CGRP upregulation in dorsal root ganglia and ileal
mucosa during Clostridium
difficile toxin A-induced enteritis
Andrew C.
Keates1,
Ignazio
Castagliuolo1,
Bosheng
Qiu1,
Sigfus
Nikulasson2,
Ashok
Sengupta1, and
Charalabos
Pothoulakis1
1 Division of Gastroenterology,
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston
02215; and 2 Department of
Pathology, Boston University School of Medicine, Boston,
Massachusetts 02118
 |
ABSTRACT |
We have previously
reported that pretreatment of rats with capsaicin (an agent that
ablates sensory neurons) or CP-96345 (a substance P receptor
antagonist) dramatically inhibits fluid secretion and intestinal
inflammation in ileal loops exposed to Clostridium difficile toxin A. The aim of this study was to
determine whether calcitonin gene-related peptide (CGRP), a
neuropeptide also found in sensory afferent neurons, participates in
the enterotoxic effects of toxin A. Administration of toxin A was also
found to increase CGRP content in dorsal root ganglia and ileal mucosa
60 min after toxin exposure. Furthermore, immunohistochemical studies
demonstrated increased neuronal staining for CGRP 2 h after toxin A
treatment. Pretreatment of rats with CGRP-(8
37), a specific CGRP
antagonist, before instillation of toxin A into ileal loops
significantly inhibited toxin-mediated fluid secretion (by 48%),
mannitol permeability (by 83%), and histological damage. We conclude
that CGRP, like substance P, contributes to the secretory and
inflammatory effects of toxin A via increased production of this
peptide from intestinal nerves, including primary sensory afferent
neurons.
intestine; sensory nerves; neurotransmitters; calcitonin
gene-related peptide
 |
INTRODUCTION |
IT IS WELL RECOGNIZED that Clostridium
difficile toxin A mediates intestinal inflammatory
responses in animals (4, 21, 25) and humans (18). The mechanism by
which toxin A induces its acute inflammatory effects in the intestine
involves toxin A binding to specific receptors on intestinal epithelial
cells (26), which leads, via unknown signaling pathways, to the
activation of enteric nerves and immune cells present in the lamina
propria (4, 5). It has been suggested that bacterial enterotoxins exert
their effects, at least in part, by activating neuronal circuits within
the intestine (20). In support of this view, we and others (4, 23, 25)
have previously reported that pretreatment of rats with capsaicin or
with specific nonpeptide substance P receptor antagonists dramatically
inhibited fluid secretion, mucosal permeability, and intestinal
inflammation in ileal loops exposed to toxin A. Because capsaicin
treatment functionally ablates sensory afferent neurons and substance P
is a major neurotransmitter in such nerves, these findings suggest that
activation of sensory afferents plays a key role in toxin A-induced
intestinal secretion and inflammation.
Over the last 10 years or so the concept of neurogenic (i.e., sensory
neuropeptide-mediated) inflammation has received considerable support.
Antidromic stimulation of sensory neurons has been shown to stimulate
release of calcitonin gene-related peptide (CGRP) and substance P and
to produce an intense inflammatory response in the skin of various
animals (see Ref. 7 for review). Furthermore, depletion of
neurotransmitters in sensory nerves with capsaicin abolishes the
inflammatory response induced by antidromic stimulation (16). These
findings are complemented by numerous studies that suggest that CGRP
and substance P have proinflammatory properties and contribute to
inflammatory changes associated with arthritis (13, 30).
CGRP I, or
-CGRP, is a 37-amino acid peptide that was originally
detected as an alternative splice product of the calcitonin gene (2).
It is now known that a closely related gene encodes CGRP II (
-CGRP),
which possesses almost identical biological properties to CGRP-I and
which in the rat differs by a single conservative amino acid
substitution (1). In addition to its functions as a
neurotransmitter/neuromodulator in the central nervous system, CGRP is
also an important neurotransmitter in the enteric nervous system, where
it has been localized to both primary sensory afferent neurons and
intrinsic neurons (33).
Several experimental observations suggest that CGRP participates in
immune and inflammatory responses (see Ref. 29 for review). First, CGRP
is a vasodilator and potentiates vascular permeability and neutrophil
recruitment induced by interleukin-1, platelet-activating factor,
histamine, and substance P. Second, binding sites for CGRP have been
detected on rat and mouse lymphocytes, rat macrophages, and canine
mesenteric lymph nodes. Binding of CGRP to its receptor stimulates
histamine release from mast cells and inhibits T lymphocyte proliferation and eosinophil chemotaxis. CGRP also inhibits antigen presentation and interferon-
-induced
H2O2
production by macrophages. Finally, antibodies to CGRP have been shown
to ameliorate inflammation induced by arthritis and topical treatment
with mustard oil in rats.
In contrast to the above findings, studies investigating the role
played by CGRP in animal models of colitis suggest that sensory nerves
function in an anti-inflammatory capacity. In the rabbit immune complex
model of colitis, tissue CGRP content was found to be reduced by 80%
48 h after induction of inflammation (12). Decreases in colonic CGRP
content have also been reported in rats treated with
trinitrobenzenesulfonic acid and Formalin to induce inflammation (11,
24). Furthermore, in the immune complex- and trinitrobenzenesulfonic
acid-induced colitis, ablation of sensory neurons with capsaicin
increases the severity of inflammation (27, 28).
Because primary sensory neurons and substance P participate in toxin
A-mediated enteritis, we sought to determine whether CGRP also
contributes to the secretory and inflammatory effects of
C. difficile toxin A. To accomplish
this we used the rat ileal loop model, in which toxin A produces a
reproducible acute inflammatory response (25). We demonstrate that
toxin A-induced fluid secretion, mucosal permeability, and inflammation
in ileal loops are markedly attenuated by CGRP-(8
37), a specific CGRP
antagonist. We also show that instillation of toxin A into ileal loops
induces an early increase of CGRP content in lumbar dorsal root
ganglia, which is followed by increased levels of CGRP in the ileal
mucosa.
 |
MATERIALS AND METHODS |
Male Wistar rats weighing 150-200 g were obtained from Charles
River Breeding Laboratories (Wilmington, MA). All rats arrived at the
animal facility at least 4 days before the experiment and were housed
under standardized environmental conditions. Pentobarbital sodium was
obtained from Abbott (Chicago, IL).
[3H]mannitol (30 Ci/mmol) was obtained from New England Nuclear (Boston, MA). The rat
CGRP antagonist CGRP-(8
37) was obtained from Peninsula Laboratories
(Belmont, CA). CGRP-(8
37) was dissolved in phosphate-buffered saline
(PBS) immediately before use and injected intravenously via a catheter.
Toxin A was purified to homogeneity from broth culture supernatants of
C. difficile strain 10,463 as
previously described (26). Enterotoxicity and cytotoxicity of toxin A
were assessed as previously described (25). A dose of 5 µg of
purified toxin A was used in all experiments, since previous studies
showed this dose to stimulate fluid secretion, increase mannitol
permeability, and cause an acute inflammatory infiltrate when injected
into rat ileal loops (25). Protein concentrations were determined by
the bicinchoninic acid protein assay (Pierce Laboratories, Rockford,
IL).
Measurement of mannitol permeability and fluid secretion in rat
ileal loops.
Two days before the experiment, rats were anesthetized by
intraperitoneal injection of pentobarbital sodium, and polyethylene catheters (1.27 mm in diameter; Clay Adams, Parsippany, NJ) were placed
in the right jugular vein and subcutaneously exteriorized in the
intrascapular region. After implantation of the catheters, animals were
kept under continuous observation until the day of the
experiment.
Two days after surgery, fasted rats were anesthetized by
intraperitoneal injection of pentobarbital sodium (40 mg/kg ip), and a
laparotomy was performed. Both renal pedicles were ligated, and 10 µCi of [3H]mannitol
were injected into the inferior vena cava. Two 5-cm closed loops were
then formed in the distal ileum, with at least 5 cm distance separating
each loop. Five minutes before instillation of toxin A [5 µg in
0.4 ml tris(hydroxymethyl)aminomethane (Tris) · HCl, pH
7.4] or buffer into ileal loops, rats were treated with an equal
volume (40 µl) of either PBS or CGRP-(8
37) (80 nmol/kg body wt)
administered via the jugular vein catheter. A similar dose of
CGRP-(8
37) was found to significantly inhibit neurogenic vasodilation
in rat paw skin after intraplantar injection of sodium nitroprusside
(17). The same amount of PBS or CGRP-(8
37) was also administered 25, 55, and 85 min after injection of toxin A. Multiple doses of
CGRP-(8
37) were given to minimize possible in vivo degradation of the
antagonist over the course of the experiment. Animals were maintained
under general anesthesia for the duration of the experiment with
pentobarbital sodium (20 mg/kg ip) given ~20 min after the end of the
operation. Animals were also placed on a heating pad to keep their body
temperatures at 37-38°C. After 4 h, the animals were killed,
the ileal loops were removed, and the weights and lengths were
measured. Intestinal fluid secretion (weight-to-length ratio; mg/cm)
and mucosal
[3H]mannitol
permeability [disintegrations per minute (dpm) per centimeter
loop] were determined as described previously (25).
Histological evaluation.
Ileal tissues were fixed in Formalin, embedded in paraffin, and stained
with hematoxylin and eosin for light microscopy. All sections were
graded in a blinded fashion by a gastrointestinal pathologist (S. Nikulasson), taking into account the following features:
1) epithelial cell damage,
2) hemorrhagic congestion and edema
of the mucosa, and 3) neutrophil
margination and tissue infiltration, as described previously (25). A
score of 0-3, denoting
increasingly severe abnormality, was assigned to each parameter. The
effect of CGRP-(8
37) on toxin A-induced histological damage was
assessed after 4 h, since full-blown enteritis requires at least 2 h
exposure to toxin A to develop (3).
Measurement of CGRP content in ileal mucosa and dorsal root
ganglia.
Ileal loops were prepared as described above and exposed to 5 µg of
toxin A. After 0, 30, 60, and 120 min, animals were killed and the
loops were removed, opened, and washed in ice-cold Hank's balanced
salt solution (Sigma). The mucosa was then scraped from the underlying
muscle layers and homogenized in 2.5 ml of ice-cold 0.2 M HCl for 20 s.
The homogenate was centrifuged at 12,000 g for 15 min at 4°C, and the
supernatant was collected and stored at
80°C until use. To
collect lumbar dorsal root ganglia, the spinal column was dissected
bilaterally and, with the use of ultrafine forceps, the lumbar dorsal
root ganglia (6-8 each animal) were removed from each side of the
animal. In separate experiments thoracic dorsal root ganglia
(T9-T12)
were isolated in a similar manner. Dorsal root ganglia from each animal
were pooled separately and processed as described above for intestinal
mucosal scrapings.
CGRP levels in mucosal scrapings and lumbar dorsal root ganglia were
determined with the use of a commercially available radioimmunoassay (RIA; Phoenix Pharmaceuticals, Mountain View, CA). Samples were prepurified on C18 reverse-phase
cartridge columns (Waters, Cambridge, MA). Briefly, columns were first
washed with 5 ml methanol and then equilibrated with 0.1% (vol/vol)
trifluoracetic acid (TFA). Samples diluted 1:1 with 0.2% (vol/vol) TFA
were slowly loaded onto the column, and the column was then washed with
10 ml of 0.1% (vol/vol) TFA. Adsorbed peptides were then eluted with
1.5 ml of 75% (vol/vol) acetonitrile and freeze-dried. Samples were
reconstituted in 0.5 ml of RIA sample buffer, and the CGRP content was
determined according to the manufacturer's protocol. Results are
expressed as picomoles CGRP per milligram protein.
Immunohistochemistry.
Ileal loops were prepared as described above and exposed to toxin A for
0, 1, and 2 h. At the indicated time points, loops were removed, opened
along their length, and washed in ice-cold PBS (Sigma). Samples were
then fixed in Zamboni's fixative for 18 h at 4°C. After fixation,
tissues were washed exhaustively in ice-cold PBS containing 0.015%
(wt/vol) sodium azide, oriented, frozen in optimal cutting temperature
compound (Miles, Elkhart, IN), and cryosectioned onto gelatin-coated
slides. Control and test sections were mounted onto each slide to
ensure accurate comparison between samples. Slides were incubated with
rabbit anti-rat CGRP antiserum (Peninsula Laboratories) at a dilution of 1:500 or nonimmune rabbit serum at the same dilution for 1 h at room
temperature. After washing, primary antibodies were detected by the
avidin-biotin peroxidase staining system, using a Vectastain Elite ABC
kit (Vector Laboratories, Burlingame, CA). Sections were then
counterstained using hematoxylin and eosin.
Data analysis.
Statistical analyses were performed using SigmaStat for Windows version
2.0 (Jandel Scientific Software, San Rafael, CA). Analysis of variance
followed by protected t-test was used
for intergroup comparisons, except for the histological grades, for which the nonparametric Kruskal-Wallis analysis of variance on ranks
was used.
 |
RESULTS |
Effect of CGRP-(8
37) on toxin A-induced intestinal secretion and
mucosal permeability.
As previously reported (4, 25), exposure of rat ileal loops to 5 µg
of highly purified C. difficile toxin
A significantly increased intestinal fluid secretion (3.6-fold;
P < 0.01) compared with loops from
control animals (Fig. 1). Instillation of
toxin A into ileal loops also caused a prominent elevation in mucosal [3H]mannitol
permeability (35.5-fold, P < 0.01)
compared with untreated loops (Fig. 2).
Intravenous injection of the CGRP antagonist CGRP-(8
37) significantly
inhibited intestinal fluid secretion in response to toxin A (by 48%,
P < 0.01; Fig. 1). Administration of
the same dose of CGRP-(8
37) also markedly reduced toxin A-mediated
increases in mucosal
[3H]mannitol
permeability (by 83%, P < 0.01;
Fig. 2). A similar dose of CGRP-(8
37) given in the absence of toxin A
had no effect on basal intestinal fluid secretion or mucosal
permeability (Figs. 1 and 2).

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Fig. 1.
Inhibition of toxin A-induced intestinal fluid secretion by calcitonin
gene-related peptide-(8 37) [CGRP-(8 37)]. Rat ileal
loops were injected with 0.4 ml Tris buffer alone or with Tris buffer
containing 5 µg purified C. difficile toxin A. Test animals were pretreated with
either phosphate-buffered saline (PBS) or PBS containing CGRP-(8 37)
(80 nmol/kg iv) 5 min before and 25, 55, and 85 min after toxin A
administration. After 4 h, ileal loops were harvested and intestinal
fluid secretion was estimated as the loop weight (mg)-to-length (cm)
ratio. Results are means ± SE from 7 loops.
 P < 0.01 compared
with control; ** P < 0.01 compared with toxin A alone.
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Fig. 2.
Inhibition of toxin A-mediated mucosal mannitol permeability by
CGRP-(8 37). Experiments were performed as described in Fig. 1. After
4 h, ileal loops were excised and intestinal permeability to
[3H]mannitol was
estimated by scintillation counting of aliquots of loop fluid. Results
are means ± SE from 7 loops.
 P < 0.01 compared
with control; ** P < 0.01 compared with toxin A alone.
|
|
Effect of CGRP-(8
37) on toxin A-induced histological damage.
Histological examination of ileal tissues from loops exposed to toxin A
showed characteristic epithelial cell damage with disruption of villus
architecture compared with buffer-treated control loops (Fig.
3). Pretreatment of animals with
CGRP-(8
37) dramatically reduced histological damage induced by toxin
A (Fig. 3). Quantitative histological scores for all three parameters studied (epithelial damage, mucosal congestion and edema, and neutrophil infiltration) were each significantly decreased in CGRP-(8
37)-treated animals compared with animals treated with toxin A
alone (Fig. 4).

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Fig. 3.
Inhibition of toxin A-induced enteritis by CGRP-(8 37). Rat ileal
loops were injected with 0.4 ml Tris buffer alone or with Tris buffer
containing 5 µg purified C. difficile toxin A. Test animals were pretreated with
either PBS or PBS containing CGRP-(8 37) (80 nmol/kg iv) 5 min before
and 25, 55, and 85 min after toxin A administration. After 4 h, loops
were removed, full-thickness samples were fixed in Formalin, and
sections were stained with hematoxylin and eosin.
A: buffer-treated ileal loop showing
normal mucosa. B: ileal loop exposed
to toxin A showing disruption of villus architecture, goblet cell
discharge, and tissue necrosis. C:
ileal loop pretreated with CGRP-(8 37) before challenge with toxin A,
showing almost complete prevention of the intestinal effects of toxin
A. Magnification, ×140.
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Effect of toxin A on CGRP content in intestinal mucosa and lumbar
dorsal root ganglia.
Because the results (Figs. 1-4) indicate that CGRP participates in
the intestinal responses to toxin A, we next measured the CGRP
content of the ileal mucosa from loops exposed to toxin A. Treatment of
ileal loops with toxin A resulted in a time-dependent increase in the
CGRP content of the mucosa as measured by specific radioimmunoassay (Fig. 5).
Mucosal CGRP content was unaltered 30 min after ileal instillation of
toxin A. One hour after toxin A administration mucosal CGRP content was
increased approximately threefold compared with control (Fig. 5), and
mucosal CGRP content remained elevated after 2 h.

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Fig. 4.
Inhibition of toxin A-mediated enteritis by CGRP-(8 37). Tissue
sections from control and toxin-treated animals were prepared as
described in Fig. 3 legend. Histological severity of enteritis was
graded in each section by a score of
0-3, using the following
parameters: 1) epithelial damage,
2) hemorrhagic congestion and
mucosal edema, and 3) neutrophil
margination and tissue infiltration. Results are means ± SE from 5 loops. * P < 0.05 compared
with toxin A alone.
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Previous studies from our laboratory indicate that sensory afferent
neurons play an important role in the mechanism of action of
C. difficile toxin A (4, 25). Several
publications have shown that sensory afferent neurons innervating the
terminal ileum have their cell bodies in either lower thoracic or upper
lumbar dorsal root ganglia (6, 34). Because we have recently shown that
the substance P content in lumbar dorsal root ganglia is increased 30 min to 1 h after toxin A treatment (3), we next investigated whether
administration of toxin A into ileal loops altered the CGRP content in
these dorsal root ganglia. Our results show that the CGRP content in
lumbar dorsal root ganglia was increased approximately twofold
(P < 0.01) 1 h after toxin A
administration compared with the content of this peptide in dorsal root
ganglia from control animals (Fig. 6).
However, 2 h after treatment of ileal loops with toxin A, the CGRP
content of lumbar dorsal root ganglia had returned to control levels
(Fig. 6). In contrast, the CGRP content of thoracic ganglia from the
T9-T12
level (n = 5 animals) was not
significantly altered 30 min (3,910 ± 324 pg/mg protein), 1 h
(4,786 ± 1,323 pg/mg protein), or 2 h (4,876 ± 1,140 pg/mg protein) after exposure to toxin A compared with CGRP levels in
ganglia from control animals (5,101 ± 560 pg/mg protein).

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Fig. 5.
Effect of toxin A (TxA) on CGRP content of ileal mucosa. Rat ileal
loops were formed and injected with 0.4 ml Tris buffer containing 5 µg purified C. difficile toxin A. After 30, 60, and 120 min, loops were harvested, and mucosal CGRP
content was determined as described in MATERIALS AND
METHODS. Results are means ± SE from 5-6
loops. ** P < 0.01 compared
with control.
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|
Immunohistochemical studies.
To determine the cell(s) of origin of increased CGRP production in
ileal loops exposed to toxin A, immunohistochemical staining was
performed using tissue sections from ileal loops exposed to toxin A
(Fig. 7). CGRP immunoreactivity in control
ileum was primarily associated with nerve terminals around myenteric
and submucosal neurons and the villus plexus in the lamina propria.
CGRP immunoreactivity was unaltered after 1 h exposure to toxin A. However, treatment of ileal loops for 2 h with toxin A markedly
increased CGRP staining intensity compared with control. Elevated CGRP
levels appeared to be associated with nerve fibers, particularly in the
lamina propria.

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Fig. 6.
Effect of toxin A (TxA) on CGRP content of lumbar dorsal root ganglia.
Rat ileal loops were formed and injected with 0.4 ml Tris buffer
containing 5 µg purified C. difficile toxin A. After 30, 60, and 120 min, lumbar
dorsal root ganglia were harvested and CGRP content was determined as
described in MATERIALS AND METHODS.
Results are means ± SE from 5-10 loops.
* P < 0.05 compared with
control.
|
|
 |
DISCUSSION |
The principal finding of this study is that instillation of purified
C. difficile toxin A into rat ileal
loops induces an early increase in the CGRP content of dorsal root
ganglia and intestinal mucosa. Furthermore, CGRP-(8
37), a CGRP
inhibitor, almost completely abolished mucosal permeability to
[3H]mannitol and
substantially inhibited intestinal fluid secretion and histological
damage induced by toxin A in rat ileum. These findings indicate that
CGRP participates in the mechanism of action of C. difficile toxin A in the rat intestine.
Retrograde tracing studies have demonstrated that the primary afferent
nerves innervating the distal ileum of the rat have cell bodies
localized in the lower thoracic and upper lumbar dorsal root ganglia
(6, 34). Increased levels of CGRP in lumbar dorsal root ganglia were
observed 1 h after treatment with toxin A. Furthermore, in a similar
experiment, increased levels of substance P in lumbar dorsal root
ganglia were evident 30 min after toxin A administration (3). These
findings suggest that activation of sensory neurons is an early event
in toxin A-induced enteritis, since upregulation of CGRP and substance
P in lumbar dorsal root ganglia precedes increases in intestinal fluid
secretion, mucosal permeability, and histological damage mediated by
toxin A (4). Interestingly, the CGRP content of thoracic ganglia
(T9-T12)
was unaltered when toxin A was applied to ileal loops. These data suggest either that toxin A only activates sensory neurons that have
cell bodies in lumbar dorsal root ganglia or, alternatively, that toxin
A may activate sensory neurons indirectly, possibly via activation of
enteric nerves.
Increases in the CGRP and substance P content of lumbar dorsal root
ganglia have also been reported in rat adjuvant-induced arthritis. For
example, Donnerer et al. (9) reported a 30-40% increase in CGRP
and substance P content of the lumbar dorsal root ganglia 5 days after
induction of inflammation in rat hindpaws. Moreover, increases in CGRP
levels in lumbar dorsal root ganglia were present as early as 12 h
after injection of adjuvant, suggesting that activation of
CGRP-containing sensory nerves is also an early event in this model. In
a separate study Hanesch et al. (15) reported that the number of
CGRP-positive cell bodies in the dorsal root ganglia increased
significantly (30%) 2 days after adjuvant challenge.
In this study we show that increased levels of CGRP are associated with
acute intestinal inflammation. Increased immunohistochemical staining
of CGRP was apparent 2 h after exposure to toxin A in the intestinal
mucosa and lamina propria. An important source of CGRP is
capsaicin-sensitive sensory afferent neurons, which are distributed
throughout the intestine. These nerves have their cell bodies in spinal
dorsal root ganglia and regulate a number of immune and inflammatory
intestinal functions (29). CGRP is also contained within intrinsic
nerves and may be produced by intestinal immune cells (29). Although
CGRP may be released from multiple sites in the intestine, our results
in Fig. 7 and previous studies (4, 25) are
consistent with release of this peptide from enteric neurons. Given the
rapid time course (1-2 h) of CGRP upregulation in response to
toxin A stimulation, it would seem unlikely that these changes are the
result of de novo protein synthesis in neuronal cell bodies. A more
likely explanation for the observed changes in CGRP staining is that
toxin A increases axonal transport and secretion of CGRP. Although
axonal transport of CGRP has been demonstrated in sensory afferent
fibers innervating the gastrointestinal tract (36), the role of this
process in acute intestinal inflammation is poorly understood. However,
increased axonal transport of CGRP has been reported in the sciatic
nerve after adjuvant-induced inflammation in rat hindpaw (10).

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Fig. 7.
CGRP immunohistochemistry in normal and toxin A-treated ileum. Rat
ileal loops were formed and injected with 0.4 ml Tris buffer containing
5 µg purified C. difficile toxin A. After 60 and 120 min, loops were harvested and CGRP
immunohistochemistry was performed as described in
MATERIALS AND METHODS. In normal ileum
CGRP immunoreactivity is mainly associated with neurons present in the
lamina propria and the submucosa (Fig.
7A). No change in ileal CGRP
immunoreactivity was observed after treatment with toxin A for 1 h
(data not shown). Instillation of toxin A into ileal loops for 2 h
markedly increased CGRP immunoreactivity compared with control.
Increased staining is particularly apparent in lamina propria neurons
(Fig. 7B). Control ileal sections
processed in parallel but incubated with rabbit nonimmune serum showed
no immunostaining (Fig. 7C).
Magnification, ×200.
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|
Although numerous studies implicate sensory neuropeptides in
gastrointestinal pathophysiology, evidence for neurogenic inflammation in the gastrointestinal tract remains controversial. Levels of substance P have been reported to be increased in the jejunum of rats
infected with Trichinella spiralis
(35) and in the colon of patients with ulcerative colitis (22). In
contrast, CGRP and substance P levels in the trinitrobenzenesulfonic
acid and immune complex models of colitis are either unchanged or
decreased (11, 12, 24, 27, 28). The discrepancy between these studies
and the results of this investigation may be explained by the use of
different models (bacterial toxin mediated vs. chemical/immune mediated), time courses of inflammation (acute vs. chronic), and animals (rats vs. rabbits).
The site of action of CGRP in the toxin A model of intestinal secretion
and inflammation cannot be elucidated from our results. Previous
anatomic studies suggest that primary sensory afferent neurons
predominantly terminate in the myenteric and submucosal plexii and
around submucosal blood vessels in the gastrointestinal tract (29).
Indeed, localization of CGRP and substance P receptors to myenteric
neurons has been demonstrated using quantitative receptor
autoradiography and confocal microscopy (14, 23). Interestingly, Mantyh
et al. (23) recently reported internalization of substance P receptors
on enteric neurons after administration of toxin A to ileal loops.
These data suggest that sensory neurons activated by toxin A may exert
their effects, at least in part, by regulating the function of other
nerves.
CGRP receptors may also be present on nonneuronal cells located in the
lamina propria. The close apposition of sensory afferent neurons and
intestinal mast cells suggests that neuropeptides such as CGRP and
substance P may directly interact with these cells (31, 32). In support
of this hypothesis, we have shown that pretreatment of rats with the
substance P receptor antagonist CP-96,345 can abolish release of rat
mast cell protease II from ileal explants exposed to toxin A (25).
Although CGRP receptors have not been identified on mucosal mast cells,
this peptide can induce release of histamine from peritoneal mast cells
(19). Another site of action of CGRP may be intestinal enterocytes. In
a recent report, Cox and Tough (8) indicated that the human intestinal
epithelial lines Col-29 and HCA-7 express functional CGRP receptors.
Vascular cells regulating vasodilation and vascular permeability are
also likely to interact with CGRP.
In this study we demonstrate for the first time increased production of
CGRP in response to acute intestinal inflammation induced by
C. difficile toxin A. We also show
that luminal application of toxin A leads to an early activation of
CGRP-containing sensory neurons in lumbar dorsal root ganglia.
Furthermore, in vivo administration of a CGRP antagonist to rats
significantly reduces the intestinal response to this toxin. These
findings and previous studies suggest that primary sensory afferent
nerves containing CGRP and substance P play an important role in the
pathogenesis of toxin A-induced enteritis.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-47343 and DK-34583. I. Castagliuolo is a recipient of a Research Fellowship Award from the
Crohn's and Colitis Foundation of America.
 |
FOOTNOTES |
Address for reprint requests: C. Pothoulakis, Division of
Gastroenterology, Beth Israel Deaconess Medical Center, Dana 601, Boston, MA 02215.
Received 28 January 1997; accepted in final form 13 October 1997.
 |
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