ARTICLE
Auteur(s) : Gergely Klausz1, Andrea
Tiszai2, László Tiszlavicz3, Zsófia
Gyulai1, Zsuzsa Lénárt2, János
Lonovics2, Yvette Mándi1
1 Department of Medical Microbiology and
Immunobiology,
2 1st Department of Internal Medicine,
3 Department of Pathology, Faculty of Medicine,
University of Szeged, Szeged, Hungary
Introduction
Helicobacter pylori infection almost invariably causes
chronic gastritis, but only a proportion of the infected subjects
develop peptic ulcers [1]. The host immune response might be of
importance as regards the outcome of the infection [2]. The local
inflammation in H. pylori infection is characterized by the
infiltration of neutrophils and lymphocytes into the gastric mucosa
and by an increased production of the proinflammatory cytokines
IL-1β, IL-6, IL-8 and TNF-α [2, 3]. In particular, IL-8 as a
neutrophil chemotactic and activating factor has been suspected of
playing an important role in H. pylori-associated disease.
H. pylori strains carrying the pathogenicity island
including the gene encoding the cytotoxicity-associated protein
(CagA) are more commonly isolated from duodenal ulcer (DU) patients
[4], and these CagA positive strains display an increased
IL-8 – inducing capacity [5]. The effects of proinflammatory
cytokines however, might be counteracted by locally produced IL-10,
a TH2 cytokine which has anti-inflammatory effects [6, 7].
H. pylori infection could lead to increased nitric oxide
(NO) production by activated macrophages and neutrophils in the
gastric mucosa. INOS is increased in symptomatic and asymptomatic
humans infected with H. pylori, and a decrease in staining
for iNOS and catalase was observed after H. pylori
eradication [8] In the presence of oxygen free radicals, NO can
form a genotoxic peroxynitrite, which can contribute to further
destruction of the mucosa. Nitrotyrosine as a stable end product of
peroxynitrite, can be used for the detection of NO and
peroxynitrite formation [9, 10].
The aim of this study was an investigation of the mucosal
production of TNF-α, IL-6, IL-8, IL-10 and nitrotyrosine in H.
pylori-infected patients. We determined the concentrations of
these cytokines in gastric antral mucosal specimens from H.
pylori-positive DU patients and compared them with those in
mucosal specimens from H. pylori-negative subjects. In
addition, we investigated whether the tissue levels of the
cytokines correlated with the peripheral cytokine levels, and the
CagA status of patients.
Methods
Patients
Forty H. pylori-positive patients with DU (mean age
47.6 years, male/female 10/30) were studied. Twelve H.
pylori-negative subjects (mean age 36.8 years, male/female
8/4) from whom biopsies were taken for different reasons served as
control. The project was approved by the Clinical Ethical Committee
of the Medical Faculty of Szeged University and informed consent
was obtained all from of the patients. Multiple biopsy specimens
were taken during upper gastrointestinal endoscopy from adjacent
sites of the gastric antrum and corpus for histology, for rapid
urease test (Controloc) and for PCR. The presence of H.
pylori was confirmed, and the severity of gastritis was graded
with the Sydney Classification System [11]. Only individuals who
gave positive histology and urease tests were regarded as infected
with H. pylori. Additionally, the PCR assays with
oligonucleotide primers homologous to a portion of the urease C
gene of H. pylori [12] were performed with DNA extracted
from biopsy materials [13], and results demonstrated 100%
specificity. Additionally, two biopsy specimens were taken for
cytokine ELISA and nitrotyrosine Western blotting.
For serological investigations, the sera of the 40 DU
patients patients were compared with those of 100 apparently
healthy blood donors.
Cytokine assays
Biopsy specimens were immediately placed in RPMI
1640 medium and transported to the laboratory. Specimens were
homogenized in 0.5 ml of 3.3 mM CaCl2. Aliquots of the
homogenate were assayed for total proteins by the Lowry method
[14], specimens were stored at – 70°C until assayed.
Concentrations of cytokines in homogenates were determined by
ELISA assays, with the following ELISA kits: TNF-α BIOSOURCE,
IL-6 BIOSOURCE, IL-8 BIOSOURCE, and IL-10 BIOSOURCE. The
procedures were performed in full accordance with the instructions
of the manufacturer. The mucosal levels of cytokines were expressed
as pg/mg biopsy protein.
Nitrotyrosine detection
Nitrotyrosine was detected by an ECL Western blot technique with
the application of anti-nitrotyrosine monoclonal antibody (HM 11,
HyCult Biotechnology). This highly specific monoclonal antibody
reacts with proteins containing nitrotyrosine.
Total protein from biopsy samples was solubilized after
ultrasonication, with subsequent processing in Laemmli buffer [15],
and individual protein samples (10 µg/lane) were fractionated
on 15% SDS-polyacrylamide gels. After electrophoresis, proteins
were transferred to nitrocellulose blotting membrane (Trans Blot
BioRad) using a semi-dry HOEFER transfer system. Membranes were
blocked in 5% nonfat dried milk. Immunoblotting was performed with
anti-human nitrotyrosine MoAb HM 11 at a dilution of 1:500. A
goat anti-mouse immunoglobulin G antibody conjugated to horse
radish peroxidase (Bio-Rad) was used to form antigen-antibody
complexes, which were detected via a chemiluminescent reaction (ECL
Amersham), followed by exposure to X-ray film (KODAK BIOMAX).
The presence of nitrotyrosine in tissue homogenate was regarded
as positive in the event of the detection of at least two or three
positive bands, indicating the nitrosylation of tyrosine residues.
For positive and negative controls, peroxynitrite-treated and
control AGS (human gastric epithelial cells #CRL –1739; American
Type Culture Collection) cell lysates were processed in the same
way. To confirm the specificity of the signal for Nytr the
following procedures were included: a) omission of the primary
antibody, b) chemical reduction of Nytr with 100 mM dithionite
before the addition of primary antibody.
Serology
For the simultaneous evaluation of H. pylori positivity
and CagA status, sera from the 40 patients with DU and sera
from the 100 blood donors were processed with a Western blot
assay elaborated by MICROGEN GmbH Germany. In this test, the strips
are incubated with 1:100 dilution of human serum, and at the
end of Western assay individual sharp bands demonstrate the
presence of antigen-antibody complexes. This test allows the
detection of different antibodies against bacterial antigens (UreB,
HspA, HspB, UreA, and Fla), VacA, and CagA. The molecular weights
and names of the antigens are provided by the manufacturer.
In vitro cytokine production in peripheral blood
samples
For the in vitro cytokine induction experiments, blood
was drawn with anticoagulant (EDTA), For whole blood cultures,
1 ml blood was mixed with 1 ml RPMI
1640 supplemented with 10% fetal calf serum (FCS), and
incubated with 109 H. pylori 26695 for 18 h
at 37°C. Supernatants of whole blood cultures were thereafter
tested for the presence of TNF-α, IL-6 and IL-8 by ELISA. For the
coculture experiments Cag A positive H. pylori
26695 strain was used, which was a kind gift from D.E. Berg
(Department of Molecular Biology and Genetics, Washington
University Medical School, St. Louis, USA).
Bacteria were maintained on Brain Heart infusion agar containing
10% horse blood, and incubated in a microaerophilic atmosphere
[16]. Inocula for coculturing were diluted from suspensions that
had been prepared from 72 h subcultures and adjusted by
comparison of the absorbance with standards.
Statistics
All statistical calculations were performed with the GraphPad
Prism statistical program. Differences between levels of cytokine
production were computed with the Mann-Whitney, and Student
tests.
Results
Mucosal TNF-α, IL-6, IL-8 and IL-10 production from
biopsy specimens
DU patients infected with H. pylori exhibited a higher
antral cytokine production than did H. pylori-negative
patients. Results are summarized in Figure 1: TNF-α:
median = 10.55 (range 1.2-23) pg/mg protein, versus 0.85
(range 0.0-2.7) pg/mg protein, p < 0.001; IL-6:
median = 425 (range 50.0-900.0) pg/mg protein, versus 25
(range 6.0-55) pg/mg protein, p < 0.001; IL-8:
median = 1890 (range 350-3200) pg/mg protein, versus 485
(range 50-1200) pg/mg protein, p < 0.001; and IL-10:
median = 3.95 (range 0.9-36.0) pg/mg protein, versus 2.4
(range 1.5-3.8) pg/mg protein, p < 0.05.
There was an inverse correlation between the levels of TNF-α and
the anti-inflammatory cytokine IL-10 (Figure 2), which clearly
demonstrates the imbalance between TH1 and TH2 cytokines in DU
patients [17]. The highest TNF-α content was measured in those
specimens, in which the lowest IL-10 concentrations were
detected.
Mucosal nitrotyrosine formation
Nitrotyrosine (Nytr) is formed in tissues in the presence of the
active metabolite NO Nitrotyrosine is a stable end-product of
nitrosylation of tyrosine and can be detected by histology on
paraffin sections and by Western blot [9, 10]. ECL Western blotting
was applied for the immunodetection of nitrated proteins in protein
lysates of biopsy samples. Figure 3 reveals a
representative result of immune detection of nitrotyrosine, showing
several distinct bands, especially in the lower molecular range
between 20 and 47.5 kDa. (Lanes 2 and 3). By comparison, no
specific bands were obtained in biopsy specimens of H.
pylori-negative subjects (Lane 1). Lane 4 demonstrates a
negative result obtained with a sample from one of the H.
pylori-positive DU patients. AGS cells (gastric epithelial
cells) treated with peroxynitrite (100-200 µM) served as a
positive control. Western blotting gave positive results only with
the peroxynitrite-treated cells (data not shown). In 23 of the
40 biopsy samples, positive results were obtained with Western
blotting, as in the representative experiment illustrated in Figure 3. None of the
biopsy specimens originating from the H. pylori-negative
controls was positive in this test. In a comparison of the
nitrotyrosine assay results with the Sydney scores, all of these
23 samples were evaluated at grade 3, and their mean
concentrations of TNF-α and IL-8 were
17.5 ± 2.1 pg/mg and
2540 ± 650 pg/mg, respectively.
Comparison of serum cytokine levels and ex vivo inducible
cytokine release in whole blood from DU patients and healthy blood
donors
We investigated whether the high cytokine production in H.
pylori-infected patients was characteristic only locally, or
whether the gastric infection could lead to an elevated cytokine
production at the periphery too. For this purpose, the cytokine
concentrations in the sera and the in vitro cytokine
production of peripheral white blood cells were determined. Table 1 summarizes the results on such
patients and on healthy blood donors attending the the South
Hungarian Regional Blood Bank of National Transfusion Service,
Szeged. These subjects were age- and sex-matched (mean age
51.2 years, male/female 25/75), with no gastrointestinal
disorders or symptoms, and without a history of gastrointestinal
disease or any other relevant illness. TNF-α is rarely measurable
in healthy humans, and was almost undetectable in DU patients.
There was no difference in mean IL-6 and IL-8 concentrations
in the sera of the DU patients and the blood donors. H.
pylori 26695 induced moderate TNF-α and IL-6 production
when whole blood cultures were coincubated with
H. pylori for 18 h. There was no significant
difference between the amounts of these cytokines in supernatants
of cultures of whole blood from the DU patients and healthy blood
donors. The in vitro H. pylori infection caused a
greater IL-8 production in the DU patients than in the
controls
(mean ± SD = 46954 ± 26100 pg/ml
versus 36484 ± 22411 pg/ml,
p < 0.01) (Table 1). More details
concerning in vitro IL-8 production in individual whole
blood cultures (WBC) are presented in Figure 4. Despite the great
variation in IL-8 production, the DU patients exhibited
significantly greater IL-8 production at the periphery than
that of the healthy blood donors. There was no further difference
in the in vitro cytokine-producing capacity between the
H. pylori-positive Cag A+ and CagA– healthy blood donors, or
between the H. pylori-positive and negative blood donors. It
is interesting, that in DU patients not only in the H.
pylori-stimulated, but also in the basal, nonstimulated WBC
culture supernatants, relatively higher IL-8 concentrations
were measured, but statistically the differences were not
significant (3.024 ± 1.933 ng/ml versus
2.45 ± 1.257 ng/ml). (Figure
4).
Table 1. Serum cytokine levels and ex
vivo cytokine release capacity* in whole blood from
DU patients and healthy donors
|
in
sera |
in
WBC** |
in
sera |
in
WBC** |
in
sera |
in
WBC** |
| DU patients |
3.5 ± 0.2 |
5 ± 1.25 × 102 |
20 ± 5 |
5 ± 0.5 × 103 |
75 ± 58 |
4.6 ± 2.61 × 104*** |
| Controls |
3.1 ± 0.3 |
4.8 ± 1.8 × 102 |
15 ± 10 |
4.9 ± 0.45 × 103 |
68 ± 65 |
3.6 ± 2.24 × 104 |
*Whole blood samples from DU patients (n = 40)
and healthy donors (n = 100) was diluted 1:1 with RPMI
1640 + 10% FCS (whole blood cultures**) and
was incubated in the presence of 109H.
pylori26695 for 18 h at 37 C. Cytokine levels in cell-free
supernatants were measured by ELISA. Results are expressed as
means ± SD statistical significance was detected by
Student's t test.***p < 0.01.
Cag A status
The MICROGEN Western blot kit revealed that 39 (97.5%) of the
40 DU patients were CagA-positive. Among the 100 healthy
blood donors, 58 were H. pylori-positive: serum samples
from the latter, in a dilution of 1:100, reacted with at least
4 different H. pylori antigens, and 41 of them (70.7%)
had antibodies against CagA antigen too. Our data are consistent
with earlier observation, that CagA-positive bacteria are more
frequently able to cause ulcerative diseases [4, 18]. However, the
frequency of CagA positivity even in the healthy control group is
high, and we therefore suggest that CagA positivity alone cannot
explain ulcer development in the H. pylori-positive DU
group.
Discussion
In H. pylori infection both bacterial virulence factors
and the inflammatory response of the host contribute to mucosal
damage [2, 19, 23]. The role of cytokines in the initiation and
modulation of gastrointestinal inflammatory responses is crucial
[3]. We set out to investigate the local cytokine activation and
the concomitant production of nitrating agents in the mucosa,
comparing the results with the peripheral cytokine pattern in H.
pylori-positive patients with duodenal ulcer.
In antral biopsy specimens from the DU patients, significantly
higher levels of TNF-α, IL-6 and IL-8 were observed than in
the H. pylori-negative subjects. H. pylori infection
is characterized by a marked neutrophil infiltration. The
activation and recruitment of neutrophils by IL-8 at the site
of inflammation can lead to an increased formation of oxygen free
radicals. At the same time,H. pylori infection can lead to
the expression of iNOS and the sustained production of NO by host
macrophages. Oxygen free radicals and NO can form potentially
genotoxic nitrating species such as peroxynitrite [20, 21]. We
examined the presence of nitrotyrosine as a marker of peroxynitrite
formation in biopsy specimens. Nitrotyrosine was detected in those
samples, in which the highest TNF-α and IL-8 cytokine
concentrations were measured. Accordingly, we concluded that these
cytokines, which activate inflammatory cells, will result in the
generation of nitrating and oxidizing agents. This can lead to
increased rates of DNA damage in the gastric mucosa, inducing NO
production and the production of oxygen free radicals, and
resulting overall in peoxynitrite formation. Considerable evidence
indicates that tyrosine residues in proteins become nitrated
preferentially at sites of inflammation-induced tissue injury,
leading to the suggestion that nitrotyrosine may be deleterious or
serve as a biomarker for the effects of reactive nitrogen oxides
[21]. These free radicals are of pathophysiological importance in
the destruction of the gastric mucosa. The apoptotic effect of
peroxynitrite is extremely important in this process.
IL-10 inhibits the secretion of proinflammatory cytokines
and chemokines from macrophages and polymorphs, thereby potentially
reducing neutrophil activation and the generation of damaging
oxygen metabolites [6, 7]. Hence, IL-10 is an important
counter-regulatory cytokine, and could damp down local inflammation
in some H. pylori-positive DU patients. Despite the
individual variation in the levels of IL-10, the gastric tissue of
the DU patients displayed relatively low levels of IL-10 (Figure
1), and the diference was only marginally significant (p < 0.05)
as compared with samples from H. pylori-negative patients.
Our study revealed a negative correlation between local IL-10 and
TNF-α production, indicating an imbalance between the induction of
the proinflammatory cascade and inhibitory cytokines, disturbing
the mucosal homeostasis [17].
The circulating TNF-α level was almost undetectable not only in
the healthy controls, but also in the DU patients. The mucosal
production of TNF-α and proinflammatory cytokines at the site of
challenge can therefore be considered as occurring separately from
the systemic circulation with respect to cytokine production. In
our in vitro experiments, the TNF-α and IL-6-inducing
ability of the H. pylori 26695 CagA + strain
was similar, regardless of whether the white blood cells originated
from the patients with DU or from the apparently healthy blood
donors, irrespectively of the H. pylori status. It is
noteworthy, however, that the peripheral blood cells from the DU
patients produced a moderately higher level of IL-8 (Table 1, Figure
4). IL-8 is one of the most important cytokines in the
pathogenesis of local inflammation at the site of H. pylori
multiplication [5, 18], where not only inflammatory cells, but also
gastric epithelial cells themselves can be the source at least of
IL-8 production [22]. It is not known, why the
IL-8 production capacity is higher in H.
pylori-positive DU patients, whereas there is no significant
difference in TNF-α and IL-6 production. The secretion of IL-8
is unlikely to be exclusively dependent on TNF-α, and the relation
between these cytokines is complex.
A considerable number of data indicate that CagA-positive
strains are more virulent, and are able to induce more
IL-8 [5, 19]. In our study, 39 of the 40 DU patients had
a CagA-positive status. In the non-ulcer group, the CagA positivity
among the H. pylori-positive subjects was as high as 70.7%.
This frequency of CagA positivity in a healthy control group is
relatively high. We therefore suggest that CagA positivity alone
can not explain the development of ulcer in the H.
pylori-positive DU group. Thus, besides the bacterial
virulence, host factors appear to be important in the outcome of
the infection [23]. Among them, the intensity of the local cytokine
response, with a consequent elevation of nitrating agents, can
contribute to the development of mucosal destruction. Further
studies are required to elucidate whether the higher IL-8-producing
ability of the peripheral white blood cells in duodenal ulcer
patients and the intensity of the local IL-8 response are
connected. IL-8 is a chemokine, that is mainly secreted in
digestive epithelial cells after bacterial interaction.
Accordingly, its role in DU patients is obviously local. It is
therefore tempting to speculate that there may be a difference in
in vitro IL-8 production by the peripheral white blood
cells in DU patients and H. pylori-positive healthy blood
donors. The H. pylori-positive and CagA-positive normal
blood donors (with no history of ulcerative disease) exhibited the
same basal in vitro, inducible IL-8-producing capacity as
that of the H. pylori-negative controls (Figure 4). It may be that a
predisposition to a higher IL-8 response to a given bacterial
stimulus might also be a predisposing factor for ulcerative
processes. It is obvious, however, that merely this one factor
cannot determine the final outcome of H. pylori infection.
Further investigations of the connections between the genetic
determination of an IL-8-producing capacity and the frequency of
ulcer diseases could be informative, and are therefore
mandatory.
Acknowledgements.
The authors are grateful to Mrs. Györgyi Mller for the excellent
technical assistance. They thank Douglas E. Berg (Department of
Molecular Biology and Genetics, Washington University Medical
School, St. Louis, USA) for the kind gift of H. pylori
strain 26695 and for his advice, and Dr Klára Vezendy and her staff
(South Hungarian Regional Blood Bank of National Transfusion
Service, Szeged) for blood samples. This work was supported by
Hungarian ETT grant T-05034 Grant and FKFP grant
0105/2000.
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TNF-α pg/ml
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IL-6 pg/ml
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IL-8 pg/ml
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