Role of mucosal mast cells in early vascular permeability
changes of intestinal DTH reaction in the rat
Aletta D.
Kraneveld,
Thea
Muis,
Andries S.
Koster, and
Frans P.
Nijkamp
Department of Pharmacology and Pathophysiology, Utrecht Institute
for Pharmaceutical Sciences, Utrecht University, 3508 TB Utrecht, The
Netherlands
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ABSTRACT |
Previously, it was shown that depletion and
stabilization of the mucosal mast cell around the time of challenge
were very effective in reducing delayed-type hypersensitivity (DTH)
reactions in the small intestine of the rat. The role of mucosal mast
cells in the early component of intestinal DTH reaction was further investigated in this study. In vivo small intestinal vascular leakage
and serum levels of rat mast cell protease II (RMCP II) were determined
within 1 h after intragastric challenge of rats that had been
sensitized with dinitrobenzene 5 days before. A separate group of rats
was used to study vasopermeability in isolated vascularly perfused
small intestine after in vitro challenge. To investigate the effects of
mast cell stabilization on the early events of the DTH reaction,
doxantrazole was used. The influence of sensory nerves was studied by
means of neonatal capsaicin-induced depletion of sensory neuropeptides.
Within 1 h after challenge, a significant increase in vascular
permeability was found in vivo as well as in vitro. This was associated
with a DTH-specific increase in RMCP II in the serum, indicating
mucosal mast cell activation. In addition, doxantrazole treatment and
caspaicin pretreatment resulted in a significant inhibition of the
DTH-induced vascular leakage and an increase in serum RMCP II. These
findings are consistent with an important role for mucosal mast cells
in early vascular leakage changes of intestinal DTH reactions. In
addition, sensory nervous control of mucosal mast cell activation early
after challenge is demonstrated.
small intestinal vascular permeability; capsaicin
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INTRODUCTION |
CONSIDERABLE EVIDENCE supports a role for mast cells in
immunologic inflammatory processes (6). Also, in
cell-mediated delayed-type hypersensitivity (DTH) reactions a role for
mast cells has been postulated (9, 25). DTH reactions in the
gastrointestinal tract have been proposed to represent some of the
features prevalent in inflammatory bowel diseases (IBD); ongoing
responses have been associated with an increased vascular permeability
and enhanced lymphocyte infiltration into the inflamed intestinal
tissue (10, 13, 31). Most of the studies investigating the role of mast cells in DTH reactions have been done in the intestine, lung, and skin
of Trichinella spiralis-infected mice
and picryl chloride contact-sensitized mice (9, 25-27). It has
been suggested that on contact sensitization with picryl chloride or
after primary helminth infection, DTH-initiating cells in lymphoid
organs are induced to release antigen-specific factors that bind
systemically to mast cells (4, 15, 22, 36). On local challenge with the
antigen, the armed mast cells are activated to release serotonin. Activation of serotonin receptor on vascular endothelium induces a
local increase in vascular permeability that facilitates the entry into
the tissue of the classical, lymphokine-producing DTH effector T cells
(TDTH) (15, 22, 36). These
TDTH cells recruit a perivascular
leukocyte infiltrate characteristic of DTH reactions and induce mast
cell proliferation. Both the T cell factor-dependent early phase
component (0-2 h) and the classical late (24-48 h) delayed
component of DTH reactions are expressed by an increase in
vasopermeability of challenged cutaneous, lung, and intestinal sites
(4, 35, 36).
We previously showed (18) that small intestinal DTH responses to the
contact sensitizer 2,4-dinitro-1-fluorobenzene (DNFB) in the rat were
characterized by an inflammatory response, intestinal mucosal mast cell
activation, and tissue accumulation of mucosal mast cells 48-72 h
after local challenge. In addition, pharmacological manipulation of the mucosal mast cell before and at time of challenge, either by depletion or stabilization, was very effective in reducing the DTH-specific increase in small intestinal vascular leakage and
mucosal mast cell degranulation found at 48 h (20). Using mast
cell-deficient mice, we demonstrated (21) that mast cells contribute
significantly to changes in vascular permeability associated with small
intestinal DTH reaction. These results suggested that the mucosal mast
cell is an important cell in the initiation of contact
sensitizer-induced DTH reactions in the small intestine of the rat.
Sensory neurons densely innervate the gastrointestinal tract and are
found in close association with lymphocytes and mucosal mast cells (3,
8, 33, 34). The effects of neuropeptides on immunologic functions
involved in DTH reactions are clearly described: directly via
lymphocyte proliferation, maturation, and activation and indirectly via
activation of mucosal mast cell or via effects on the vascular
endothelium (30, 32). Substance P has the ability to activate mucosal
mast cells, and substance P-induced inflammatory responses are shown to
be mast cell dependent (12, 37). Moreover, we have demonstrated that
sensory nerves are involved in the development of
dinitrobenzene-induced small intestinal DTH reactions in the mouse
(17). The results were consistent with an initiating role of sensory
neuropeptides, especially tachykinins, in dinitrobenzene-induced
intestinal DTH reactions.
The relevant biological actions together with the close association
between mast cells and sensory nerves in the gastrointestinal tract
suggest a possible function for mast cell-sensory nerve interaction in
the early component of DTH reactions. Taking the above-described
studies together, it can be postulated that at the time of challenge
the mucosal mast cell is activated via sensory neuropeptides. This
early phase process is necessary for the development of the late-phase
inflammatory reaction (edema formation, tissue damage, and mucosal mast
cell activation and accumulation). In the present study, the role of
mucosal mast cells in the early phase of the dinitrobenzene-induced DTH
reaction in the rat small intestine was examined in detail. First, we
investigated whether mucosal mast cells are activated shortly after the
challenge. Rat mast cell protease II (RMCP II), a unique protease
located within the granules of intestinal mucosal mast cells, was used as a serum marker for mast cell degranulation. Second, we examined whether this mucosal mast cell activation leads to early phase vascular
permeability changes, which in turn facilitate the entry into the
tissue of classical DTH effector cells. The vascular permeability
changes were studied from 0 to 1 h after the challenge both in vivo and
in vitro in the isolated vascularly perfused small intestine (19). The
effects of the mast cell stabilizer doxantrazole on the early
DTH-induced changes in small intestinal vascular permeability and RMCP
II serum levels were investigated in vivo. Finally, to investigate
whether the DTH-induced mucosal mast cell activation and early phase
vascular permeability changes were under sensory nervous control, the
influence of capsaicin-sensitive nerves on the early phase events of
the small intestinal DTH response were studied. Neonatal pretreatment
with capsaicin was used to deplete sensory neuropeptides. The effect of
this neuropeptide depletion was examined in vivo on DTH-induced changes
in small intestinal vascular permeability and mucosal mast cell
activation observed early after the challenge.
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METHODS |
Male Wistar rats (Utrecht × Wistar Unilever, 200-250 g; Utrecht
University, Utrecht, The Netherlands) that had free access to food
(Rmh-tm-11/10; Hopefarm, Woerden, The Netherlands) and tapwater
were used. The experiments were approved by the
Animal Care Committee of Utrecht University.
Sensitization and challenge procedure.
DNFB (5 mg/ml, 300 µl) or vehicle (acetone/olive oil, 4:1, 300 µl)
was applied epicutaneously to the shaven dorsal skin of anesthetized
rats (60 mg/kg body wt pentobarbital sodium; Nembutal, Sanofi, France;
n = 5/group) on
day 0 and day
1. On
day
5, the animals, which were previously
fasted for 16 h, were challenged with an intragastric administration of
dinitrobenzene sulfonic acid (DNBS, the water-soluble form of DNFB; 1 mg/ml, 1 ml) or vehicle (1 ml saline). DNFB and olive oil
were purchased from Sigma Chemical (St. Louis, MO). DNBS was purchased
from Eastman Kodak (Rochester, NY).
All animals used in this study, except those involved in the in vitro
vascular permeability study, received a topical challenge on one ear
with DNFB (2 mg/ml, 40 µl). Skin reactions demonstrated that all rats
sensitized with DNFB developed an ear swelling response to topical DNFB
challenge. From this, we concluded that the animals were sufficiently
sensitized to DNFB.
Treatment of experimental animals.
To investigate the effect of mucosal mast cell stabilization,
doxantrazole (10 mg/kg body wt doxantrazole monohydrate; a gift from
Wellcome, Beckenham, Kent, UK) or vehicle [0.5% sodium
bicarbonate (Sigma Chemical) in sterile saline] was injected
intraperitoneally (1 ml). The doxantrazole treatment was applied 30 min
before and at the time of challenge. This procedure was followed to be
sure that at the time of challenge a sufficiently high amount of
doxantrazole was present in the rats to stabilize mucosal mast cells in
the small intestine. The dose and the route of administration were obtained using the method of Perdue and Galli (28).
Capsaicin (Fluka Chemika, Buchs, Switzerland) was used to deplete
neuropeptides from unmyelinated sensory C fibers. Neonatally, rats
pretreated with the analgetic agent flunixine (1 mg/kg body wt ip)
received subcutaneous injections of capsaicin (50 mg/kg body wt) on 2 consecutive days. Controls were treated with vehicle (alcohol/Tween 80/saline, 2:1:7) alone. The rats were used 6-7 wk
after pretreatment with capsaicin (body wt, 200-225 g). The degree
of sensory neuropeptide depletion was checked by verifying that in
vitro capsaicin-induced relaxations of isolated carbachol-precontracted tracheas of the capsaicin-pretreated rats were reduced to <10% of
control values of tracheas of vehicle-pretreated rats.
In vivo small intestinal vascular leakage.
To assess vascular leakage in the small intestine in vivo, Evans blue
dye (20 mg/kg body wt in sterile saline; Fluka Chemika) was injected
intravenously into anesthetized animals (60 mg/kg body wt pentobarbital
sodium) 30 min after the challenge. Heparin (1,000 IU/rat; Leo
Pharmaceutical Products, Ballerup, Denmark) was administered
intravenously 5 min before the rats were killed. At
t = 60 min, the animals were killed
(with an overdose of pentobarbital sodium) and a blood sample was
taken. The intestines were perfused with warm saline (30 ml) to
eliminate the excess of vascular Evans blue. Perfusion was performed
via cardiac puncture, and the perfusate was allowed to depart via a cut
vein in the hind paw. Accumulated Evans blue dye in the small intestine
was extracted overnight using formamide (Merck, Darmstadt, Germany) at
40°C. Evans blue dye (in µg) in extracts and plasma samples was
determined using a spectrophotometer (double-beam spectrophotometer UV
150-02, Shimadzu) at 620 nm (38). Over the time interval of 30-60
min after the challenge, the small intestinal vascular leakage was measured. The amount of Evans blue was expressed as nanograms of Evans
blue per milligrams of small intestinal dry weight and corrected for
the plasma concentration (ng Evans blue/mg dry wt). The dry weight of
the pieces of small intestine was determined by drying the parts in a
stove at 60°C for ~10 days. If no changes in weight occurred over
the 2 following days, this weight was taken as the dry weight.
In vitro small intestinal vascular permeability changes.
In vitro assessment of vascular permeability was measured in the
isolated vascularly perfused small intestine. On
day 5 the entire small intestine from DNFB- or sham-sensitized rats was isolated as described previously (19). Briefly, after cannulation of
the superior mesenteric artery and the portal vein, the vascular bed
was perfused with medium. This medium consisted of a buffered salt
solution containing 20% (wt/vol) perfluorotributylamine (FC-43; 3M,
Leiden, The Netherlands), 2.46% (wt/vol) poly(propylene
glycol):polyethylene glycol (1:4) (Synperonic F-68; Sevra
Feinbiochemica, Heidelberg, Germany), 103 mM NaCl, 4.56 mM KCl,
1 mM MgCl2, 1.9 mM
CaCl2, 25 mM
NaHCO3, 10 mM glucose, 1%
(wt/vol) albumin, and 0.6 mM glutamine and was gassed with 95%
O2-5%
CO2. Perfusion through the
vascular bed was recirculated at a rate of 5 ml/min. The lumen was
perfused single pass at a rate of 0.5 ml/min. The luminal perfusate was a 0.9% NaCl solution. The small intestine was excised and transferred to a tissue bath, which was kept at 37°C. Thirty minutes after the
intestine was isolated, the luminal perfusate was changed to a solution
of DNBS (1 mg/ml in 0.9% NaCl) for in vitro challenge of the
preparation. Four tracer injections were administered 0 (i.e., before
DNBS challenge), 15, 30, and 60 min (i.e., after DNBS challenge) after
the start of the DNBS infusion. The following tracers were studied:
tetramethylrhodamine-conjugated dextran (TMRD3; 3.0 kDa, 0.03 mg/ml),
Cascade blue-conjugated dextran (CBD10; 10 kDa, 0.10 mg/ml), Texas
red-conjugated dextran (TRD70; 70 kDa, 0.27 mg/ml), and FITC-conjugated
Ficoll (FF400; 400 kDa, 0.27 mg/ml) (Molecular Probes, Eugene, OR). All
compounds were dissolved in the vascular perfusate. After the perfusion
through the vascular bed was changed from recirculating to single pass, a mixture of the four tracers (200 µl) was injected rapidly into the
arterial cannula and total venous drainage was collected in a series of
10 tubes (~300 µl each). The venous samples were weighed to
determine volume and collection time. After centrifugation (10 min at
15,000 gmax)
the concentrations of the four tracers in the supernatants were
measured fluorometrically in a 96-well plate reader (LS50B; Perkin
Elmer, Gouda, The Netherlands) with TMRD3 at 555/580 nm, CBD10 at
400/425 nm, TRD70 at 591/612 nm, and FF400 at 495/520 nm for excitation
and emission wavelength, respectively.
Pharmacokinetic analysis, based on statistical moment theory, of the
tracer (i) outflow concentration-time curve was used to assess
vasopermeability: extraction ratios
(Ei), volumes of distribution
(Vd,i), and intrinsic clearance
values (Clint,i). This involves
analysis of the complete venous outflow concentration-time curve
(dilution curve). The outflow concentrations of the tracers were
normalized by expressing the concentration in each sample relative to
the concentration in the original injectate
(Ci). Subsequently, a relative
concentration-time outflow curve was obtained for each tracer used.
The first two moments of the relative concentration-time curve of each
tracer on a single pass through the isolated vascularly perfused small
intestine were determined for the calculation of the area under the
concentration-time curve (AUCi)
and the mean transit time
(ti).
A vascular tracer (vrs) is a tracer that is not eliminated and
retained in the intravascular space. Thus, theoretically, the
extraction of a vascular tracer is 0. The extraction
ratio for diffusible tracers was calculated by
where
AUCvrs is the area under the
concentration-time curve of the vascular tracer.
The intrinsic clearance (in ml/min) according to the parallel tube
model is calculated by
where
Q is the perfusate flow rate.
The volume of distribution of each tracer
(Vd,i) is calculated (in ml)
from the intrinsic clearance by
All
the data are expressed as either a percentage
(Ei), in milliliters
(Vd,i), or in milliliters per
minute (Clint,i) per small
intestine.
Measurement of RMCP II.
The sera were collected via orbital puncture in the anesthetized rats
(sham/DNBS and DNFB/DNBS) 15, 30, and 60 min after the challenge, and
the samples were snap frozen in liquid nitrogen and stored at
80°C. From a separate group of nonsensitized and nonchallenged (naive) rats, serum samples were collected at
t = 0, 15, 30, and 60 min. The RMCP II
ELISA (Moredun Animal Health, Edinburgh, UK) was used for measurements
of RMCP II in the serum of the groups of rats described above. The
results were expressed as nanograms of RMCP II per milliliter of serum.
Data analysis.
Results are presented as means ± SE. Data were analyzed by ANOVA,
and the statistical significance of difference between means was
determined using the Newman-Keuls test.
P < 0.05 was considered to reflect a
statistically significant difference.
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RESULTS |
In vivo small intestinal vascular leakage.
Figures 1 and 2
demonstrate that 30-60 min after challenge in vivo a significant
increase in small intestinal vascular permeability was observed in
DNFB-sensitized and DNBS-challenged rats compared with control groups
(P < 0.05, ANOVA). The
DNBS challenge alone did not influence basal vascular leakage (Figs. 1
and 2).

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Fig. 1.
Effects of doxantrazole on in vivo delayed-type hypersensitivity
(DTH)-induced changes in vascular leakage in the rat small intestine
30-60 min after challenge. Small intestinal vascular leakage is
expressed as amount of Evans blue (EB) tissue accumulation (ng EB/mg
small intestinal dry wt) in 2,4-dinitro-1-fluorobenzene
(DNFB)-sensitized (solid bars) rats and sham-sensitized rats (hatched
bars) after intragastric challenge with dinitrobenzene sulfonic acid
(DNBS, the water-soluble form of DNFB) or in naive rats (open bars).
Treatments consisted of doxantrazole (10 mg/kg body wt ip) or vehicle
(0.5% sodium bicarbonate) 30 min before and at time of challenge.
Leakage responses are means ± SE for
n = 5 rats/group.
* P < 0.05 compared with
control groups; # P < 0.05 compared with vehicle-treated animals (ANOVA).
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Fig. 2.
Effects of capsaicin-induced sensory neuropeptide depletion on in vivo
DTH-induced changes in vascular leakage in the rat small intestine
30-60 min after challenge. Small intestinal vascular leakage is
expressed as amount of EB tissue accumulation (ng EB/mg small
intestinal dry wt) in DNFB-sensitized (solid bars) rats or
sham-sensitized rats (hatched bars) after intragastric challenge with
DNBS or in naive rats (open bars). Rats were treated with capsaicin (50 mg/kg body wt sc) neonatally (Caps) or vehicle (alcohol/Tween
80/saline, 2:1:7) (Veh). Leakage responses are means ± SE for
n = 5 rats/group.
* P < 0.05 compared with
control groups; # P < 0.05 compared with vehicle-treated animals (ANOVA).
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In vitro small intestinal vascular permeability changes.
The isolated fluorocarbon vascularly perfused small intestine, a
preparation with physiological viability, was used to measure microvascular permeability changes from 15 to 60 min after in vitro
DNBS challenge in detail using tracer molecules of different sizes
(1.4- to 6-nm radius). FF400 (vrs) was used as the vascular tracer
(Clint,vrs = 0 ml/min). The volume
of distribution of FF400 (Vd,vrs = 1.45 ± 0.05 ml) reflects the volume of the mesenteric vasculature
between injection and sampling sites. Under control conditions (before
in vitro DNBS challenge), the volume of distribution and the intrinsic
clearance value of the diffusible tracer TRD70, a molecule that
resembles plasma albumin in size, did not differ significantly from the
vascular tracer. When the molecular radii of the tracers (CBD10 > TMRD3) decrease, an increase in volumes of distribution and intrinsic
clearance values was observed. Under control conditions, no
significantly differnt permeability values were found for CBD10
and TMRD3 (Table 1 and Fig.
3). Figure
3A and Table 1 show that in vitro DNBS
challenge in sham-sensitized intestinal preparations did not
significantly change the vascular permeability characteristics of all
the tracers studied. In addition, DNBS challenge in isolated vascularly
perfused small intestines of DNFB-sensitized rats did not induce
changes of the volume of distribution of the vascular tracer FF400.
However, intraluminal DNBS challenge in DNFB-sensitized preparations
after 30-60 min significantly increased the volumes of
distribution of the diffusible tracers TRD70, CBD10, and TMRD3 (Table
1). The volumes of distribution of CBD10 and TMRD3
(Vd,CBD10 vs.
Vd,TMRD3, 3.07 ± 0.23 vs. 3.13 ± 0.20 ml, respectively) were two times the
vascular volume (Vd,vrs = 1.59 ± 0.03 ml) 30 min after the DNBS challenge. Figure
3B shows that 15 min after the DNBS
challenge the vascular permeability for all diffusible tracers started
to rise. The increase in vascular permeability peaked 30-60 min
after the challenge. At t = 15 min, the sensitized and challenged mesenteric vasculature was most permeable
for the smallest tracer, TMRD3 (Fig.
3B). However, 30-60 min after
the DNBS challenge the clearance values for CBD10 and TMRD3 were not
statistically different from each other (Fig.
3B). The increase in permeability is
seen earlier for the smaller molecules but is more extensive for the
larger tracer (TRD70). Intraluminal DNFB challenge did not have an
effect on the basal vascular pressure (~90 mmHg) in either sham- or
DNFB-sensitized intestinal preparations (data not shown). These results
clearly demonstrate that vascular permeability changes early after mast
cell activation can be detected using tracer molecules of varying
sizes.
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Table 1.
Extraction ratios and volumes of distribution of tracers in the rat
isolated vascularly perfused small intestine
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Fig. 3.
In vitro small intestinal vascular permeability of sham-sensitized
(A) or DNFB-sensitized
(B) rats. Intrinsic clearance values
(Clint,i; in ml/min) of the
diffusible tracers: TRD70 (circles), CBD10 (diamonds), and TMRD3
(triangles) under control conditions and 15, 30, and 60 min after in
vitro DNBS challenge in isolated vascularly perfused small intestinal
preparations. Results are means ± SE for
n = 4 preparations. If no
error bar is shown, SE was smaller than the symbol used. Under control
conditions, there was a statistically significant difference between
the vascular tracer FF400 and the diffusible tracers CBD10 and TMRD3 at
all time points measured (P < 0.05, ANOVA). * P < 0.05, significant differences between control (before challenge) and after
DNBS challenge (ANOVA).
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RMCP II release in serum.
RMCP II was determined in serum samples from naive rats as well as from
sham- or DNFB-sensitized and DNBS-challenged rats. Basal RMCP II level
in the serum of naive rats was ~100 ng RMCP II/ml serum. No
differences were observed in RMCP II basal levels when the samples were
obtained at t = 15, 30, and 60 min.
Figure 4 shows that DNBS challenge alone
significantly enhanced the RMCP II levels 15 and 30 min after the
challenge. However, in DNFB-sensitized and DNBS-challenged rats, a more
profound fourfold increase in RMCP II serum levels was found, which was
significantly different from levels of sham-sensitized and
DNBS-challenged rats. This elevation of serum RMCP II returned to basal
levels 120 min after the DNBS challenge (Fig. 4).

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Fig. 4.
In vivo DTH-induced changes in levels of rat mast cell protease II
(RMCP II) in rat serum 0-60 min after challenge. RMCP II serum
levels are expressed as ng RMCP II/ml serum in sham-sensitized rats
( ) or DNFB-sensitized rats ( ) after intragastric challenge with
DNBS or in naive rats ( ). Results are means ± SE for
n = 5 rats/group. If no error bar is
shown, SE was smaller than the symbol used.
* P < 0.05 compared with
control groups; # P < 0.05 compared with naive rats (ANOVA).
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Effect of doxantrazole treatment on small intestinal DTH reaction.
Mast cell stabilization induced by doxantrazole injection 30 min before
and at time of challenge significantly inhibited the DTH-induced
increase in small intestinal vascular permeability 30-60 min after
the challenge (Fig. 1). In addition, analysis of RMCP II levels in the
serum of DTH rats after doxantrazole pretreatment showed that this
compound was effective in reducing the DTH-induced elevation of serum
RMCP II (Fig. 5).

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Fig. 5.
Effect of doxantrazole on in vivo DTH-induced changes in levels of RMCP
II in rat serum 0-60 min after challenge. Treatment consisted of
doxantrazole (10 mg/kg body wt ip) or vehicle (0.5% sodium
bicarbonate) 30 min before and at time of challenge. RMCP II serum
levels are expressed as ng RMCP II/ml serum after intragastric
challenge with DNBS in sham-sensitized, vehicle-treated ( ) or
doxantrazole-treated ( ) rats or DNFB-sensitized, vehicle-treated
( ) or doxantrazole-treated ( ) rats. Results are means ± SE
for n = 5 rats/group. If no error bar
is shown, SE was smaller than the symbol used.
* P < 0.05 compared with
control groups; # P < 0.05 compared with vehicle-treated animals (ANOVA).
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Effect of sensory neuropeptide depletion on small intestinal DTH
reaction.
Figure 2 shows that neuropeptide depletion completely inhibited the
increase in small intestinal vascular permeability observed 30-60
min after challenge in DNFB-sensitized rats. No effects of capsaicin
pretreatment were found on basal vascular leakage. Furthermore,
depletion of neuropeptides completely abolished the DTH-induced
increase in serum RMCP II levels observed 30 min after challenge (Fig.
6).

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Fig. 6.
Effect of capsaicin-induced sensory neuropeptide depletion on in vivo
DTH-induced changes in levels of RMCP II in rat serum 0-60 min
after challenge. Treatment consisted of capsaicin (50 mg/kg body wt sc)
neonatally or vehicle (alcohol/Tween 80/saline, 2:1:7). RMCP II serum
levels are expressed as ng RMCP II/ml serum after intragastric
challenge with DNBS in sham-sensitized, vehicle-treated ( ) or
capsaicin-treated ( ) rats or DNFB-sensitized, vehicle-treated ( )
or capsaicin-treated ( ) rats. Results are means ± SE for
n = 5 rats/group. If no error bar is
shown, SE was smaller than the symbol used.
* P < 0.05 compared with
control groups; and # P < 0.05 compared with vehicle-treated animals (ANOVA).
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DISCUSSION |
The aim of this study was to investigate the role of the mucosal mast
cell in the early vascular permeability changes of
dinitrobenzene-induced DTH reactions in the small intestine of the rat.
DTH reactions are local T lymphocyte-dependent immune responses
manifested by an inflammation (35). It has been suggested that such a
cell-mediated response plays an important role in the pathogenesis of
IBD (13).
DTH reactions are characterized by an early (<2 h after the
challenge) and a late component (24-48 h after the challenge). It
is hypothesized that the early DTH response is induced by
antigen-specific factors released by T lymphocytes within 1-2 days
of sensitization, which arm mast cells systemically (26). On local
challenge, the T cell factor-armed mast cells release vasoactive
mediators, which cause a local increase in vascular permeability (27). First, we have measured DTH-induced small intestinal vascular leakage
within 2 h after the challenge. In vivo as well as in vitro a
significant increase in small intestinal vascular permeability was
found 30-60 min after DNBS challenge of DNFB-sensitized rats. Experiments with isolated vascularly perfused small intestines of
DNFB-sensitized rats revealed that the DTH-induced increase of
vasopermeability had already started 15 min after the DNBS challenge.
Because the volume of distribution of the vascular tracer (FF400) did
not change during the DTH reaction, it can be concluded that the DTH
reaction does not give a greater exchange surface by opening up
capillaries (Table 1). These in vitro results clearly suggest that
changes in vascular peremability, expressed as
Clint,i, can be detected using
tracer molecules of varying sizes, although it cannot be formally
excluded that changes in Clint,i
[or the permeability- surface area (PS) product]
are the result of changes in available surface area rather than changes in permeability per se. It is unlikely that capillary
recruitment and/or increased perfusion of mucosal vascular bed
contributes significantly to an increase of PS product.
Furthermore, it has been shown previously (19) that diffusion
limitation, rather than flow limitation, applies to the tracers used.
Our findings are consistent with the early vascular permeability
changes in the skin DTH reaction to picryl chloride described by Van
Loveren and co-workers (35) and in the intestinal helminth infections
described by Parmentier and co-workers (26). Furthermore, it was
demonstrated that mice intravenously injected with purified T cell
factor developed an antigen-specific early cutaneous response after
local challenge (4).
There are several lines of evidence that favor a role for the mast cell
in the early phase of DTH reactions. First, the reactions are elicited
preferentially at sites enriched in mast cells, such as the intestinal
tract, lung, buccal mucosa, and skin (1, 9, 25). The release of the
mast cell mediator serotonin during contact sensitization in the skin,
early after the challenge, has been reported (14, 16, 35). This
vasoactive amine can act locally by increasing vascular permeability
and inducing vasodilatation, thereby facilitating cellular infiltration
(2, 4). In addition, corticosteroid-induced mucosal mast cell depletion
and treatment with mast cell stabilizers or serotonin antagonists
suppressed DTH reaction in the skin and small intestine of the mouse
and the rat (2, 4, 14). Defective DTH responses have been observed in
strains of mast cell-deficient mice (4, 23). However, no reports have
described direct assessment of mast cell activation during the early
events of DTH reactions in the small intestine. In this study, we have
monitored in vivo mucosal mast cell activation up to 120 min after the
challenge by measurement of RMCP II. RMCP II is a protease specific for
intestinal mucosal mast cells that appears in the serum of rats after
mast cell degranulation (24). Our results demonstrate
that mucosal mast cells are activated 15-30 min after DNBS
challenge in DNFB-sensitized rats. This mucosal mast cell activation
slightly preceded the DTH-induced small intestinal vascular leakage
changes found in vivo 30-60 min after the challenge. It even
suggests that the DNBS challenge alone has a direct irritating effect
on intestinal mucosal mast cells, because of the small rise in RMCP II
found in sham-sensitized animals. This DNBS-induced mucosal mast cell
activation was not accompanied by changes in vascular permeability in
the small intestine after only DNBS challenge. It can be concluded that
a more profound mucosal mast cell activation is necessary to result in
an increase in vascular permeability.
To further investigate the role of the mucosal mast cell in the early
component of small intestinal DTH reactions, rats were pretreated
before the challenge with the mast cell stabilizer doxantrazole. This
compound is able to prevent antigen-induced histamine release from
gut-associated mucosal mast cells and to protect sensitized rats
against ovalbumin-induced anaphylactic reactions (5, 28). We have found
that doxantrazole pretreatment inhibited the DTH-induced increase in
small intestinal vascular permeability and RMCP II elevation in the
serum. This is consistent with an important initiating role of the
mucosal mast cell in the onset of dinitrobenzene-induced DTH reactions
in the small intestine of the rat.
Few investigators have examined the role of sensory neuropeptides in
DTH reactions. The close association of sensory nerves with mucosal
mast cells and lymphocytes found in the gastrointestinal tract and the
relevant biological actions suggest a possible function for
neuropeptides in DTH reactions (3, 8, 32-34). Substance P induces
mediator release from different mast cell types in vitro (5). The
observation of release of airway mast cell mediators in vivo by
substance P and neurokinin A supports the growing belief that mast
cells are under nervous control (12). In this study, it was
demonstrated that depletion of neuropeptides from sensory nerves by
neonatal pretreatment with capsaicin resulted in significant inhibition
of the early vascular leakage DTH response as well as in reduced
mucosal mast cell activation. These results confirm the inhibitory
effects of neuropeptide depletion and blockade of the neurokinin-1
receptor observed on dinitrobenzene-induced small intestinal DTH
reaction in the mouse (17). However, it is unclear whether sensory
neuropeptides act directly on the mucosal mast cell in the DTH reaction
or indirectly on T cell factor-producing lymphocytes. The tachykinins
substance P and neurokinin A predominantly stimulate the proliferation,
migration, and activation of lymphocytes (32, 33), whereas vasoactive
intestinal peptide, calcitonin gene-related peptide, and somatostatin
have inhibitory activities (32). In addition, receptors for tachykinins
have been found on lymphocytes (7). Thus neuropeptide depletion could
result in a direct inhibition of DTH-induced mucosal mast cell
activation or in an inhibition of proliferation and activation of T
cell factor-producing lymphocytes, which in turn will lead to less T
cell factor-armed mast cells. Neonatal treatment with capsaicin could
also affect the number of mucosal mast cells in the small intestine.
Recently, Gottwald and colleagues (11) have demonstrated that 3 mo
after neonatal capsaicin administration 28% fewer intestinal mucosal
mast cells were found in treated rat jejunum compared with littermate
controls. In this study, the rats were used at the age of 7-8 wk.
It is not likely that at this age neonatal capsaicin pretreatment
results in a complete depletion of intestinal mucosal mast cells and
that the effects of this treatment found on DTH-induced vascular
permeability changes and mucosal mast cell activation are only the
result of a lowering of the number of mast cells.
In conclusion, the results of this study are consistent with an
important role of the mucosal mast cell in the early vascular events in
the dinitrobenzene-induced DTH reaction in the small intestine of the
rat. In addition, we have demonstrated an interaction between sensory
nerves and mucosal mast cell activation during the initiation phase of
the small intestinal DTH response.
 |
ACKNOWLEDGEMENTS |
A. D. Kraneveld was supported by a Glaxo (Ware, UK) Ph.D.
scholarship.
 |
FOOTNOTES |
Address for reprint requests: A. D. Kraneveld, Dept. of Pharmacology
and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences,
Utrecht Univ., PO Box 80.082, 3508 TB Utrecht, The Netherlands.
Received 3 September 1996; accepted in final form 23 January 1998.
 |
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