* Department of Experimental Pathology, Institute of Medical Biology, University of Tromsø, Norway
Department of Evolutionary Immunobiology, Institute of Zoology, Jagiellonian University, Krakow, Poland
Correspondence: Barbara Plytycz, Department of Evolutionary Immunobiology, Institute of Zoology, Jagiellonian University, R. Ingardena 6, PL-30-060 Krakow, Poland. E-mail: plyt{at}zuk.iz.uj.edu.pl
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, IL-1ß, and IL-6) measured at
6 h of inflammation. All of those factors were fully restored
after selective intraperitoneal reconstitution of W/Wv mice
with bone marrow-derived mast cells from their control +/+
counterparts. Cromolyn pretreatment of Balb/c mice reduced exclusively
the early plasma exudation and histamine influx. Blocking of histamine
receptors inhibited not only the early plasma exudation but also
temporarily diminished primary leukocyte influx and levels of MCP-1 and
IL-1ß. In conclusion, mast cells play an important role in the
initiation of zymosan-induced peritonitis and modulate its further
course.
Key Words: peritonitis • cromolyn • mepyramine • histamine receptors • exudate leukocytes
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(TNF-), which can be released immediately during the
initial stages of acute inflammation, and various other factors can be
rapidly produced de novo upon mast cell stimulation.
Inducible factors include vasoactive and chemotactic mediators like
platelet-activating factor (PAF), leukotrienes, complement factors, and
proinflammatory and growth-promoting cytokines [21
,
22
].
Inhibition of inflammation-related factors has been recorded
consistently in genetically mast cell-deficient WBB6F1-W/Wv
(W/Wv) mice with a defect of the hemopoietic stem cells
(because of lack of the c-kit receptor) leading, among others, to an
age-related depletion of the connective-tissue mast cells
[23
24
25
26
]. Plasma protein permeation was significantly
lower in W/Wv mice than in their congenic controls during
the reverse passive Arthus reaction [27
]. A significant
delay in polymorphonuclear leukoctye (PMN) influx was described in
W/Wv mice during thioglycollate-induced [10
]
and immune complex-induced peritonitis, the latter case accompanied by
a reduced peritoneal TNF- level [12
]. In the above
models, the defects of inflammation were corrected by adoptive transfer
of mast cells from the bone marrow of the congenic controls
[10
, 12
]. The comparison of inflammation in
W/Wv mice and their congenic (+/+) controls led to the
conclusion that mast cells participate in neutrophil recruitment by the
release of preformed and stored TNF- very early during the immune
complex-induced peritonitis [12
]. The importance of mast
cells and/or stem cell factors (required for mast cell maturation) in
the regulation of cytokine production in the course of zymosan-induced
peritonitis was also established in stem cell factor-deficient
(WCB6F1/J-Sl/Sld) mice. The peritoneal lavage fluid of
Sl/Sld mice showed a decreased level of interleukin (IL)-4,
a markedly increased IL-1ß, and unaffected levels of TNF- and IL-6
[28
]. Both strains of mast cell-deficient mice,
W/Wv and Sl/Sld, were used for investigations
of the role of tissue mast cells in polyacrylamide gel-induced,
skin-pouch inflammation. It was confirmed that mast cells play a
prominent role in the PMN influx, TNF- production, and eicosanoid
formation [11
].
In the present studies, some inflammation-related parameters were
compared in zymosan-stimulated W/Wv mice with those in the
+/+ controls and mast cell-reconstituted W/Wv mice
(W/Wv+MC). In concordance with the results of other
investigators, all the recorded parameters were decreased in animals
with the genetic defects, and the intraperitoneal transfer of +/+ bone
marrow-derived mast cells restored all the impaired factors fully.
Furthermore, we investigated the involvement of mast cells in the
zymosan-induced peritoneal inflammation in the Balb/c mice without any
genetic defects. For this purpose, we have established the normal time
course of the main hallmarks of the inflammatory process (such as an
increased vascular permeability, exudatory leukocyte influx,
accumulation of plasma and exudate chemoattractants and proinflammatory
cytokines, TNF-, IL-1,ß, and IL-6) during 24 h following
irritant administration. Then we focused on the selected time points
(30 min, and 2, 6, and 24 h) when we compared these parameters in
the control and drug-pretreated animals.
Pretreatment with cromolyn, a well-known mast cells stabilizer protecting against degranulation [29 , 30 ], caused a significant impairment of plasma exudation at 30 min of inflammation corresponding to a significantly decreased level of histamine, one of the most potent vasoactive factors released from activated mast cells [1 ]. Cromolyn action, however, was short-lasting because the level of histamine returned to the control level as soon as 2 h after induction of peritonitis. In contrast, the pharmacological blockade of histamine receptors impaired not only the early plasma exudation (at 30 min) but also exudatory leukocyte influx (at 2 h). Also, the levels of monocyte chemoattractant protein-1 (MCP-1) and IL-1ß were reduced significantly at 2 and 6 h of inflammation, respectively.
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Reconstitution of mast cells in W/Wv mice
Bone marrow cells harvested from femurs of male +/+ mice were
cultured for 3 weeks in WEHI 3-conditioned medium as described
previously [31
]. Mast cells (>98% purity,
1x107 cells/mouse) were injected intraperitoneally (i.p.)
into W/Wv mice 3 weeks before induction of peritonitis
[10
, 12
, 13
]. Selective
administration of mast cells (but not bone marrow cells) restores only
the mast cell deficiency [32
].
Induction of peritonitis
Peritoneal inflammation was induced according to Doherty
et al. [2
]. Zymosan A (Sigma Chemical Co.,
St. Louis, MO) was prepared freshly (2 mg/ml) in sterile, 0.9% w/v
saline, and 0.5 ml was injected i.p. At the selected time points,
animals were killed by cervical dislocation. The peritoneal cavity was
lavaged with 1.5 ml saline, and after a 30-s gentle manual massage, 1
ml exudate was retrieved, centrifuged at 3000 g for 3 min,
and frozen at -20°C prior to cytokine or chemotactic-factor
assessment. Alternatively, 1.5 ml exudate was collected and used
freshly for cell counts and cytospin preparations.
Vascular permeability
Evans blue (Sigma) was suspended in saline (10 mg/ml) and
injected i.v. into the caudal vein (0.3 ml/mouse). Thirty minutes
later, the animals were killed, and the peritoneal cavity was lavaged
with 1.5 ml saline as described above. The lavage fluid was
centrifuged, and the absorbance of dye in the supernatant was measured
at 650 nm with a Titer-Tech, 96-well multiscanner following
transfer of 200 µl aliquots to a 96-well plate [3
].
Histamine content
Histamine content in the peritoneal fluid was measured by a
Histamine ELISA kit (ICN Pharmaceuticals, Inc., Cost Mesa, CA). The
assay was carried out as indicated by the manufacturer.
Cell counts
Total and differential cell counts were done with a
hemocytometer following staining with Turk’s solution (0.01% crystal
violet in 3% acetic acid) and on May-Grunwald Giemsa-stained cytospin
preparations. Mast cell staining with alcian blue (stains connective
tissue and mucosal mast cells) and safranin O (specific for connective
tissue mast cells; Sigma) was performed as described previously
[10
].
Plasma collection
Blood was obtained from the interior vena cava and immediately
centrifuged at 300 g for 10 min. Then the plasma was
collected and stored at -20°C prior to evaluation of the leukocyte
chemoattractant activity.
Cytokine content
The peritoneal exudate content of mouse TNF- and IL-1ß was
measured by DuoSet ELISA (Genzyme Corp., Cambridge, MA), and the
immunoreactive MCP-1 was measured by an immunoassay it (BioSource
International, Camarillo, CA). The assays were carried out as
indicated by the manufacturers.
Cytokine bioactivity
Bioassays for IL-1 and ß, TNF-, and IL-6 were performed
on WEHI 164 clone 13, D-10.G.4 and B9 cell lines, respectively,
according to the well-established procedures [33
34
35
].
Chemotaxis assay
A 48-well microchemotaxis chamber (Neuro Probe, Inc.,
Gaithersburg, MD) was used to assess the leukocyte chemoattractant
activity [36
]. The lower wells of the apparatus were
filled with 27 µl control culture media or samples of plasma (in
triplicates) from intact or zymosan-treated animals and covered with a
nitrocellulose filter (nucleopore membrane; Neuro Probe), 5 µm pore
size. The upper wells were filled with 50 µl bone marrow leukocyte
suspension washed out from femurs of intact mice (1.5x106
cells/ml). The bone marrow leukocytes were used for chemotaxis assay,
because they are the main source of murine leukocytes
[37
]. Following 3 h incubation at 37°C, the cells
remaining on the upper surface of the filter were removed, the filter
was fixed in 4% buffered formaline, stained with Harris’ hamatoxyline
(40 min), and cleared in xylene (15 min). The cells accumulated within
the filter were counted per high-power field (40x) on three to four
levels and pooled. The procedure was repeated in three independent
fields, and the mean value was calculated for each well. The mean
values from triplicate wells of each sample (within the same filter)
were used for statistical analysis.
Evaluation of the effects of drug pretreatment
Mast cell stabilizer
Sodium cromoglycate (cromolyn; Sigma) [29
,
30
] is used commonly for mast cell stabilization at
various doses and routes of administration. Our pilot experiments
(unpublished results) led to selection of a dose (one for each route)
that inhibited zymosan-induced vascular permeability by approximately
50% (ED50). The ultimate chosen doses were as follows:
subcutaneously (s.c.; 160 mg/kg; 39 mM), i.p. (160 mg/kg; 39 mM), i.p.
(400 mg/kg; 97 mM), or intravenously (i.v.; 200 mg/kg; 48 mM)
[38
]. Cromolyn was dissolved in saline and administered
(200 µl/mouse) 30 min before i.p. injection of zymosan, followed by
i.v. injection of Evans blue when necessary.
Histamine receptor antagonists
Mepyramine and cimetidine (Sigma) were administrated s.c. in a
vol of 1 ml/kg [39
40
41
42
]. Cimetidine base was dissolved
in a small quantity of 0.1 N HCl, neutralized with NaOH, and made up to
the required volume. Triprolidine and ranitidine (ICN Pharmaceuticals)
were administrated i.p. in a volume of 10 ml/kg [41
].
Antagonists were administered 30 min before i.p. injection of zymosan
followed by i.v. injection of Evans blue when necessary.
Based on dose studies (see Fig. 5), 2 mg/kg (5 µM) mepyramine was chosen for further investigations.
Statistical analysis
All values are shown as means ± SE.
Kinetic changes of each parameter were analyzed by one-way analysis of
variance (ANOVA), comparing the values recorded at the individual time
points with that at time 0 (in intact animals). Differences between
treated and control animals were analyzed by Student’s
t-test. Differences were considered statistically
significant at p < 0.05.
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Figure 1. Kinetics of zymosan-induced peritoneal inflammation in Balb/c mice.
Animals were i.p. injected with zymosan (1 mg in 0.5 ml) and sacrificed
at selected time points after treatment; time 0 represents the
untreated control animals. The blood plasma and peritoneal exudate were
retrieved and analyzed for the amount of soluble factors or exudate
cell numbers. a) Changes of vascular permeability (solid symbols)
measured by albumin-bound Evans blue leakage into peritoneal cavity 30
min after i.v. dye injection and changes of histamine level (open
symbols). b) Exudate cell numbers: solid square symbols, total number
of PTL; open square symbols and broken line, number of PMN; solid oval
symbols and broken line, number of monocytes/macrophages (MO/MF). c)
Level of plasma chemotactic factors measured as the number of bone
marrow cells (per high-power field) from intact mice responding to
tested plasma from zymosan-treated animals in a 48-well chemotactic
chamber (solid symbols); MCP-1 content in the peritoneal exudate fluid
estimated by ELISA (open symbols and broken line). d) Bioactivity of
proinflammatory cytokines (TNF-, IL-1, and IL-6) in the peritoneal
exudate. All results are shown as means ± SE in
groups of 5–6 mice. a–c) Mean values not sharing letters are
statistically significant according to ANOVA.
|
Histamine level in the peritoneal fluid
Histamine content was negligible in control mice and sharply
increased after zymosan stimulation with a peak at 30 min but was still
above the control level 24 h after stimulation (Fig. 1a)
.
Accumulation of exudate leukocytes
The number of peritoneal leukocytes (PTL) decreased slightly
immediately after zymosan injection, perhaps because of their increased
adherence to the peritoneal lining, a phenomenon also recorded in other
studies [2
, 43
]. Following this initial
drop, the number of PTL increased gradually with a maximum at 6 h
after zymosan injection and then decreased slightly but was still far
above the control level 24 h after injection (Fig. 1b)
and even 7
days after induction of peritonitis (unpublished results). Among
exudate PTL, the PMN dominated at the early stages of inflammation and
then were gradually replaced by mononuclear leukocytes (Fig. 1b)
.
Chemotactic factors
The cell recruitment at the inflammatory focus correlated with the
presence of chemotactic factors (total and MCP-1) in the peritoneal
fluid (Fig. 1c)
.
Proinflammatory cytokines
Peritoneal exudate fluid was also a rich source of proinflammatory
cytokines, which appeared in a characteristic sequence, with TNF-,
IL-1 and ß, and IL-6 reaching maximum bioactivity at 4, 6, and
12 h after zymosan injection, respectively (Fig. 1d)
. The TNF-
level measured by enzyme-linked immunosorbent assay (ELISA) showed two
distinct peaks, the first one almost immediately after zymosan
injection and the second one 2–4 h later (Fig. 2a
). The IL-1ß kinetics measured by ELISA also showed two peaks
and a lower level than that measured by bioassay, which detects IL-1
and IL-1ß (compare Fig. 2b
with Fig. 1d
).
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Figure 2. Kinetics of TNF- (a) and IL-1ß (b) content in the peritoneal
exudate fluid at selected time points after i.p. zymosan injection (1
mg in 0.5 ml). Mean values ± SE.
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Figure 3. Effects of cromolyn on vascular permeability (a) and histamine content
in the peritoneal fluid (b) during zymosan-induced peritonitis in
Balb/c mice. For evaluation of vascular permeability (a), animals were
pretreated s.c., i.p., or i.v. with cromolyn 30 min before i.p.
injection of zymosan (1 mg in 0.5 ml) followed by i.v. injection of
Evans blue. The amount of blue dye was spectrophotometrically estimated
at 650 nm in the peritoneal exudate retrieved 30 min after zymosan/dye
treatment. For evaluation of histamine content (b), mice were i.p.
injected with saline (VEH) or cromolyn (CRO; 160 or 400 mg/kg, i.e., 39
and 97 mM, respectively) 30 min before i.p. injection of zymosan and
sacrificed at the selected time points. All results are shown as
means ± SE in groups of 5–6 mice. Differences of
mean values not sharing letters are statistically significant according
to ANOVA.
|
Cromolyn doses used here (160 and 400 mg/kg, i.p.) had no effects on
PTL number, the level of plasma chemoattractants, and TNF-, IL-1ß,
and IL-6 levels measured 2, 6, and 24 h after zymosan stimulation
(Fig. 4
).
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Figure 4. Effects of cromolyn on selected parameters of zymosan-induced
peritoneal inflammation. Thirty minutes before zymosan injection (1 mg
in 0.5 ml), Balb/c males were i.p. injected with saline (VEH) or mast
cell stabilizer cromolyn (CRO; 160 or 400 mg/kg, i.e., 39 and 97 mM,
respectively). At 2, 6, or 24 h after zymosan injections, some
parameters were compared between the control vehicle-treated (open
bars, VEH) and cromolyn-treated experimental animals (hatched bars,
CRO). a) Exudate PTL numbers; b) PMN numbers; c) plasma chemoattractant
level measured as the number of bone marrow cells (per high-power
field) responding to plasma from zymosan-treated animals; d–f)
ELISA-estimated amounts of proinflammatory cytokines (MCP-1, TNF-,
IL-1ß, respectively) in exudate fluid; g) bioactivity of IL-6. All
results are shown as means ± SE in groups of 5–8
mice. ND, not determined.
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Figure 5. Effects of antagonists of histamine receptors on early vascular
permeability during zymosan-induced peritonitis in Balb/c mice. Animals
were pretreated with H1-receptor antagonists (mepyramine or
triprolidine) or H2-receptor antagonists (cimetidine or
ranitidine) 30 min before i.p. injection of zymosan (1 mg in 0.5 ml)
followed by i.v. injection of Evans blue. The amount of blue dye was
estimated at 650 nm in peritoneal exudate retrieved 30 min after
zymosan/dye injection. All results are shown as means ±
SE in groups of 6–8 mice. *p < 0.5 when
compared with the saline-pretreated control (zymosan only).
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Figure 6. Effects of blocking histamine H1 receptors on the selected
parameters of zymosan-induced peritoneal inflammation. Thirty minutes
before zymosan injection (1 mg in 0.5 ml), Balb/c males were s.c.
injected with saline or mepyramine, an antagonist of
H1-histamine receptor (H1; 2 mg/kg, 5 µM). At
2, 6, or 24 h after zymosan injections, some parameters were
compared between the vehicle-treated control (open bars, VEH) and the
mepyramine-treated (hatched bars, H1) experimental animals.
a) Exudate PTL numbers; b) PMN numbers; c) plasma chemoattractant level
measured as number of bone marrow cells (per high-power field)
responding to plasma from zymosan-treated animals; d–f)
ELISA-estimated amounts of proinflammatory cytokines (MCP-1, TNF-,
IL-1ß, respectively) in exudate fluid; g) bioactivity of IL-6. All
results are shown as means ± SE in groups of 5–6
mice. Some differences between the corresponding control and
experimental groups are significant at 0.05 > p > 0.01 (*) or 0.01 > p > 0.001 (**). ND, not
determined.
|
In the course of zymosan-induced peritonitis in mast cell-deficient
W/Wv mice, all investigated inflammation-related factors
were decreased significantly when compared with +/+ congenic controls
and reached the level specific for +/+ mice in mast cell-reconstituted
W/Wv animals (W/Wv+MC). Basal levels in the
vehicle-treated mice were the same in all three groups of animals
(Figs. 7
and 8
). Protein leakage and histamine
content in the peritoneal fluid at 30 min of inflammation were
significantly lower in W/Wv animals than in +/+ and
W/Wv + MC mice (Fig. 7a
and 7b)
. At 6 h after zymosan treatment, the number of exudate
cells (total PTL and PMN) and the levels of cytokines (TNF-,
IL-1ß, and IL-6) were reduced significantly in W/Wv
compared with those in +/+ and W/Wv + MC mice (Fig. 8)
.
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Figure 7. Vascular permeability (a) and histamine content in the peritoneal fluid
(b) during zymosan-induced peritonitis in WBB6F1 mice. Control (+/+,
solid bars), mast cell-deficient (W/Wv, open bars), and
mast cell-reconstituted W/Wv mice (W/Wv+MC,
dotted bars) were i.p. injected with saline (vehicle, VEH) or zymosan
(1 mg in 0.5 ml, Z). Vascular permeability was measured as the amount
of blue dye spectrophotometrically estimated at 650 nm in the
peritoneal exudate retrieved 30 min after i.v. injection of Evans blue.
All results are shown as means ± SE in groups of 6–8
mice. Some differences between W/Wv versus +/+ or
W/Wv + MC versus +/+ groups are significant at
p < 0.5 (*).
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Figure 8. Selected parameters of zymosan-induced peritoneal inflammation in
WBB6F1 mice: control (+/+), mast cell-deficient (W/Wv), and
mast cell-reconstituted W/Wv (W/Wv+MC). At time
0 (0 h, intact mice) and 6 h after zymosan injections (6 h,
animals with peritonitis), some parameters were compared between the
control (+/+, solid bars), mast cell-deficient (W/Wv, open
bars), and mast cell-reconstituted W/Wv mice
(W/Wv+MC, dotted bars). a) PTL numbers; b) PMN numbers; c)
plasma chemoattractant level measured as the number of bone marrow
cells (per high-power field) responding to plasma from zymosan-treated
animals; d and e) ELISA-estimated amounts of proinflammatory cytokines
(TNF-, IL-1ß, respectively) in exudate fluid; f) bioactivity of
IL-6. All results are shown as means ± SE in groups
of 6–8 mice. Some differences between W/Wv versus +/+ or
W/Wv + MC versus +/+ groups are statistically
significant at 0.05 > p > 0.01 (*), 0.01 >
p > 0.001 (**), or p < 0.001 (***).
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followed by
IL-1ß and IL-6; Figs. 1
and 2
)—a pattern of inflammation typical of
zymosan-induced peritonitis. As recorded previously by Lokesh et
al. [44
], the accumulation of serum proteins in the
peritoneum is considerably more rapid and stronger after zymosan
injection than when induced by some other stimulants given i.p. A
gradual replacement of PMN by mononuclear cells was recently described
in detail [6
, 45
, 46
]. The two
peaks of TNF- activity recorded in our studies were also detected by
Zhang et al. [12
], who concluded that the
early influx of TNF- originated from mast cell granules (containing
preformed TNF-), and the second peak probably represented newly
synthesized TNF- of neutrophilic origin. In our samples, the second
peak of TNF- estimated by a bioassay was slightly delayed versus
that detected by ELISA, perhaps because the presence of TNF- soluble
receptors could mask its bioactivity at low concentrations
[47
].
Cromolyn inhibition of mast cell degranulation
The cromolyn blockade of zymosan-induced mast cell degranulation
in Balb/c mice led to significant (but not total) inhibition of early
vascular permeability (Fig. 3a)
and peritoneal histamine levels at 30
min of inflammation (Fig. 3b)
. However, at the later stages of
peritonitis (2, 6, and 24 h), the histamine content and all
measured parameters (the number of peritoneal leukocytes and level of
proinflammatory cytokines) were the same in drug-treated animals and
their vehicle-treated counterparts (Fig. 4
and Table 1
).
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Table 1. Effects of Mast Cells/Histamine on Zymosan-Induced Peritonitis
|
Tanizaki et al. [51 ] demonstrated that the mode of action of cromolyn is connected with the inhibition of influx of Ca2+ into the mast cells, which in turn suppresses the release of histamine. In our experiments, such a phenomenon seems to be reflected by the significantly decreased histamine level correlated with the decreased vascular permeability in the cromolyn-treated mice at 30 min of inflammation (Fig. 3a and 3b) . Nevertheless, it is interesting that cromolyn blocks only certain activities of mast cells because they can produce many mediators without degranulation [1 , 19 , 20 ]. Furthermore, clinical studies show that cromolyn is short-lived [52 ], and this feature might explain the lack of the apparent effects at the later stages of inflammation.
Mepyramine blockade of histamine receptors
Zymosan-induced vascular permeability was reduced significantly in
animals pretreated with antagonists of H1-histamine
receptors (mepyramine and triprolidine) or with antagonists of
H2-histamine receptors (cimetidine and ranitidine; Fig. 5
).
The H1-histamine receptors are involved mainly in smooth-muscle contraction and vascular permeability [53 ], and H2 receptors mediate gastric-acid secretion [54 ], although both participate in vasodilatation [55 ]. Therefore, mepyramine was chosen for the subsequent experiments because it is the principal selective antagonist (KD=0.8 nM) of H1-type histamine receptors [56 ].
The pharmacological blockade of H1-histamine receptors (mepyramine 2 mg/kg; 5 µM) inhibited almost completely the vascular permeability (Fig. 5) and had a significant impact on further stages of peritonitis, including decreased exudate cell counts (PTL and PMN) and cytokine (MCP-1, IL-1ß, and IL-6) levels (Fig. 6) . This indicates that the effects of histamine (detected here by the blockade of H1 receptors) extend beyond induction of vascular leakage (Table 1) , maybe through activation of H2-type histamine receptors.
We are unaware of other studies on the effects of pharmacological blockade of histamine receptors on murine peritonitis. Nevertheless, our results correspond with those by Woodward and Owen [57 ] on another model of inflammation, namely cutaneous inflammation in guinea pigs, where mepyramine and cimetidine, alone or in combination, had pronounced effects on the initial phase of inflammation [57 ].
Pharmacological treatments
Histamine is known to initiate early vascular changes during
inflammatory reactions by inducing vasodilatation and partial
dissociation of the endothelium [58
]. In our
experiments, the cromolyn-induced inhibition of mast cell histamine
release seems to have been incomplete and short-lasting (Fig. 3b)
, and
the effects of mepyramine (H1-receptor antagonist) were
stronger and longer lasting. It is possible that in the
cromolyn-treated mice, the early vascular changes were induced by
histamine originated from other sources than the mast cells (e.g., the
vascular wall cells [59
]). Moreover, the involvement of
other vasoactive factors such as serotonin, eicosanoids, certain
clotting factors, their derivatives, and bradykinins cannot be excluded
[27
, 60
, 61
].
A reduced number of PMN and a decreased level of MCP-1 at 4 h of zymosan-induced peritonitis were recently demonstrated by Ajuebor et al. [7 ] in Swiss mice depleted of intact peritoneal mast cells by the pretreatment of the animals 3 days earlier with a degranulating agent (compound 48/80). In our experimental system, such effects were induced by mepyramine blocking of H1-type histamine receptors but were absent after cromolyn-induced mast cell stabilization. Taken together, these data support the previous statement that cromolyn effects are short-lasting and insufficient for a total blocking of histamine release. Another explanation could be that histamine from other sources or other factors like serotonin (of platelets origin) are sufficient to promote the subsequent steps of inflammation in cromolyn-treated animals.
In humans, histamine enhances IL-1-induced IL-1ß synthesis at the
level of transcription [62
] as well as IL-1-induced IL-6
gene expression and protein synthesis [63
]. This
corresponds with our results that the level of IL-1 was reduced in mice
with the blocked histamine receptors.
In conclusion, in the normal organism mast cells and histamine are involved mainly in the early stages of zymosan-induced peritonitis.
Peritonitis in mast cell-deficient mice
Zymosan-induced peritonitis was also induced in WBBF1 males,
controls +/+ and their mast cell-deficient W/Wv littermates
with several other genetic defects [23
,
64
65
66
]. Additionally, some W/Wv mice were
reconstituted selectively with mast cells of +/+ bone marrow origin. It
has been shown previously that histamine content in the whole body of
W/Wv mice is about 5–10% that of +/+ mice and in some
organs like spleen, liver, and stomach, can reach up to 8, 15, and
34%, respectively [67
]. In our experiments,
zymosan-treated W/Wv animals showed a strong impairment of
histamine level, plasma exudation, and all other inflammation-related
factors during the course of inflammation (Figs. 7
and 8
, and Table 1 ).
It should be noted that the levels of these factors in the untreated or
vehicle-treated W/Wv and +/+ animals are similar (Figs. 7
and 8)
, and the number of granulocytes and platelets in the blood of
adult W/Wv mice is normal [68
,
69
].
Despite the lack of full differentiation of +/+ bone marrow-derived mast cells into connective tissue mast cells, the course of inflammation was restored in W/Wv + MC mice, because the histamine content and all other parameters were similar in +/+ and W/Wv + MC animals. Complete inhibition of vascular permeability in mast cell-deficient mice was expected because mast cells produce and store a wide range of vasoactive factors [19 20 21 22 ]. The partial inhibition observed here might be attributed to the presence of histamine of another origin or, most likely, to the contribution of vasoactive factors originating from other cell types.
Restoration of all investigated parameters observed in W/Wv + MC animals indicates that presence of peritoneal mast cells is essential for the maximal response at the early as well as later stages of the zymosan-induced peritonitis. The mast cell-mediated impact on the later steps of the inflammation seems to be a consequence of the optimal early stage. Therefore, most probably, mast cell deficiency affects the later steps of the inflammation as a consequence of the impairment of its early phase.
Received August 30, 1999; revised August 9, 2000; accepted September 20, 2000.
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