptosis (Fig. 6B), even at a concentration of 500 µM (data not
shown). Confirmation by TUNEL assay that arginase and ODC regulate H. pylori-stimulated macrophage apoptosis
The effects of arginase and ODC inhibition on TUNEL staining are
shown in Fig. 7. While cells exposed to
H. pylori alone (Fig. 7B) had frequent cellular
changes consistent with apoptosis, in the presence of norNOHA (Fig. 7C) or DFMO (Fig. 7D) there was a marked
reduction of morphologic features of apoptosis. When quantified (Fig. 7E), the percentage of apoptotic cells was increased 2-fold
above control levels with H. pylori alone; this increase in
apoptosis was inhibited by 146 and 85% with norNOHA and DFMO,
respectively.
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Up-regulation of arginase II in RAW 264.7 cells was also observed
when H. pylori were separated from macrophages by a filter
support (Fig. 8A). These data
suggest that H. pylori-derived effector molecules are potent
activators of arginase II. In addition, factors released from H.
pylori are capable of reproducing the same levels of apoptosis
(Fig. 8B) as the bacteria not physically separated from the
cells, previously shown in Fig. 3. Thus, contact with H.
pylori is not required to induce macrophage apoptosis. In
addition, H. pylori above the Transwell filter
also stimulated apoptosis by an arginase- and ODC-dependent mechanism,
because both norNOHA and DFMO significantly inhibited H.
pylori-induced macrophage apoptosis (Fig. 8B).
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To confirm the in vivo relevance of the H.
pylori-stimulated arginase II expression that we have demonstrated
in vitro, we assessed arginase II levels in both mouse and human
H. pylori gastritis tissues. As shown in Fig. 9A, in mouse antral tissues
assessed 4 mo after inoculation with H. pylori SS1, there
was a marked and consistent increase in arginase II mRNA levels
compared with control tissues from mice inoculated with nonpathogenic
E. coli. Additionally, there was basal expression of
arginase I in control tissues, which was actually decreased in the
H. pylori-infected tissues. We confirmed the arginase II
up-regulation at the protein level by Western blot analysis (Fig. 9B). We also assessed arginase mRNA expression in human
tissues (Fig. 9C) and found that arginase II was absent in
normal tissues and increased in H. pylori gastritis, while
arginase I expression was generally very low, with 40 PCR cycles
required to detect transcripts and no significant increase compared
with control tissues.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is important to realize that generation of L-ornithine as a substrate for ODC in our studies occurs mainly through the arginase metabolic pathway, because L-ornithine is not present in DMEM culture medium. Our data show that both arginase and ODC activities are necessary to observe polyamine-dependent macrophage apoptosis. Although ODC activity is also increased by H. pylori, our data suggest that arginase II expression is rate limiting in the H. pylori-induced apoptosis, because inhibition of arginase, which is upstream of ODC in the metabolic pathway, effectively abolishes the apoptosis. In addition, we have observed that DFMO inhibits basal macrophage apoptosis, while arginase inhibition had no such effect, indicating that constitutive ODC expression and activity are sufficient to facilitate apoptosis once L-ornithine levels are increased by induction of arginase II. The primary importance of arginase in polyamine synthesis has been previously reported in macrophages (29) and rat smooth muscle cells (30). It should be noted that while arginase I is located in the cell cytosol, where ODC is also located, facilitating polyamine synthesis from ornithine, arginase II is a mitochondrial enzyme that could preferentially enhance proline or glutamate synthesis from ornithine, because ornithine aminotransferase is also located in the mitochondria (31). However, it has been shown that proline can be converted to ornithine (31) and that ornithine can be transported from the mitochondria to the cytosol (32).
Consistent with our findings for H. pylori stimulation,
up-regulation of arginase II has been reported for RAW 264.7 cells
activated by LPS (33) or cAMP (34) and in
peritoneal macrophages stimulated by LPS (35). Arginase I
expression was not increased in macrophages stimulated by H.
pylori, even though this gene has been shown to be induced in
macrophages by a cAMP-related pathway (34) involving
activation of PKA (36). The signal transduction mechanisms
leading to arginase II expression are less well understood. Induction
of arginase II by LPS in rat alveolar macrophages was prevented by two
NF-B inhibitors, including pyrrolidine dithiocarbamate (PDTC)
(37). However, arginase II expression has also been
reported to be unaffected by PDTC in RAW 264.7 macrophages stimulated
with LPS (12). In our experiments H.
pylori-stimulated arginase II mRNA levels were decreased by 25%
when 20 µM PDTC was used (data not shown), but a complete inhibition
of arginase II expression occurred with the more specific NF-B
inhibitor, MG-132. These findings were specific, because inhibitors of
PKA and PKC had no effect. Consistent with reported activation of
NF-B by H. pylori (38), our data suggest
that macrophage arginase II expression in response to H.
pylori involves NF-B activation.
Numerous reports have investigated the involvement of NO in apoptosis.
The type of cells, the time of exposure to NO or to its different
derivatives, and the type of stimulus is a nonexhaustive list of the
parameters that can modulate apoptotic events (39). NO can
have paradoxical effects on programmed cell death. It has been reported
to have proapoptotic effects on epithelial cells (40) and
macrophages (11, 41). It also has been shown to have
antiapoptotic activity by inactivating enzymes that mediate apoptosis,
such as IL-1-converting enzyme, cysteine protease protein
(42), caspases, and tissue-transglutaminase
(43), or by inhibiting ceramide formation
(44). In murine macrophages stimulated by LPS alone,
induction of both iNOS and arginase II has been reported
(12), and apoptosis is NO independent under these
conditions (45). However, when arginase was inhibited by
addition of IFN-
to LPS (12), macrophage apoptosis was
NO dependent (11, 41). From our experiments three findings
support NO-independent macrophage apoptosis in response to H.
pylori: 1) apoptosis was not affected by iNOS inhibition; 2)
apoptosis occurred at the same levels in macrophages from
iNOS-deficient mice as in those from WT mice; and 3) apoptosis was
increased by spermidine and spermine, polyamines that inhibited
iNOS-derived NO production by H. pylori. Consistent with our
finding that polyamines inhibited iNOS, potentiation of LPS-induced
iNOS activity by DFMO has been described (46). It is
possible that the viability of peritoneal macrophages is decreased by
H. pylori-induced NO production, but this would not affect
our results, because the apoptosis ELISA we used includes all cellular
material in the culture wells from both viable and nonviable cells.
Although the role of NO in apoptosis has been extensively studied, the
effect of arginase in this process remains elusive. In agreement with
our findings of a proapoptotic effect of arginase activity, it was
recently reported that transfection and overexpression of p53 in
bladder carcinoma cells resulted in up-regulation of arginase II that
paralleled the induction of apoptosis (47). In contrast,
it has also been described that long-term inhibition of arginase
induces apoptosis of human breast cancer lines expressing high basal
levels of arginase (48). In addition, overexpression of
arginase in RAW 264.7 cells by transfection or stimulation with
dexamethasone and cAMP decreased NO generation and related apoptosis in
cells stimulated with IFN- plus LPS (16). In our
studies iNOS and arginase are also both expressed, but the mechanism
described herein differs from the previous report (16) by
the mode of macrophage stimulation and by the fact that ODC activity
was increased in response to H. pylori, while dexamethasone
inhibits ODC expression and activity (49).
Although there is a consensus that polyamines stimulate cell
proliferation (50) and migration (18), the
role of polyamines in apoptosis is more controversial. In different
systems accumulation of polyamines has been linked to both induction of
cell apoptosis (51, 52, 53) and protection against apoptosis
(53, 54). In intestinal epithelial cells, for example,
polyamines have been shown to either prevent or induce apoptosis
depending on the nature of the death stimulus (53). In the
latter report TNF--mediated apoptosis was blocked by DFMO and
facilitated by spermidine (53), similar to the current
findings with H. pylori-stimulated macrophages. The levels
of polyamines that we have added to induce apoptosis are similar to
those used in previous studies (53). Additionally, the
concentrations of polyamines used in Fig. 6 have in vivo relevance. We
used a range of 5–50 µM, which converts to 2.5–25 nmol/mg
macrophage protein. We have measured polyamine levels in H.
pylori-infected murine gastric tissues and found concentrations of
spermine and spermidine in the range of 10–12 nmol/mg protein, which
is in the same range as the concentrations we used in the in vitro
studies herein. Our finding that putrescine lacked the ability of the
other biogenic polyamines to induce apoptosis has been previously
described in murine leukemia L1210 cells (55). An
important link between polyamine levels and apoptosis appears to be the
regulation of NF-B, because polyamine depletion can induce NF-B
activation (56), and NF-B has been shown to protect
against apoptosis in a variety of cell types, including macrophages
(57) and lymphocytes (58). However, it should
be noted that H. pylori-stimulated gastric epithelial cell
apoptosis has been attributed to activation of NF-B
(59).
Our data suggest that analysis of arginase expression and activity in mucosal inflammation may provide new insights into understanding the often ambiguous effects of iNOS-derived NO. In support of the relevance of our in vitro data linking arginase II to H. pylori infection, we have demonstrated that arginase II expression is up-regulated in both murine and human H. pylori gastritis tissues. Therefore, experiments are now underway in our laboratory to elucidate the role of the balance of iNOS and arginase activities in murine H. pylori gastritis in vivo. Intriguingly, H. pylori itself possesses a constitutively expressed arginase (60), which we have shown inhibits macrophage NO production by iNOS under conditions of low arginine availability, thus enhancing H. pylori survival (15). It is highly unlikely, however, that the arginase inhibitors used in the present study decreased macrophage apoptosis by affecting H. pylori arginase, because isogenic mutant strains of H. pylori lacking rocF, the gene encoding for bacterial arginase, have normal viability under conditions of physiologic pH (61), as in the present study. It should be noted that we observed that rocF mutant H. pylori strains lacking bacterial arginase induce macrophage arginase II expression and apoptosis to an equal degree as WT H. pylori strains; in addition, treatment of cocultures of macrophages and rocF mutant strains with arginase inhibitors resulted in the same reversal of macrophage apoptosis as with WT strains (data not shown). These findings indicate that under the experimental conditions in the present study macrophage apoptosis is not altered by potential effects of arginase inhibitors on H. pylori arginase. It should also be recognized that H. pylori can release L-ornithine (60) and produce polyamines, such as agmatine (62), histamine, and spermidine (63). Thus, H. pylori may modulate the immune response by facilitating host polyamine synthesis or by producing the polyamines directly. In summary, our data strongly implicate arginase II in the pathogenesis of H. pylori infection, because it acts as the rate-limiting enzyme for polyamine synthesis and macrophage apoptosis, providing an important clue to the ineffective mucosal immune response to this gastric pathogen.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Keith T. Wilson, University of Maryland School of Medicine, 22 South Greene Street, Room N3W62, Baltimore, MD 21201. E-mail address: kwilson{at}umaryland.edu
3 Abbreviations used in this paper: iNOS, inducible NO synthase; DFMO, -difluoromethylornithine; Gö 6983, 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide; H89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinoline-sulfonamide; L-NIL, N-iminoethyl-L-lysine; MG-132, Z-Leu-Leu-Leu-CHO; MOI, multiplicity of infection; NOHA, N-hydroxy-L-arginine; norNOHA, N-hydroxy-nor-L-arginine; ODC, ornithine decarboxylase; PDTC, pyrrolidine dithiocarbamate; PKA, protein kinase A; PKC, protein kinase C; WT, wild type.
Received for publication November 26, 2001. Accepted for publication February 20, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-hydroxy derivatives, and N-hydroxynor-L-arginine. Biochemistry 37:10453.[Medline]
B in gastric epithelial cells. Gastroenterology 113:1099.[Medline]
B, inducible nitric oxide synthase and apoptosis by Helicobacter pylori infection. Free Radical Biol. Med. 31:355.[Medline]
-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J. Exp. Med. 185:601.-induced apoptosis by reducing the generation of ceramide. Proc. Natl. Acad. Sci. USA 97:5480.-dependent and -independent mechanisms. J. Biol. Chem. 276:30043.-hydroxy-L-arginine selectively inhibits cell proliferation and induces apoptosis in MDA-MB-468 cells. Cancer Res. 60:3305.B activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells. Am. J. Physiol. 280:G992.B activation in IEC-6 cells. J. Biol. Chem. 5:5.
B in preventing TNF--induced cell death. Science 274:782.-induced apoptosis by NF-B. Science 274:787. suppresses nuclear factor B-mediated apoptosis induced by Helicobacter pylori in gastric epithelial cells. J. Biol. Chem. 276:31059.This article has been cited by other articles:
|
|
 |
|
  M.-Y. Tsao, T.-L. Lin, P.-F. Hsieh, and J.-T. Wang The 3'-to-5' Exoribonuclease (Encoded by HP1248) of Helicobacter pylori Regulates Motility and Apoptosis-Inducing Genes J. Bacteriol., April 15, 2009; 191(8): 2691 - 2702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Chaturvedi, M. Asim, N. D. Lewis, H. M. S. Algood, T. L. Cover, P. Y. Kim, and K. T. Wilson L-Arginine Availability Regulates Inducible Nitric Oxide Synthase-Dependent Host Defense against Helicobacter pylori Infect. Immun., September 1, 2007; 75(9): 4305 - 4315. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Johann, V. Barra, A.-M. Kuhn, A. Weigert, A. von Knethen, and B. Brune Apoptotic cells induce arginase II in macrophages, thereby attenuating NO production FASEB J, September 1, 2007; 21(11): 2704 - 2712. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Gobert, M. Vareille, A.-L. Glasser, T. Hindre, T. de Sablet, and C. Martin Shiga Toxin Produced by Enterohemorrhagic Escherichia coli Inhibits PI3K/NF-{kappa}B Signaling Pathway in Globotriaosylceramide-3-Negative Human Intestinal Epithelial Cells J. Immunol., June 15, 2007; 178(12): 8168 - 8174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Lasbury, S. Merali, P. J. Durant, D. Tschang, C. A. Ray, and C.-H. Lee Polyamine-mediated Apoptosis of Alveolar Macrophages during Pneumocystis Pneumonia J. Biol. Chem., April 13, 2007; 282(15): 11009 - 11020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. T. Pullan, M. D. Gidley, R. A. Jones, J. Barrett, T. M. Stevanin, R. C. Read, J. Green, and R. K. Poole Nitric Oxide in Chemostat-Cultured Escherichia coli Is Sensed by Fnr and Other Global Regulators: Unaltered Methionine Biosynthesis Indicates Lack of S Nitrosation J. Bacteriol., March 1, 2007; 189(5): 1845 - 1855. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. M. S. Algood and T. L. Cover Helicobacter pylori Persistence: an Overview of Interactions between H. pylori and Host Immune Defenses Clin. Microbiol. Rev., October 1, 2006; 19(4): 597 - 613. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Kusters, A. H. M. van Vliet, and E. J. Kuipers Pathogenesis of Helicobacter pylori Infection Clin. Microbiol. Rev., July 1, 2006; 19(3): 449 - 490. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Talaue, V. Venketaraman, M. H. Hazbon, M. Peteroy-Kelly, A. Seth, R. Colangeli, D. Alland, and N. D. Connell Arginine Homeostasis in J774.1 Macrophages in the Context of Mycobacterium bovis BCG Infection J. Bacteriol., July 1, 2006; 188(13): 4830 - 4840. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. I. Bussiere, R. Chaturvedi, M. Asim, K. L. Hoek, Y. Cheng, J. Gainor, A. Scholz, W. N. Khan, and K. T. Wilson Low Multiplicity of Infection of Helicobacter pylori Suppresses Apoptosis of B Lymphocytes. Cancer Res., July 1, 2006; 66(13): 6834 - 6842. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Niklaus and J. W. Pollard Mining the Mouse Transcriptome of Receptive Endometrium Reveals Distinct Molecular Signatures for the Luminal and Glandular Epithelium Endocrinology, July 1, 2006; 147(7): 3375 - 3390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T. Schwartz and L.-A. H. Allen Role of urease in megasome formation and Helicobacter pylori survival in macrophages J. Leukoc. Biol., June 1, 2006; 79(6): 1214 - 1225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Das, G. Suarez, E. J. Beswick, J. C. Sierra, D. Y. Graham, and V. E. Reyes Expression of B7-H1 on Gastric Epithelial Cells: Its Potential Role in Regulating T Cells during Helicobacter pylori Infection. J. Immunol., March 1, 2006; 176(5): 3000 - 3009. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Desnues, M. Ihrig, D. Raoult, and J.-L. Mege Whipple's Disease: a Macrophage Disease Clin. Vaccine Immunol., February 1, 2006; 13(2): 170 - 178. [Full Text] [PDF]
|
|
|
|
 |
|
 D. Pinto-Santini and N. R. Salama The Biology of Helicobacter pylori Infection, a Major Risk Factor for Gastric Adenocarcinoma Cancer Epidemiol. Biomarkers Prev., August 1, 2005; 14(8): 1853 - 1858. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Cheng, R. Chaturvedi, M. Asim, F. I. Bussiere, H. Xu, R. A. Casero Jr., and K. T. Wilson Helicobacter pylori-induced Macrophage Apoptosis Requires Activation of Ornithine Decarboxylase by c-Myc J. Biol. Chem., June 10, 2005; 280(23): 22492 - 22496. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. H. Bergman, K. D. Passalacqua, R. Gaspard, L. M. Shetron-Rama, J. Quackenbush, and P. C. Hanna Murine Macrophage Transcriptional Responses to Bacillus anthracis Infection and Intoxication Infect. Immun., February 1, 2005; 73(2): 1069 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. I. Bussiere, R. Chaturvedi, Y. Cheng, A. P. Gobert, M. Asim, D. R. Blumberg, H. Xu, P. Y. Kim, A. Hacker, R. A. Casero Jr., et al. Spermine Causes Loss of Innate Immune Response to Helicobacter pylori by Inhibition of Inducible Nitric-oxide Synthase Translation J. Biol. Chem., January 28, 2005; 280(4): 2409 - 2412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Xu, R. Chaturvedi, Y. Cheng, F. I. Bussiere, M. Asim, M. D. Yao, D. Potosky, S. J. Meltzer, J. G. Rhee, S. S. Kim, et al. Spermine Oxidation Induced by Helicobacter pylori Results in Apoptosis and DNA Damage: Implications for Gastric Carcinogenesis Cancer Res., December 1, 2004; 64(23): 8521 - 8525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Chaturvedi, Y. Cheng, M. Asim, F. I. Bussiere, H. Xu, A. P. Gobert, A. Hacker, R. A. Casero Jr., and K. T. Wilson Induction of Polyamine Oxidase 1 by Helicobacter pylori Causes Macrophage Apoptosis by Hydrogen Peroxide Release and Mitochondrial Membrane Depolarization J. Biol. Chem., September 17, 2004; 279(38): 40161 - 40173. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Galgani, I. Busiello, S. Censini, S. Zappacosta, L. Racioppi, and R. Zarrilli Helicobacter pylori Induces Apoptosis of Human Monocytes but Not Monocyte-Derived Dendritic Cells: Role of the cag Pathogenicity Island Infect. Immun., August 1, 2004; 72(8): 4480 - 4485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Gobert, Y. Cheng, M. Akhtar, B. D. Mersey, D. R. Blumberg, R. K. Cross, R. Chaturvedi, C. B. Drachenberg, J.-L. Boucher, A. Hacker, et al. Protective Role of Arginase in a Mouse Model of Colitis J. Immunol., August 1, 2004; 173(3): 2109 - 2117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zabaleta, D. J. McGee, A. H. Zea, C. P. Hernandez, P. C. Rodriguez, R. A. Sierra, P. Correa, and A. C. Ochoa Helicobacter pylori Arginase Inhibits T Cell Proliferation and Reduces the Expression of the TCR {zeta}-Chain (CD3{zeta}) J. Immunol., July 1, 2004; 173(1): 586 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Duleu, P. Vincendeau, P. Courtois, S. Semballa, I. Lagroye, S. Daulouede, J.-L. Boucher, K. T. Wilson, B. Veyret, and A. P. Gobert Mouse Strain Susceptibility to Trypanosome Infection: An Arginase-Dependent Effect J. Immunol., May 15, 2004; 172(10): 6298 - 6303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Menaker, P. J. M. Ceponis, and N. L. Jones Helicobacter pylori Induces Apoptosis of Macrophages in Association with Alterations in the Mitochondrial Pathway Infect. Immun., May 1, 2004; 72(5): 2889 - 2898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Meyer, K. S. Ramanujam, A. P. Gobert, S. P. James, and K. T. Wilson Cutting Edge: Cyclooxygenase-2 Activation Suppresses Th1 Polarization in Response to Helicobacter pylori J. Immunol., October 15, 2003; 171(8): 3913 - 3917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Gobert, B. D. Mersey, Y. Cheng, D. R. Blumberg, J. C. Newton, and K. T. Wilson Cutting Edge: Urease Release by Helicobacter pylori Stimulates Macrophage Inducible Nitric Oxide Synthase J. Immunol., June 15, 2002; 168(12): 6002 - 6006. [Abstract] [Full Text] [PDF]
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) or iNOS-/- (
) mice were cocultured with
H. pylori in DMEM-BSA in the presence or the absence of
norNOHA, a specific arginase inhibitor. At 24 h apoptosis levels
were measured by ELISA. Each bar represents the mean ± SEM of
data collected from duplicate experiments with four mice. **,
p < 0.01 (vs control macrophages); 
