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Dig Dis Sci (2006) 51:18101817

DOI 10.1007/s10620-006-9085-5

ORIGINAL PAPER

Neuroimmune Link in the Mucosa of Chronic Gastritis


with Helicobacter pylori Infection
Pongor L. P. Chen
G. Sipos K. Altdorfer E.
E. Feher

Received: 29 August 2005 / Accepted: 5 October 2005 / Published online: 16 September 2006
C Springer Science + Business Media, Inc. 2006


Abstract It is suggested that different neuropeptides regulate gastric mucosal integrity and participate in the development of chronic gastritis. The aim of this study was to examine the roles and changes of immunoreactive (IR) nerves
and immunocompetent cells in human gastritis. Immunohistochemical, immunocytochemical, and confocal laser microscopic methods were used. All investigated nerve fibers
were found in different quantities in the mucosa of both control and gastritis samples. The number of SP, NPY, and VIP
IR nerve fibers increased significantly (P < 0.05) in gastritis. No IR immunocompetent cells (lymphocytes, plasma
cells, mast cells) were found in the control, however, some
showed NPY (16.8%) and SP (9.4%) immunoreactivity in
chronic gastritis. The distance between nerve fibers and immunocompetent cells was 200 nm to 1 m. In conclusion,
the increased number of SP, NPY, and VIP IR nerves and IR
immunocytes suggests that they participate in development
of neurogenic inflammation, repairing processes of chronic
gastritis.
Keywords Neuropeptides . Gastritis .
Neuroimmunomodulation .
Immunohistochemistry
G. Sipos
Department of Gastroenterology, Uzsoki Teaching Hospital,
Budapest, Hungary
Pongor L. P. Chen E. Feher ()
K. Altdorfer E.
Department of Anatomy, Histology and Embryology,
Semmelweis University,
Tuzolto u. 58, P.O. Box 95,
Budapest, H-1450 Hungary
e-mail: feher@ana.sote.hu

Springer

Introduction
Chronic inflammation of the gastric mucosa (chronic gastritis) is caused by Helicobacter pylori or bacterial overgrowth
in the hypochlorhydric stomach [1, 2]. In some patients,
chronic gastritis eventually leads to persistent acid hypersecretion and duodenal ulcers [3]. In gastritis, chronic and
recurrent inflammation is associated with a breakdown of
host defense mechanisms that leads to local and systemic
infection. The actual minute-to minute behavior of the stomach, either normal or disordered, is determined by integrative
functions of the enteric nervous system. The released sensory neuropeptides from both extrinsic and intrinsic sensory
nerves are involved in the regulation of gastric acid secretion
and they may mediate mucosal protection in humans [4, 5].
Certain centrally and/or peripherally injected neuropeptides
influence both acid secretion and gastric mucosal resistance
to injury [6]. It was demonstrated that calcitonin gene-related
peptide (CGRP) and substance P (SP) nerve fibers innervate
the mucosal and submucosal vessels of the upper gastrointestinal tract [7, 8]. SP and CGRP are thought to be the
mediators of neurogenic inflammation [911]. Peptides synthesized in primary sensory neurons are released through
the axon reflex from small-diameter fibers in the peripheral
tissues near blood vessels and they may act as neurogenic
mediators of inflammation and pain. When this pathway is
impaired (such as by ablation of sensory afferent nerves with
capsaicin), the mucosa does not mount an appropriate hyperemic response. As a result, exposure to mild irritants results
in the development of extensive mucosal necrosis [12, 13].
In response to psychological stress as well as certain physical stressors, an inflammatory process may occur by release
of neuropeptides, especially SP, or other inflammatory mediators from sensory nerves, and they activate mast cells or
other inflammatory cells [14].

Dig Dis Sci (2006) 51:18101817

A growing body of data has demonstrated innervation of


immunocompetent cells of gut-associated lymphoid tissues
(GALT) and direct interactions between mucosal mast cells
and other immunocompetent cells and nerves [1518]. It is
widely accepted that some neuropeptides (SP, CGRP, neuropeptide Y [NPY], vasoactive intestinal polypeptide [VIP],
and somatostatin) are important regulators of the immune
system in a number of tissues. However, their function in the
stomach has traditionally been associated with their ability
to regulate gastric acid secretion, although they may also
play an important role in regulation of the innate immune
system during gastritis [19]. Several cell types, including
polymorph nuclear leukocytes, lymphocytes, macrophages,
dendrocytes, and mast cells, are present in continuously varying numbers in the mucosa of the gastrointestinal tract. Each
of these cell types can be found in close spatial association with the neuronal elements [20, 21], suggesting that the
different neuropeptides may have important effects on immunity and play a possible role in various diseases. It was
also demonstrated [22] that neuropeptides can induce typical
and atypical cytokine secretion from T cells and intestinal
epithelial cells so they might have important implications for
numerous physiological and pathological conditions, including chronic inflammation and neoplasia.
NPY, SP, CGRP, VIP, and somatostain represent the neuropeptides that are most involved in neuroimmunomodulation [23]. Detailed knowledge of interactions between
nervous and immune systems may represent an important
basis for the development of strategies for treating diseases/pathologic conditions in which altered neuroimmune
crosstalking may be involved. Therefore, the goal of the
present study was to determine whether these neuropeptides are involved in the pathogenesis of gastritis. We have
analyzed changes in the density of different neuropeptidecontaining nerve fibers and immunocytes in human gastritis
and the relationship of immunoreactive (IR) nerve fibers to
immunocompetent cells in human gastritis.

Materials and methods


Endoscopic biopsies of gastric antrum were obtained from
10 Helicobacter pylori (HP)-positive patients (4 male, 6 female; ranging from 30 to 67 years of age) who underwent
esophagogastroduodenoscopy for dyspeptic symptoms over
a period of 1 year. Antral samples of 10 functional dyspeptic patients (5 male, 5 female; ranging from 27 to 58
years of age) with endoscopically and histologically normal HP-negative stomach were used as controls. These patients first underwent the classic medical physical examinations and, thereafter, laboratory, ultrasonographic, and endoscopic examinations. For the control group, the results of
the examinations were all negative. HP positivity was as-

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sessed by rapid urease test and subsequently confirmed by


histology and serology in all patients. Biopsies from HPpositive patients showed histology of chronic gastritis with
moderate or severe activity according to the Sydney system
[24].
All patients and healthy volunteers gave their informed
consent according to Semmelweis University guidelines for
ethics in human tissue experiments. Permission for patient
studies was given by our local authorities.
Immunohistochemical analysis
Biopsy materials were fixed in Zambonis fixative containing 4% paraformaldehyde, 0.1% glutaraldehyde in 250 ml
0.4 M phosphate buffer, 150 ml picric acid (pH 7.3) for 6
hr and placed overnight in glutaraldehyde-free fixative containing 20% sucrose at 4 C. Sections (40 m thick) were
treated for 1 hr with 1% Triton-X 100 to increase membrane
permeability and for 15 min with 3% hydrogen peroxide to
remove endogenous peroxidase activity. Specificity, working
dilution, and sources of the primary antibodies used in this
work are summarized in Table 1.
Antibodies were incubated with primary antisera for 48 hr
at 4 C. Avidinbiotin immunoperoxidase technique was employed using a commercially available kit (Vectastain Elite
ABC; Vector Laboratories, Peterborough, UK) for immunostaining. All steps were performed at room temperature. Immunoreactivity was visualized by diaminobenzidine (DAB)
chromogen reaction (0.025% 3,3-DAB, 0.0015% H2 O2 in
0.05 M TrisHCl buffer, pH 7.5) for 8 to 10 min, at room
temperature. For light microscopic examination the sections
were mounted on gelatinized slides, air-dried, cleared, and
covered with Depex.
Confocal laser microscopic analysis
For double-staining the sections were also examined by
confocal laser microscope (Nikon Eclipse 800 microscope,
Nikon, Japan; Radiance 2100, Bio-Rad, and LaserSharp2000
Software, Bio-Rad House, Hertfordshire, UK). Frozen
Table 1

Primary antisera used for immunohistochemistry

Antiserum

Abbreviation

Species Dilution

Source

Substance P
Vasoactive
intestinal
polypeptide
Neuropeptide Y
Somatostatin
Galanin
Tyrosine
-hydroxylase

SP
VIP

Rabbit
Rabbit

1:10,000
1:10,000

Daifuku
T. Gorcs
(Budapest)

NPY
SOM
GAL
TH

Rabbit
Rabbit
Rabbit
Rabbit

1:10,000
1:2,000
1:8,000
1:1,000

T. Gorcs
Amersham
Peninsula
Incstar

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sections were washed in PBS at room temperature and permeabilized for 20 min in PBS (2 NaCl) containing 0.3%
Triton X-100 and 2% normal serum; the same solution was
used to dilute the antibodies. Afterward, they were sequentially incubated with anti-SP antiserum at a 1:10,000 dilution overnight. Slides were washed in phosphate-buffered
saline (PBS) and then incubated for 3 hr at room temperature with a secondary fluorescein (FITC; 1:100)-conjugated
donkey anti-rabbit IgG antibody (Jackson ImmunoResearch,
West Grove, PA, USA). After sections were washed with
buffer and incubated with anti-VIP antiserum (1:5.000) for
24 hr, followed by secondary antiserum raised in donkey
(florescein-labeled anti-rabbit IgG, Alexa 594, diluted 1:500;
Molecular Probes, Eugene, OR, USA) for 3 hr, they were
mounted in antifade medium (Vectashield; Vector Laboratories, Peterborough, UK) and stored at 20 C until
needed. IR nerve fibers and immunocytes could be traced
when scanned with a confocal laser microscope equipped
with a kryptonargon laser. Fluorescent signals from FITC
(green) and Alexa 594 (red) were sequentially detected using
a Bio-Rad MicroRadiance confocal laser system (Bio-Rad
MRC1024).
Immunocytochemical analysis
Small pieces of the same biopsy materials were placed
overnight in glutaraldehyde-free fixative containing 10%
sucrose and 4 C. Thirty-micrometer sections were cut by
Vibroslices and plunged rapidly into liquid nitrogen, thenthawed in 0.1 M PBS. From this point on, immunocytochemistry was performed as described above with the exceptions
that Triton X-100 was omitted from all solutions and the
sections were incubated for 48 hr at 4 C in the antibody.
Furthermore, the sections were osmicated in a 0.5% OsO4 containing PBS solution for 1 hr and dehydrated by ascending alcohols and propylene oxide. The sections were then
flat-embedded in Epon. The selected sections were reembedded, and ultrathin sections were obtained with an ultramicrotome using a diamond knife. The sections were counterstained using uranyl acetate and lead citrate. The materials were observed using a Jeol 100 electron microscope. All
morphological examinations were performed by the same
investigator, E.F.
For quantitative analysis the numbers of IR nerve fibers
and immunocompetent cells in a 15- to 20-mm2 tissue area
were counted and calculated for a 1-mm2 tissue area as the
average. For analysis 40 magnification was used with a
graduated eyepiece graticule and the entire section was assessed. Microphotographs were also taken (n = 1525 per
patient), digitalized, and then analyzed using the PC-based
image analyzing software IMAN (beta) 2.0 (MFA, Budapest,
Hungary). The studies were carried out by two investigators
as a double-blind trial.
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Dig Dis Sci (2006) 51:18101817

Control experiments
To verify the specificity of immunohistochemical findings,
nonimmune serum or specific antigen in microgram quantities was added to the primary antibody solution (preabsorbtion) and applied on serial sections where no immunostaining
appeared.
Statistical analysis
Analysis of variance (ANOVA) was employed to determine overall differences in quantities of IR nerve terminals. To evaluate the statistical significance of differences
in materials from healthy controls versus gastritis patients,
the data were also analyzed with Students two-sample
t-test. A value of P < 0.05 was considered statistically
significant.

Results
Gastric hyperemia was the main feature of chronic gastritis
at endoscopy. In all investigated materials the surface epithelium was intact, and the lamina propria exhibited only
moderate edema and was infiltrated by lymphocytes, plasma
cells, mast cells, and a moderate number of neutrophil granulocytes. The distribution pattern of IR nerve fibers in the
mucosa was similar to that of the controls.
All neuropeptides investigated were present in the mucosa among the glands in different quantities and distributions (Fig. 3). Of the six neuropeptides investigated, VIP
(Fig. 1) and NPY IR nerve fibers were most pronounced.
TH IR nerve fibers were also numerous and they were found
mainly around the vessels. The number of SP- and CGRPpositive terminals was moderate, and GAL IR nerve fibers
were encountered only sporadically. None of the immunocompetent cells exhibited IR for any antisera in the control
materials (Fig. 2).
The density of the different IR nerve fibers changed variously in gastritis. The number of SP, VIP, and NPY IR nerve
fibers (Fig. 3) was increased significantly, while the number of CGRP IR nerve fibers was CGRP decreased slightly,
compared to the control.
A large number of immunocompetent cells showed immunoreactivity for NPY and SP in gastritis (Figs. 46).
Some of them were small, round cells (610 m in diameter) that proved to be lymphocytes. Confocal laser microscope investigations also showed that dense green reaction
end products were distributed throughout the cytoplasm of
some immunocytes and the red reaction was absent in these
cells (Fig. 6). Counting of all immunocompetent cells in
whole sections showed that 16.8.% of them were IR for
NPY and 9.4% for SP. Electron microscopic investigation

Dig Dis Sci (2006) 51:18101817

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Fig. 3 Changes of the IR nerve fibers in the mucosa of the stomach

Fig. 1 A part of the mucosa from the control stomach. Arrows indicate
the VIP IR nerve fibers around the glands and in the lamina propria.
Bar = 50 m

proved that these cells were lymphocytes exhibiting SP or


NPY immunoreactivity. The reaction end products were distributed in the cytoplasm and at the membranes of these cells
(Fig. 7).
Electron microscopic analysis showed that IR nerve fibers
were wrapped in Schwann cell processes, whereas in other
instances, the terminals had completely lost their association with Schwann cells and were located in a very
close association with the immunocytes (Figs. 7 and 8).
The varicosity of SP IR nerve terminals showed a large

Fig. 2 A part of the mucosa from the control stomach. Arrows show
the NPY IR nerve fibers in the connective tissue among the glands.
Note that no IR immunocytes can be found in the mucosa. Bar = 50
m

number of large granulated and small clear synaptic vesicles; electron-dense IR material outlined the membranes
of small and large granulated vesicles. The distance between IR nerve fibers and immunocytes was about 1 m
and very often less than 200 nm. In some instances, degranulated mast cells were in the vicinity of IR nerve fibers
(Fig. 7).

Discussion
During chronic gastritis we found an increase in SP, NPY,
and VIP IR nerve terminals within the mucosa. The increased
number of SP IR nerve fibers suggests that SP specifically
stimulates the chemotaxis of lymphocytes, monocytes, neutrophils, and fibroblasts [2528]. Direct interactions between
neuropeptides and immune cells have been demonstrated
in lymphoid organs and in other inflamed tissues [29, 30].
It has previously been demonstrated that neuropeptides influence the differentiation of lymphocytes in mucosa [31].

Fig. 4 A part of the inflamed mucosa (gastritis). Arrows show the


increased number of SP IR nerve fibers in the connective tissue. Arrowheads point out the immunopositive immunocytes close to the IR
nerve fibers. Bar = 50 m

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Fig. 5 A part of the inflamed mucosa (gastritis). Arrows show the


increased number of NPY nerve terminals in the mucosa. Arrowheads
show NPY IR immunocytes in very close association with IR nerve
fibers. Bar = 50 m

Dig Dis Sci (2006) 51:18101817

Fig. 7 Electron micrograph of a part of the inflamed mucosa (gastritis).


Arrowheads show dark reaction end products in a NPY immunopositive
lymphocyte (Ly). Arrow shows a NPY IR nerve terminal close to this
lymphocyte. MC indicates a degranulated mast cell in the vicinity of it.
Bar = 1 m

After activation macrophages secrete a wide variety of biologically active products (e.g., oxygen and NO metabolites, proteases, cytokines, chemotactic factors, growth factors) which, if released in an uncontrolled manner, result in
tissue injury and development of the fibrosis characteristic
of chronic inflammation. Neuropeptides such as VIP and
PACAP inhibit the in vitro and in vivo production of proinflammatory agents (IL-6, IL-12, TNF, and nitric oxide)
and stimulate the production of the anti-inflammatory cytokine IL-10 [32]. They also decrease NFB binding, which
affects numerous genes involved in inflammation [33]. NPY
also has some potent immunological effects such as differentiation of T helper cells, monocyte mediator release, natural
killer [NK] cell activation, and immune cell redistribution
[34].

In our study some of the immunocytes exhibited a very


close relationship to the IR nerve fibers in chronic gastritis, suggesting that these neuropeptide-containing nerve
fibers might be involved in neuroimmunomodulation. In
inflammation lymphocytes, plasma cells, mast cells, and
eosinophils are always present and accumulated in the inflamed area; these immune cells express specific receptors
for neurotransmitters. The activated lymphocytes produce
lymphokines and inflammatory mediators. Evidence for bidirectional communication between nervous and immune systems is well established in the alimentary tract [11, 18, 35,
36]. Changes in immune function might influence the distribution of nerves and the expression of neural transmitter
receptors in immunocytes [37].

Fig. 6 Confocal laser micrograph from a part of the inflamed mucosa (gastritis). Arrows show the SP IR nerve fibers in the mucosa.
Arrowheads point out the SP immunopositive immunocytes. Bar = 50
m

Fig. 8 Electron micrograph of inflamed mucosa (gastritis). Arrows


point out SP IR nerve terminals having a large number of synaptic
vesicles close to an immunonegative lymphocyte. Bar = 1 m

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Dig Dis Sci (2006) 51:18101817

Our three examinations (light immunohistochemical, confocal laser microscopic, and electron microscopic) showed
that some lymphocytes were also IR for NPY and SP, suggesting that in inflammation these cells produce these neuropeptides and they are active in local immune responses
of the stomach. Previously it was demonstrated in other tissues that certain conditions, e.g., inflammation, can induce
the secretion and release of different neuropeptides from
immunocytes (lymphocytes, macrophages, eosinophils) [18,
3840]. Human monocytes and lymphocytes express the SP
gene at both the mRNA and the protein level [41, 42], as well
as CGRP [43]. The study of purified B and T cells confirmed
that only activated lymphocytes were able to synthesize NPY
[44] and TH [45]. Immunopositive mast cells were found in
higher amounts in lesional atopic dermatitis and in chronic
ulcerative colitis [46]. To date, at least 27 different neuroendocrine mediators have been demonstrated to be produced
by cells of lymphoid organs. These neuropeptides then might
stimulate further leukocyte recruitment, thereby amplifying
the inflammatory response [47].
The demonstration that some immunocytes produce SP
and NPY led to the hypothesis that SP and NPY not only
act as a mediator of the crosstalk between the nervous and
the immune systems but also are biologically involved in
the direct interaction between immune cells in a paracrine
and/or autocrine fashion, independent of sensory nerves or
neurogenic inflammation [38, 41]. In the thymus medulla
and in the spleen VIP-containing nerve fibers were detected
closely associated with VIP IR lymphocytes [4851]. A close
association was also observed between SP IR nerve fibers
and SP-positive lymphocytes and macrophages at the corticomedullary junction [52].
The balance between pro- and anti-inflammatory factors
plays an essential role in the successful control of inflammation. VIP/PACAP has been proposed as an efficient therapeutic treatment for several disorders, especially inflammatory
and autoimmune diseases (such as septic shock, rheumatoid arthritis, Crohns disease, and autoimmune diabetes),
via down-regulation of both inflammatory and autoimmune
components of the disease [53, 54]. VIP also prevented
stress-induced gastric ulceration in the rat when given intraperitoneally. The protective effect may be due to inhibition of mast cell degranulation and protection of gastric
tissue from lipid peroxidation [55]. Dietary chili intake was
shown to increase mucosal defense and protection from peptic ulcer disease [56]. The capsaicin-sensitive afferent nerves
increase the resistance of the gastric tissue to injury, facilitating the repair of damaged tissue [5759]. Antagonists of
pro-inflammatory peptides such as SP and CRH may control
inflammatory diseases or processes in which these peptides
have a primary pathogenic role [60].
We postulate that repeated episodes of acute or chronic
psychogenic stress may produce chronic inflammatory

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changes in the stomach. In these sites, lymphocyte extravasations are facilitated by neurogenic inflammation and plasma
extravasations that are dependent on the release of SP from
capsaicin-sensitive primary sensory nerve endings via an
axon reflex. Therefore, it can be concluded that better treatment/control of disease activity and of pain can be achieved
by blocking the cascade leading to initiation and/or amplification of the inflammatory process. We therefore propose
that neuropeptides in gastritis act as endogenous factors
that regulate immune homeostasis; the physiological consequence of this action is that the immune microenvironment
depends on the timing of neuropeptide release and the activation stage of neighboring immune cells. Inflammatory
mediators in this tissue can further facilitate or modulate
the release of neurotransmitters from both nerve fibers and
immunocytes.
Acknowledgements The authors would like to thank Ms. E. Burka
for assisting with the manuscript.

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