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,§
Departments of
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Pediatrics,
Internal Medicine, and
Microbiology and Immunology, and the
§
Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, TX 77555
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Urease is regarded as an important virulence factor for H.
pylori survival in gastric acid through the hydrolysis of urea.
Ammonia, a byproduct of this process, neutralizes hydrochloric acid and
thus allows H. pylori to colonize the human stomach. Urease
is one of the most abundant proteins produced by H. pylori,
representing 
5% of the total bacterial cell protein (3, 4). Isogenic mutant H. pylori strains showed that
urease is essential for colonization because the mutant bacteria were
unable to colonize gnotobiotic pigs (5). However, the
urease-negative H. pylori strains also failed to colonize
under hypochlorhydric conditions (6), which suggested a
role for urease in colonization beyond its ability to neutralize the
gastric acidity.
Adherence of H. pylori to the surface of gastric epithelial cells is undoubtedly a key step in colonization and has been shown to induce host cell protein phosphorylation and changes in the cytoskeleton (7, 8). One of the consequences of the signals initiated during H. pylori adhesion to cultured gastric epithelial cells is increased apoptosis of the epithelium, which has been noted to occur in vivo (9, 10). Thus, the bacterial structures responsible for adhesion and stimulation of host cell death may be regarded as important virulence factors. Although a number of epithelial cell molecules have been reported to contribute to H. pylori binding to the epithelium (11, 12, 13, 14, 15, 16, 17), it is not clear which cell and bacterial factors stimulate host cell apoptosis.
Class II MHC molecules are best known for their regulation of immune responses through the presentation of foreign Ags to CD4+ T cells. However, engagement of class II MHC by TCRs or bacterial superantigens also has consequences for the class II MHC-expressing cells, including apoptosis (18). We have recently shown that H. pylori uses class II MHC molecules on the surface of gastric epithelial cells as receptors (19). Following the binding of H. pylori to class II MHC, apoptosis was increased in gastric epithelial cells. The induction of gastric epithelial cell apoptosis was dependent on class II MHC expression because it was blocked by anti-class II MHC Abs, and cells deficient in class II MHC expression were refractory to apoptosis induction by H. pylori. Because urease is a major surface protein of H. pylori that facilitates the colonization of the gastric epithelium and has been shown to activate human monocyte secretion of cytokines (20, 21), we sought to examine whether urease is a bacterial receptor that binds to class II MHC molecules on gastric epithelial cells and is thus responsible for initiating apoptosis. In this report, we show that H. pylori urease binds to cells which express class II MHC and to affinity-purified class II MHC. Urease binding to solubilized membrane proteins from class II MHC+ cells is inhibited by anti-class II MHC Abs. Urease bound to a panel of human B cell lines expressing a diverse set of class II MHC alleles. Further, we show that urease induced apoptosis that depends on the expression of class II MHC molecules by the target cells. The observations presented in this report demonstrate a previously unsuspected role for urease in H. pylori pathogenesis and provide added support for the use of this bacterial product in the development of vaccination strategies to reduce infection and cellular injury by this important human pathogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The gastric epithelial cell lines, Kato-III, N87, and AGS were
obtained from American Type Culture Collection (Manassas, VA) and were
maintained in RPMI 1640 medium supplemented with 10% FCS. Additional
cell lines differing in the levels of class II MHC molecules’
expression were also used. COS-1 cells were kindly provided by Dr. M.
Falzon (University of Texas Medical Branch
(UTMB)3, Galveston,
TX). COS-1 cells transfected with the genes encoded for HLA-DR1 
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and ß-chains (1D12; 22) were provided by Dr. M. Xu (Ag
Express, Worcester, MA). COS cells were transfected with the CXCR2
receptor provided by Dr. J. Navarro (UTMB). A panel of B cell lines
expressing various class II MHC alleles in a homozygous manner, kindly
provided by the UTMB Tissue Ag Laboratory, were also included. The B
cell lines and their alleles were as follows: 9070 (DRB1*0803;
DQB1*0301), 9063 WT47 (DRB1*13; DRB3), 9064 (DRB1*14; DRB3), 9036
(DRB1*11; DRB3), and 9002 MZ070782 (DRB1*01). The hybridomas HB55,
HB145, and HB180 (American Type Culture Collection) were used as the
source of anti-human class II MHC Abs (L243, IVA12, and 9.3F10
respectively).
Purification of Fab from anti-class II MHC (IVA-12) Ab
Anti-class II MHC (IVA-12) Ab was purified through a Protein G Sepharose HR 16/5 column. One-hundred microliters of 2 mg/ml purified IgG in PBS was added into microcentrifuge tubes, which also received 100 µl of 0.1 mg/ml papain (Sigma, St. Louis, MO). The tubes were incubated for 8 h at 37°C. Iodoacetamide (0.3 M) in PBS was added into the tubes to inactivate papain, and the samples were dialyzed for 2 h at 4°C with stirring. Optimal conditions to obtain Fab from IgG were assessed following separation of the digested samples by SDS-PAGE. After papain treatment, digested IgG was injected into a Protein G Sepharose HR 16/5 column again. The unbound material (Fab) was collected and its purity was confirmed by SDS-PAGE. The concentration of Fab was estimated by the absorbance at 280 nm.
Binding of urease to the surface of gastric epithelial cells and B cells
Recombinant H. pylori urease was obtained from Orovax
(Cambridge, MA) and was biotinylated to assess its binding to the
surface of epithelial cells using flow cytometry. Briefly, gastric
epithelial cells (Kato-III, N87, and AGS) were cultured in the presence
or absence of 100 U/ml IFN-
for 48 h and then washed twice with
PBS containing 1% BSA and 0.02% sodium azide. Gastric epithelial cell
lines as well as B cell lines expressing multiple class II MHC alleles
(9070, 9064, 9063 WT47, 9036, and 9002 MZ070782) were then incubated
with biotinylated urease for an additional 45 min on ice. After washing
with PBS/BSA/azide the cells were incubated with avidin-PE to detect
the bound biotinylated urease. The cells were resuspended in 0.5 ml
paraformaldehyde (0.5% in PBS) and were analyzed by flow cytometry on
a FACScan (Becton Dickinson, San Jose CA). Competition studies were
done by preincubating the cells with the indicated proteins at the
indicated concentrations for 2 h on ice followed by incubation
with biotinylated urease for an additional 2 h on ice.
Detection of class II MHC among gastric epithelial cell-derived proteins that bound to urease-coated Sepharose beads
Recombinant H. pylori urease was coupled to cyanogen bromide-Sepharose 4B beads (Sigma), according to the manufacturer’s instructions. The urease-coated beads were used to capture gastric epithelial cell-derived proteins with affinity for urease. Beads treated under the same coupling conditions in the absence of urease were used as controls. Gastric epithelial cell proteins were metabolically labeled and solubilized as described previously (19). Briefly, the cells were metabolically radiolabeled with [35S]methionine and [35S]cysteine (0.5 mCi/108 cells). Cells were then washed and lysed in ice-cold 10 mM Tris-HCl buffer (pH 8.1) containing protease inhibitors (2 mM PMSF and 10 mM N-ethyl maleimide) for 30 min on ice. Nuclei were removed by centrifugation at 500 x g for 10 min. The supernatant was subjected to ultracentrifugation at 100,000 x g for 45 min, and microsomal membranes were obtained as a pellet. The membrane pellet was solubilized in the lysis buffer containing 0.5% Triton X-100 followed by a second ultracentrifugation at 100,000 x g for 45 min. The membrane proteins were pre-cleared by incubation with normal rabbit serum, which was followed by three passages over Staphylococcus aureus Cowen strain A and one passage over protein A-Sepharose beads (Sigma).
The solubilized membrane proteins were incubated with urease-coated beads for 16 h at 4°C. After incubation, unbound proteins were washed with PBS until the cpm of the wash reached background levels. The proteins bound to the urease-coated beads were eluted with 0.1 M glycine buffer(pH 2.2), and the corresponding cpm were measured by liquid scintillation counting to assess the level of binding. The eluted proteins were separated on 12% SDS-PAGE and were analyzed by autoradiography, as described previously (23).
To determine the extent of class II MHC binding to the urease-coated beads in relation to other proteins that could bind, the Kato-III and N87 membrane proteins were incubated with a mixture of the anti-MHC II Abs IVA12, L243, and 9.3F10 or an isotype control. Subsequently, urease-coated beads were incubated with the membrane proteins that were pretreated with anti-class II MHC Abs.
Urease binds to purified class II MHC from gastric epithelial cells
Wells in 96-well microtiter plates were coated with recombinant urease (400, 100, and 25 µg/ml), BSA, the anti-HLA-DR Ab 9.3F10, or with borate buffered saline (BBS) used as the binding buffer. Affinity-purified [35S]methionine-labeled class II MHC (19) were added to the wells and were incubated for 2 h at room temperature. After incubation, unbound proteins were washed with PBS from the wells. The bound class II MHC molecules were eluted with SDS sample buffer, run on 12% SDS-PAGE gel, and detected by autoradiography.
Cross-linking of surface class II MHC on gastric epithelial cell lines
Cells (2.5–3 x 106) were allowed to attach overnight in a 25-cm2 flask. The cells were exposed to varying concentrations of urease or to anti-class II MHC Ab (RFD1, IgM isotype at 10 µg/ml; Serotec, Raleigh, NC) for 12, 24, 48, and 72 h. At the end of incubation, the cells were harvested and the DNA fragmentation, as an indicator of apoptosis, was assayed as described below.
Detection of DNA fragmentation
DNA fragmentation was measured using a commercially available ELISA kit (Boehringer Mannheim, Indianapolis, IN), as previously described (19). After stimulating the cells with urease, low m.w. nucleosome fragments in the cytoplasm of the cells were collected as previously described (19). Wells in an ELISA plate were coated with anti-histone Ab. The nucleosome-containing samples were added to the wells and trapped by the Ab. After incubation, the unbound DNA was washed off and quantities of histone-associated DNA were estimated using an anti-DNA and an enzymatic detection system. The absorbance was measured at 405 nm by Titertek Multiskan MCC/340 (Irvine, CA) and compared with a substrate solution as a blank. The apoptotic index was calculated according to the manufacturer’s instructions by dividing the absorbance of stimulated cells by the absorbance for control cells.
Statistical analysis
Results are expressed as the mean + SEM. Data were compared using a two-tailed Student’s t test and the differences were considered significant if p values were <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that class II MHC on human gastric
epithelial cells act as receptors for H. pylori
(19). Initial efforts to identify H. pylori
proteins responsible for bacterial binding to class II MHC and
induction of apoptosis focused on outer membrane proteins from H.
pylori. The extracted outer membrane proteins were found to induce
apoptosis. Urease was detected among the extracted proteins using an
ELISA assay and Western blots (not shown). Because urease is an
abundant protein expressed on the surface of H. pylori
(21) and has been shown to stimulate cytokine secretion in
monocytes (20), we examined whether urease could
contribute to bacterial binding to the epithelium. To examine urease
binding we treated three human gastric epithelial cell lines with
either medium or IFN-, since we have previously shown that IFN-
increases both the expression of class II MHC and the binding of
H. pylori to gastric epithelial cells (19). The
cell lines were selected based on the level of class II MHC molecules
expressed on their surface. As previously shown, both Kato-III and N87
express class II MHC, and the expression is higher in the latter cell
line (19). The variant of the AGS cell line used in these
studies does not express class II MHC, even after IFN- stimulation.
The extent to which urease bound to the surface of the gastric
epithelial cell lines correlated with the level of class II MHC
expression (Fig. 1
). IFN- increased
the amount of urease binding only to the cell lines which express class
II MHC molecules.
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Because H. pylori use class II MHC on gastric
epithelial cells as receptors (19) and the extent of
urease binding coincided with the level of class II MHC expression by
the three different cell lines, we examined whether class II MHC
molecules were responsible for urease binding. To that end,
urease-coated beads were used to identify which gastric epithelial cell
membrane proteins bound to urease. Solubilized membrane proteins from
gastric epithelial cell lines (Kato-III, N87, and AGS) metabolically
labeled with [35S]methionine and
[35S]cysteine were incubated with urease-coated
beads or uncoated beads. The extent to which epithelial proteins bound
to urease was quantified by measuring the amount of radioactivity in
the proteins that were eluted from the beads (Fig. 2A). The results showed that
radioactive protein binding to the urease beads was high when class II
MHC expression was high, particularly after IFN- treatment (Fig. 2A). The proteins obtained from AGS cells, which do not
express class II MHC, demonstrated minimal binding to the urease-coated
beads compared with the binding to control beads. When the proteins
that bound to urease-beads were analyzed by SDS-PAGE, bands that
coincided in size with class II MHC - and ß-chains were detected
(Fig. 2B). Samples with low cpm did not show any bands by
autoradiography even after 3 wk of exposure. The identity of the bands
was confirmed by western blotting with a mixture of anti-class II
MHC Abs (not shown).
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, C and D). Because proteins other than
class II MHC were detected in the autoradiographs, we sought to confirm
whether they might represent proteins that are coexpressed with class
II MHC and also bind urease or proteins that coprecipitate with class
II MHC. To that end, we preincubated the gastric epithelial proteins
with an anti-class II MHC Ab mixture (IVA12, L243, and 9.3F10) or
an isotype control before they were incubated with urease-coated beads.
Blocking of class II MHC decreased the binding to the urease-coated
beads to background levels comparable to those obtained with control
beads (Fig. 2E). The high m.w. proteins were absent in all
conditions where class II MHC were absent. These proteins may represent
proteins recruited by class II MHC when they are cross-linked by urease
and which may be involved in the signaling processes leading to
apoptosis. Urease binds to affinity-purified class II MHC from gastric epithelial cells
As an independent approach to confirm that urease adheres to class
II MHC molecules on gastric epithelial cells, 96-well microtiter plates
were coated with various concentration of either urease, BSA,
anti-HLA-DR, or BBS (binding buffer). Then, metabolically labeled
and purified class II MHC molecules were added to each well. Unbound
class II MHC molecules were washed off, and bound class II MHC were
eluted and separated on a 12% SDS-PAGE gel (Fig. 3). The gel revealed that class II MHC
chains were recovered from the wells that were coated with urease (Fig. 3) whereas no class II MHC molecules were recovered from the wells
coated with either BBS or BSA. Affinity-purified class II MHC molecules
captured by the anti-HLA-DR Ab were run on the last lane as a
positive control. These data confirm that urease binds to class II MHC
molecules.
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Given that urease binds directly to class II MHC molecules, it was
important to determine whether polymorphic differences in class II MHC
could influence the binding of urease. To that end, we assessed the
binding of urease to a panel of human B cells homozygous in HLA-DR or
HLA-DQ (9070, 9064, 9063 WT47, 9036, and 9002 MZ070782). Urease bound
to the various alleles examined. The only apparent differences in
binding were due to differences in the level of class II MHC expression
by each of the cell lines (Fig. 4). The
binding to multiple class II MHC alleles is a property shared by
bacterial proteins referred to as superantigens. To determine whether a
superantigen that is known to bind to class II MHC could influence the
interaction of urease with cells expressing class II MHC, the cells
were preincubated with either staphylococcal enterotoxin B (SEB) or
urease before incubation with biotinylated urease. The binding of
biotinylated urease to the cells was detected with PE-avidin as
described in Materials and Methods. Using this approach, it
was noted that SEB, as well as unbiotinylated urease, significantly
reduced the level of urease binding (Fig. 5). A mixture containing the
anti-class II MHC Abs L243, IVA12, and 9.3F10 also reduced the
level of urease binding.
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H. pylori binding to class II MHC on gastric epithelial
cells triggers apoptosis of the epithelial cells (19).
Ligation of HLA-DR by the bacterial superantigens staphylococcal
enterotoxin A and SEB has been shown to stimulate apoptosis (18, 24, 25). Thus, to confirm whether urease acts as a superantigen
in its ability to ligate class II MHC molecules and whether it
represents the bacterial coreceptor that initiates apoptosis of gastric
epithelial cells, we examined its ability to trigger apoptosis on
epithelial cells. IgM Abs to class II MHC were used as controls in
these studies. After cross-linking with the anti-class II MHC Ab,
there was a significant increase in the apoptotic index in cells
expressing class II MHC molecules (Kato-III), but not in cells lacking
class II MHC molecules (AGS) as evaluated by ELISA (3.95 ± 0.3,
p < 0.05; Fig. 6A). No apoptosis was detected
by ELISA when the cells were cultured either in medium alone or with
isotype control Ab. When Kato-III and AGS cells were exposed to various
concentrations of urease (0.1, 1, and 10 µg/ml) for 48 h, there
was a dose-dependent induction of apoptosis by urease in class II
MHC+ Kato-III cells, but not in class II
MHC- AGS cells (Fig. 6B).
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A). The role of class II MHC
on gastric epithelial cells in the binding of urease and subsequent
induction of apoptosis was further confirmed when Fab from an
anti-class II MHC Ab inhibited apoptosis in a dose-dependent manner
(Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to contributing to colonization of the target tissue, adhesion of the bacteria to the target cells is believed to be important in pathogenesis as it allows for a direct effect of the bacterial toxins on the epithelium. Also, the release of IL-8 by gastric epithelial cells appears to be enhanced by direct contact of the bacteria with the host cells (26, 27, 28, 29). The release of IL-8 by gastric epithelial cells influences the recruitment of neutrophils, which together with other recruited inflammatory cells, mediate some of the epithelial damage. Attachment of the bacteria to the epithelial cells also induces actin polymerization and the phosphorylation of cellular proteins (7, 8), which may be reflective of the stimulation of signaling mechanisms that lead to programmed cell death. Apoptosis of the gastric epithelium has been reported to be increased in the infected mucosa (10, 30) and may be a mechanism for the epithelial damage that leads to ulcer formation or atrophy. Because H. pylori is not an invasive bacterium, the injury associated with the attachment of H. pylori to the gastric epithelium must be due to the interactions between bacterial and host cell surface proteins that are capable of delivering intracellular signals.
Gastric epithelial cells express class II MHC molecules on their surface and their expression increases in the H. pylori-infected tissue (31, 32, 33). The best known function of class II MHC is the presentation of Ags to CD4+ T cells, which are also increased in the infected gastric mucosa (34, 35). However, engaged class II MHC may also deliver intracellular signals to the cells which express them. For instance, binding of bacterial superantigens to class II MHC molecules has been shown to trigger apoptosis in APCs (18, 24, 25). We have recently shown that H. pylori uses class II MHC on gastric epithelial cells as receptors (19). H. pylori induced apoptosis of cells expressing class II MHC either constitutively or following gene transfection, and apoptosis induction by H. pylori was blocked by Abs to class II MHC. However, the bacterial proteins that attached to class II MHC molecules on gastric epithelial cells and transduce apoptotic signals were unknown.
Based on the observations that H. pylori can bind to class II MHC on gastric epithelial cells, the expression of proteins with the ability to bind human class II MHC by these bacteria was suspected. Our initial analysis by ELISA and Western blot of extracted outer membrane proteins revealed the presence of urease in extracts that were able to elicit apoptosis of human gastric epithelial cells. Urease is present on the outer membrane surface of H. pylori following adsorption of urease from other H. pylori bacteria undergoing autolysis (21). Thus, it was not surprising to find urease among the proteins in the extract; however, it was surprising to find that H. pylori urease was capable of binding to human class II MHC and inducing apoptosis.
The beads coated with urease only immunoprecipitated cellular proteins when class II MHC were present or in the absence of anti-class II MHC Abs. However, when class II MHC were precipitated by urease-coated beads, there were also unknown additional proteins of a higher m.w. that coprecipitated. These additional proteins may represent proteins that have an affinity for class II MHC and which could be involved in the intracellular signals delivered by cross-linked class II MHC. Alternatively, they may reflect proteins that normally do not have affinity for class II MHC, but when the large urease complex becomes bound by class II MHC, other sites become exposed on the urease complex which bind those additional proteins. It is also possible that those proteins represent cell surface proteins with a low affinity for urease and may explain why unbiotinylated urease did not efficiently compete biotinylated urease on the cells before fixation with paraformaldehyde as is normally done for flow cytometry. The inability of anti-class II MHC Abs to completely block the binding of urease may suggest that the Abs we used are not to the epitopes on class II MHC involved in urease binding, and the inhibition that we see is possibly due to inefficient stearic hindrance. Although urease was shown to bind to class II MHC by various independent methods and the inhibition of that interaction prevents apoptosis, there may be other structures on the cell surface to which urease binds and could not be efficiently competed. However, those additional structures may not be involved in the signaling processes triggered by urease.
The observation that urease binds to class II MHC on gastric epithelial cells may help explain why urease-negative mutants of H. pylori failed to colonize in an animal model of infection even under hypochlorhydric conditions (6). In other studies with urease-negative mutants, it was speculated that the failure to colonize was due to a requirement of the enzymatic activity to provide a more hospitable environment for H. pylori to grow (5, 36). Interestingly, one of those mutants that fails to form the enzyme complex was found to adhere to gastric epithelial cells in vitro (37). Because the recombinant urease used in these studies lacks enzymatic activity, but bound to and induced apoptosis of class II MHC+ cells, it may be inferred that the binding of urease to class II MHC may be mediated by a domain that is present in one of the individual subunits, regardless of its association as an enzymatic complex.
While inducing apoptosis of gastric epithelium, H. pylori urease may play a role in the compensatory increase in cellular proliferation and could thus contribute to an increase in DNA mutations which may give rise to gastric cancers. Although our studies with established cell lines have not shown the stimulation of cell proliferation by urease, some cell populations may be stimulated in vivo. This possibility parallels observations with the intact bacterium.
In summary, H. pylori urease may represent an important target for immunization for reasons other than what has been previously suspected. The development of Abs that would block the binding of urease to class II MHC may effectively prevent bacterial binding to the target tissue and colonization, and reduce the cellular injury. Thus, efforts are currently directed at mapping the sites of urease contact with human class II MHC molecules.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Victor E. Reyes, Children’s Hospital, Room 2.300, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555.
3 UTMB, University of Texas Medical Branch; BBS, borate buffered saline; SEB, staphylococcal enterotoxin B.
Received for publication June 28, 1999. Accepted for publication June 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-chains. J. Immunol. 146:3877.[Abstract]
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K. Hirota, K. Nagata, Y. Norose, S. Futagami, Y. Nakagawa, H. Senpuku, M. Kobayashi, and H. Takahashi Identification of an Antigenic Epitope in Helicobacter pylori Urease That Induces Neutralizing Antibody Production Infect. Immun., November 1, 2001; 69(11): 6597 - 6603. [Abstract] [Full Text] [PDF]
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