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*
Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and
Department of Pathology, Boston University Medical Center Hospital, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MIP-2, the rat homologue of the human GRO genes, was first isolated from LPS-stimulated murine macrophages (4) and subsequently identified as a potent chemotactic protein for neutrophils in vitro (4) and in vivo (5, 6, 7, 9, 10). In animal models of lung inflammation (7, 11, 12) and experimental glomerulonephritis (5), early MIP-2 expression is a necessary step in inducing neutrophil infiltration. These studies support a role for MIP-2 as a potent neutrophil chemoattractant during inflammatory processes.
Clostridium difficile, the causative agent of antibiotic-associated diarrhea and colitis in animals and man, produces two exotoxins: toxin A and toxin B (13). Both toxins possess cytotoxic activity and cause cell rounding in several cell types in vitro (14, 15). The cytotoxic effects of toxins A and B are caused by monoglucosylation of small GTP-binding rho proteins leading to depolymerization of actin microfilaments and cell rounding (16). In vivo studies using rodent intestinal loops showed that toxin A, but not toxin B, elicits fluid secretion, mucosal damage, and a prominent neutrophil infiltration in the intestinal mucosa (13, 14). Similarly, the human disease is characterized by a dense colonic inflammatory infiltrate with microabscesses and surface plaques rich in neutrophils (13). A necessary step in the pathogenesis of toxin A enteritis is binding of the toxin to its glycoprotein receptor expressed on the enterocyte surface (17, 18). Following binding, toxin A elicits an acute inflammatory response that involves early activation of substance P- and CGRP-containing sensory neurons (19, 20, 21) and release of inflammatory mediators from immune and inflammatory cells, including mast cells (22), macrophages (23), and neutrophils (24). The importance of neutrophils in the in vivo mechanism of toxin A is underscored by the substantial inhibition of toxin A-induced fluid secretion and epithelial cell damage in rabbit ileal loops when neutrophil infiltration is inhibited by i.v. injection of a mAb directed against the leukocyte adhesion molecule CD18 (24).
It is well established that intestinal epithelial cells function as an integral component of the mucosal immune system (25). Thus, human epithelial cell lines in vitro process and present Ags to T cells (26) and can be stimulated to express HLA class II and intercellular adhesion molecules (27, 28). In addition, colonic epithelial cells respond to invasive bacteria, LPS, and inflammatory agonist by expressing a specific array of proinflammatory cytokines (29, 30). Based on these observations, we hypothesized that early release of inflammatory mediators from intestinal epithelial cells is involved in initiating and regulating the mucosal inflammatory response to C. difficile toxin A in vivo. In this study, we investigated the role of the inflammatory chemokine MIP-2 in C. difficile toxin A-induced inflammation and fluid secretion and examined whether intestinal epithelial cells are sources of MIP-2 during intestinal inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[3H]mannitol (30 Ci/mmol) was obtained from DuPont NEN (Boston, MA). Sodium pentobarbital was purchased from Abbott (Chicago, IL). Protein concentrations were determined by the bicinchoninic acid (BCA) protein assay reagent (Pierce Laboratories, Rockford, IL). Toxin A was purified from broth culture supernatants of C. difficile strain VPI 10,463 (American Type Culture Collection, Rockville, MD), as previously described (18, 19, 20, 21, 22, 23). Rabbit polyclonal anti-rat MIP-2 serum was obtained from Biosource International (Camarillo, CA), and actinomycin D from Sigma (St. Louis, MO). RMPI complete medium was prepared by supplementing RMPI 1640 medium (Cellgro, Herndon, VA) with 10% heat-inactivated FCS (HyClone, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 10 mM glutamine (Sigma).
Effects of toxin A on MIP-2 and myeloperoxidase (MPO) levels in the intestinal mucosa
Fasted (16 h) male Wistar rats (Charles River Breeding
Laboratories, Wilmington, MA), 
250 g body weight, were anesthetized
by an i.p. injection of sodium pentobarbital (40 mg/kg). Two 5-cm ileal
loops were prepared and injected with either 5 µg of toxin A in 0.4
ml 50 mM Tris buffer (pH 7.4) or buffer alone. The abdomen was closed
with sutures, and rats were left to recover while their body
temperature was maintained at 37°C by a heating pad. Rats were
sacrificed at different time points by an i.p. bolus of sodium
pentobarbital (120 mg/kg). The loops were then removed and washed in
ice-cold PBS, and the mucosa was scraped from the underlying muscularis
with glass slides and placed in 2 ml of ice-cold Tris-EDTA (TE, 10 mM
Tris-HCl, and 1 mM EDTA, pH 7.4) buffer containing 0.05% sodium azide,
1% Tween-80, 2 mM PMSF, and 1 µg/ml of each of the protease
inhibitors aprotinin, leupeptin, and pepstatin A (Sigma) (31). The
mucosa was then homogenized (20 s) and centrifuged (11,000 x
g, 10 min at 4°C), and the supernatants were collected and
filtered (4.5-µm filter; Gelman Sciences, Ann Arbor, MI). MIP-2
levels were assayed by a commercially available immunoassay kit
(Biosource International). Mucosal MIP-2 content was expressed as pg/mg
of protein. To determine MPO levels, mucosal scrapings, obtained from
the same experiments described above, were placed in 1 ml of
hexadecyltrimethylammonium bromide (0.5%) in 50 mM
KH2PO4 (pH 6). Samples were then frozen
(-70°C) and thawed three times, sonicated for 10 s, and
centrifuged (14,000 x g) for 15 min. The
supernatants were assayed for MPO activity by colorimetric assay
modified from the method of Bradley et al. (32), and results were
expressed as MPO U/mg of protein.
RNA extraction and PCR (RT-PCR) amplification
Total RNA was extracted from mucosal scrapings of ileal loops
injected with either toxin A or buffer, as described above, using the
acid guanidinium thyocyanate-phenol-chloroform extraction method (33).
RNA integrity was confirmed by 1% agarose formaldehyde gel
electrophoresis, and cDNA was prepared from 1 µg of total RNA in 20
µl reverse-transcriptase buffer (23). The RT-PCR reaction was
performed at a final concentration of 1x PCR buffer, 1 µM dNTPs, 1.2
pM of each of the 5' and 3' primers, 1.5 U Amplitaq DNA polymerase, and
0.25 µl [
-32P]dCTP (3000 Ci/mmol; DuPont NEN)
in a total volume of a 50 µl using a Perkin-Elmer (Norwalk, CT)
thermal cycler (23). The PCR primer sequences and amplification
conditions for MIP-2 were as previously described (6). Rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were obtained
from Clontech (Palo Alto, CA) and used as previously described (23).
PCR reactions were overlaid with mineral oil, and, to ensure detection
of low-abundance mRNA, 35 cycles of amplification were performed.
Amplification reactions (10 µl) were fractionated on a 5%
polyacrylamide gel (19:1 acrylamide/bisacrylamide), and the gels were
then dried and exposed to Kodak X-OMAT AR film (Eastman Kodak Company,
Rochester, NY) to visualize amplification products. For
semiquantitative expression of MIP-2 mRNA, the films were replaced on
the gel, the area corresponding to the visualized bands was cut, and
radioactivity content was determined in a ß-scintillation counter by
Cerenkov counting. Results were expressed as the ratio of MIP-2 to
GAPDH-associated dpm for each sample. To exclude contamination by
genomic DNA, controls were conducted in the same conditions in samples
in which RNA-reverse transcriptase was omitted during reverse
transcription. Any sample showing PCR products in this experimental
condition was discarded.
Effects of anti-MIP-2 Ab on toxin A-mediated enterotoxicity in rat ileal loops
Fasted, anesthetized rats were injected i.v. with either saline, a rabbit polyclonal Ab directed specifically against MIP-2, or control rabbit IgG (Jackson ImmunoResearch, West Grove, PA) 30 min before toxin administration. The renal pedicles were then ligated to prevent excretion of [3H]mannitol, and 10 µCi of [3H]mannitol was injected i.v. for measurements of mucosal permeability. Two 5-cm ileal loops were then prepared and injected with either toxin A or buffer alone, and 4 h later animals were sacrificed by an i.p. bolus of pentobarbital (120 mg/kg). The loops were then removed, and the weight and length were measured. Aliquots of the ileal contents were assayed for radioactivity and MPO activity. Secretion of fluid was expressed as loop weight to length ratio (mg/cm), and intestinal permeability was quantitated as blood-to-lumen clearance of [3H]mannitol (dpm per centimeter loop), as previously described (18, 19, 20, 21, 22, 23). Neutrophil MPO activity in fluid exudate was determined as described above, and results were expressed as MPO U/cm of loop. At the end of each experiment, ileal tissue samples were fixed in Formalin and paraffin embedded, and sections were stained with hematoxylin and eosin (H&E) for light microscopy. Histologic damage was graded for three parameters: 1) epithelial cell necrosis; 2) congestion and edema of the mucosa; and 3) neutrophil margination in blood vessels and neutrophil tissue infiltration, as previously described by us (18, 19, 20, 21, 22, 23). A score of 0 to 3, denoting increasingly severe abnormalities, was assigned to each of these parameters by a single histopathologist "blinded" (S.T.N.) as to the experimental conditions. This study was approved by Beth Israel and Deaconess Medical Center Institutional Animal Care and Use Committee (Boston, MA).
Immunohistochemical detection of MIP-2
Immunohistochemistry was performed to determine the cellular localization of MIP-2 expression. Ileal loops were prepared and injected with either toxin A or buffer, as described above. After 1 h, rats were sacrificed and full thickness ileal loop samples were fixed in 4% paraformaldehyde-PBS at 4°C for 30 min. Tissues were washed in 1x PBS (30 min x 3 at 4°C), cryo-protected overnight in 30% sucrose, and embedded in OCT compound (Miles, Elkhart, IN). Sections (5 µm) were mounted on superfrost plus slides (Fisher Scientific, Pittsburgh, PA), fixed in 4% paraformaldehyde (3 min), washed in 1x TBS (0.05 M Tris-HCl containing 0.15 M NaCl, pH 7.5), and incubated for 1 h at room temperature in TBS containing 1% normal donkey serum, 3% BSA, and 50 mM NH4Cl2 to reduce nonspecific binding. MIP-2 protein was detected by incubating ileal sections (22°C for 1 h) with 5 µg/ml of the rabbit polyclonal Ab against MIP-2. In some experiments, the MIP-2 Ab was preincubated with an excess of rat rMIP-2 (Biosource International) (1 h, 22°C) before application to ileal sections. Sections were then washed (10 min x 3, at 22°C) with 1x TBS, and incubated (22°C for 30 min) with FITC-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) at a dilution of 1/100 in TBS. Subsequently, slides were washed (10 min x 3, at 22°C) in 1x TBS containing 0.1% Triton X-100 (Sigma), and then mounted in 90% glycerol and n-propyl-gallate (1 mg/ml) to reduce photobleaching. MIP-2 expression was visualized using a confocal microscope (Bio-Rad MRC-1024; Bio-Rad Microsciences, Cambridge, MA), and confocal images were digitally stored using photon-counting Bio-Rad COMOS software. For each experimental condition, four tissues, each derived from a different animal, were investigated.
Effect of toxin A on MIP-2 release from purified rat intestinal epithelial cells
Intestinal epithelial cells were purified from normal terminal
ileum, as previously described (29). Briefly, ileum was dissected, cut
in small pieces (2 x 2 mm), washed in HBSS, and incubated (5
min at 22°C) in HBSS containing 1 mM DTT (Sigma) to remove adherent
mucus. Ileal explants were then incubated (1 h at 37°C) in RMPI
complete medium containing dispase (3 mg/ml) and DNase (0.1 mg/ml)
(Boheringer Mannheim, Indianapolis, IN) with gentle shaking every 5
min. Supernatants were collected and centrifuged (600 x
g, 5 min), and epithelial cells were purified by
centrifugation through a discontinuous Percoll density gradient
(Pharmacia Fine Chemicals, Uppsala, Sweden). Percoll stock was diluted
to 60, 40, 30, and 0% final concentrations using RMPI complete medium.
The gradient was prepared by gentle sequential layering of 3 ml 60%
Percoll, 6 ml 40% Percoll, 3 ml 30% Percoll, and finally 2 ml RMPI
media (0% Percoll). This mixture was centrifuged (0.5 h at 600 x
g), and epithelial cells migrating at the interface
between 30% and 0% Percoll were collected, washed twice with PBS, and
resuspended in RMPI complete medium. Aliquots of cell suspensions were
used to determine cell number by hemocytometer counting. At the end of
each experiment, cell viability was >95%, as determined by trypan
blue exclusion. These cell suspensions contained 
95% epithelial
cells, as assessed by staining with a mouse mAb to episialin, an
epithelial cell-specific Ag (Novocastra, Newcastle, U.K.) (34), and
minimal staining with a mouse mAb recognizing the rat leukocyte common
Ag (CD-45) (Biosource International) and a 97-kDa Ag expressed by rat
macrophages (Biosource International) (23). For each experiment,
106 cells were placed into 12-well culture plates precoated
with rat tail collagen (Sigma) and cultured in 1 ml of RMPI medium at
37°C. At the indicated time, the culture medium was collected and
MIP-2 was released in the culture media measured as described above.
Results were expressed as pg of MIP-2 released per 106
cells.
Effect of actinomycin D pretreatment on toxin A-induced release of MIP-2 from purified intestinal epithelial cells
Rats were injected i.p. with actinomycin D (0.8 mg/kg) in 1 ml of normal saline, 30 min before exposing ileal loops to toxin A or buffer, as described above. After 30 min, rats were killed, intestinal epithelial cells were purified and placed in culture, and MIP-2 release was assessed after 4 h, as described above.
Effect of ablation of sensory afferent neurons by capsaicin on toxin A-induced MIP-2 release from intestinal epithelial cells
Anesthetized rats were injected s.c. with capsaicin for 3 consecutive days (20, 30, and 50 mg/kg, respectively), while control animals received only vehicle (19). Ileal loop experiments were performed 2 wk after capsaicin treatment using rats that showed functional ablation of sensory neurons, as previously described (19). Following injection of toxin A or buffer into ileal loops, epithelial cells were purified and used to determine either MIP-2 mRNA levels or MIP-2 protein release during in vitro culture, as described above.
Statistical analyses
Results are presented as means ± SEM. Statistical analyses were performed using the SIGMA-STAT professional statistics software program (Jandel Scientific Software, San Rafael, CA). Analyses of variance with protected t tests (ANOVA) were used for intergroup comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported that neutrophil infiltration of ileal
mucosa was evident 1 h after toxin A injection into rat ileal
loops, before intestinal secretory and permeability changes occurred
(23). Therefore, we determined the time course of MIP-2 mRNA in the
ileal mucosa following intraluminal toxin A administration. MIP-2 mRNA
signal was barely detectable in RNA obtained from ileal mucosa of
buffer-injected loops (Fig. 1
A). In toxin-exposed
rat ileal loops, MIP-2 mRNA levels were increased at 30 min and further
increased after 2 h (Fig. 1A). Semiquantitative
analysis of the RT-PCR data showed a 1.7-fold increase in MIP-2 mRNA 30
min after toxin A injection, which was not statistically significant as
compared with controls (n = 5 per group).
However, 1 h and 2 h after toxin A exposure, MIP-2 mRNA
levels were increased by 5.1- and 4.8-fold, respectively, compared with
levels in buffer-injected loops (p < 0.01 for
both, n = 5 per group). Mucosal levels of MIP-2 protein
were also barely detectable in buffer-injected loops and did not change
30 min after toxin A injection (Fig. 1B). However,
MIP-2 levels showed a 5.9-fold increase 1 h after toxin injection,
as compared with buffer-treated loops (p <
0.01, Fig. 1B), and remained increased after 2 h
(p < 0.01, Fig. 1A),
although at a lower level when compared with 1-h exposure. Since
previous studies indicated that neutrophil infiltration is a prominent
feature during toxin A enteritis (24), we next examined the time-course
increases in mucosal MPO activity following toxin administration. As
shown in Figure 1C, 30 min after toxin A exposure, there was
no significant increase in mucosal MPO levels as compared with
controls. However, MPO levels were increased 1 and 2 h after toxin
administration (Fig. 1C). Taken together, results in
Figure 1 indicate that, during toxin A enteritis, increases in MIP-2
mRNA and protein coincide with neutrophil infiltration.
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As expected, injection of toxin A into rat ileal loops caused a
3.5-fold increase in fluid secretion (Fig. 2A), a 28.3-fold
increase in [3H]mannitol permeability (Fig. 2B), and a 16.4-fold increase in MPO activity (Fig. 2C) in ileal exudates as compared with
buffer-injected loops. Administration of anti-MIP-2 Ab, but not
control Ab, 30 min before toxin A injection inhibited toxin-induced
secretion by 58% (Fig. 2A, p < 0.01),
mucosal mannitol permeability by 80% (Fig. 2B,
p < 0.01), and MPO activity by 85% (Fig. 2C, p < 0.01).
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). Toxin A-induced histologic damage was
reduced by pretreatment with anti-MIP-2 Ab (Table I). Histologic
examination indicated that MIP-2 Ab reduced mucosal neutrophil
infiltration and attenuated mucosal edema as well as epithelial cell
damage (Table I).
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Immunostaining was used to determine the distribution of cells
within the ileal mucosa producing MIP-2 during toxin A enteritis.
Sections from buffer-injected loops showed only a light staining in
cytoplasm of scattered cells throughout the villi (Fig. 3A). However, MIP-2
immunoreactivity was increased dramatically 1 h after intraluminal
toxin A (Fig. 3, B and C). MIP-2-positive
cells occurred throughout the epithelial layer of the villi,
predominantly in the top of the villi (Fig. 3B). At
higher magnification, MIP-2-immunoreactive staining was also present in
cells of the lamina propria (Fig. 3C). Incubation of
anti-MIP-2 Ab in vitro with an excess of MIP-2 resulted in absence
of specific staining (Fig. 3D) when applied to
toxin-exposed tissues.
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To determine whether de novo RNA synthesis is required for toxin A-induced MIP-2 release from intestinal epithelial cells, we administered rats with 0.8 mg/kg of the transcriptional inhibitor actinomycin D 30 min before exposing ileal loops to toxin A. Administration of actinomycin D had no significant effect on MIP-2 release from intestinal epithelial cells of buffer-injected loops (90 ± 8 vs 75 ± 10 pg/106 cells, n = 5 per group). However, actinomycin D almost completely normalized MIP-2 release from enterocytes obtained from toxin A-injected loops (305 ± 14 vs 110 ± 5, p < 0.01, n = 5 per group), indicating that RNA synthesis is a necessary event in toxin A-induced MIP-2 release.
Toxin A directly induces MIP-2 release from intestinal epithelial cells
To address the possibility that toxin A directly induces MIP-2
synthesis and release from intestinal epithelial cells, we performed
two sets of experiments. First, we determined the effect of ablation of
sensory nerves by chronic capsaicin treatment on toxin A-induced MIP-2
synthesis and release, since toxin A-induced intestinal inflammation is
mediated through activation of sensory afferent nerves containing
substance P (19, 20). Injection of toxin A into ileal loops of
vehicle-treated animals increased cellular MIP-2 mRNA levels (Fig. 4A) and increased MIP-2
protein release from intestinal epithelial cells as compared with
control (Fig. 4B), consistent with results shown in
Figure 3
. In addition, ablation of sensory afferent nerves by capsaicin
had no significant effect on toxin A-induced increase in MIP-2 mRNA
accumulation and protein release (Fig. 4) in epithelial cells. These
results suggest that toxin A-induced MIP-2 release from intestinal
epithelial cells is not mediated through activation of primary sensory
neurons. To determine whether toxin A directly induces MIP-2 release
from intestinal epithelial cells, we incubated purified enterocytes in
vitro for 4 h with toxin A (5 µg/ml). As expected, toxin
A-treated cells released significantly higher amounts of MIP-2 as
compared with controls (88 ± 12 vs 255 ± 24,
p < 0.01, n = 5 per group).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several cell types of the intestinal mucosa can express MIP-2,
including fibroblasts (6), endothelial cells (6), and macrophages (4, 6). We have reported recently that lamina propria macrophages are
activated during the early stages of toxin A enteritis and release
MIP-2 (35). In this work, we report that enterocytes are also a source
of MIP-2 and release this cytokine shortly after exposure to toxin A in
vivo. Our results are in agreement with previous studies showing MIP-2
expression in a rat intestinal epithelial cell line (IEC-18) after
stimulation with TNF- (6). In the past few years, it has become
apparent that gastric and colonic epithelial cells can respond to
bacterial invasion, or to toxin exposure with a coordinate expression
and secretion of a specific array of proinflammatory cytokines. For
example, human gastric epithelial cells exposed to pathogenic
Helicobacter pylori release the potent neutrophil
chemoattractant IL-8 (36). Furthermore, human colonic epithelial cells
infected with invasive strains of bacteria express several
proinflammatory cytokines (29, 37) in a specific temporal sequence to
generate a chemokine gradient during the different phases of mucosal
inflammation (38). Recent results also indicate that human colonic
adenocarcinoma cell lines and freshly isolated human colonocytes
release the proinflammatory cytokine IL-8, the human homologue of
MIP-2, in response to toxin A in vitro (39, 40). Interestingly, toxin
A-induced IL-8 release from human colonocytes occurred 3 h
following toxin exposure, suggesting that IL-8 release in response to
toxin A is a relatively early event. However, the physiologic relevance
of increased IL-8 expression by epithelial cells in the inflammatory
response to toxin A was not examined in these studies (39, 40).
Administration of an Ab to MIP-2 in rats significantly reduced the
secretory and inflammatory effects of C. difficile toxin A.
We have shown previously that toxin A-induced inflammation and diarrhea
in rat ileal loops require activation of sensory afferent neurons
containing the peptides substance P and CGRP (20, 21), and release of
inflammatory mediators from mast cells (22), and macrophages (23).
Prevention of neuropeptide release from sensory afferent neurons by
capsaicin dramatically inhibits toxin-induced intestinal secretion and
inflammation (19, 41). In this study, we show that MIP-2 expression and
release from epithelial cells in response to toxin A are not affected
by capsaicin (Fig. 4). These findings indicate that MIP-2 production is
upstream to activation of sensory afferent neurons and may be a direct
effect of toxin A on the epithelial cell. In support of this
interpretation, we demonstrate in this study that exposure of purified
intestinal epithelial cells to toxin A resulted in increased secretion
of MIP-2. Since neutralizing MIP-2 or preventing neuropeptide release
from sensory afferent neurons block both inflammation and diarrhea,
MIP-2, directly or indirectly, might play a role in activation of the
sensory neurons. Previous reports demonstrate that cytokines can
interact with sensory nerves to regulate release of proinflammatory
neuropeptides from nerve terminals. For example, IL-1ß enhances
capsaicin-induced vasodilatation in rat skin (42), and intraarticular
injection of IL-1 increased substance P efflux into synovial fluid
(43). In addition, desensitization of sensory nerves by capsaicin
administration resulted in a specific inhibition of IL-1ß-induced
neutrophil migration into skin air pouches, suggesting that endogenous
substance P released from sensory nerves is necessary in IL-1ß-driven
neutrophil recruitment (44). Alternatively, MIP-2-mediated neutrophil
recruitment during toxin A enteritis may require functional sensory
neurons that can be activated by a process that does not involve
MIP-2.
The mechanism by which C. difficile toxin A stimulates synthesis and release of MIP-2 from intestinal epithelial cells has not been examined. We reported that exposure of purified rat intestinal epithelial cells to the tyrosine kinase inhibitor tyrphostin B56 inhibited toxin A-induced MIP-2 release (45). Furthermore, recent results indicate that toxin A stimulates inositol triphosphate production in human duodenal biopsies (46), indicating a possible role of protein kinase C in the signaling cascade activated by toxin A binding. Although other studies (47) also suggested that MIP-2 expression is dependent on activation of tyrosine kinase(s), the signal-transduction pathway(s) by which this potent neutrophil chemoattractant is induced in response to toxin A remains to be elucidated.
In summary, the present studies demonstrate that the neutrophil chemoattractant MIP-2 is released from rat intestinal epithelial cells during the early stages of toxin A enteritis by a mechanism that involves a direct effect of toxin A on enterocytes. We also show that MIP-2 participates in the inflammatory cascade triggered by toxin A, since an Ab to this chemokine inhibits fluid secretion and neutrophil transmigration in response to toxin A in rat ileum. These results may be relevant to the pathophysiology of C. difficile colitis in humans, in which colonic mucosal neutrophil infiltration is a prominent feature of patients with this disease.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Charalabos Pothoulakis, Beth Israel Hospital, Harvard Medical School, Division of Gastroenterology, Dana 601, 330 Brookline Avenue, Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MPO, myeloperoxidase; TBS, Tris-buffered saline.
Received for publication November 25, 1997. Accepted for publication February 12, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in gastric epithelial cells. Gastroenterology 113:1099.[Medline]
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T Goode, J O'Connell, P Anton, H Wong, J Reeve, G C O'Sullivan, J K Collins, and F Shanahan Neurokinin-1 receptor expression in inflammatory bowel disease: molecular quantitation and localisation Gut, September 1, 2000; 47(3): 387 - 396. [Abstract] [Full Text] [PDF]
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H. Stubbe, J. Berdoz, J.-P. Kraehenbuhl, and B. Corthesy Polymeric IgA Is Superior to Monomeric IgA and IgG Carrying the Same Variable Domain in Preventing Clostridium difficile Toxin A Damaging of T84 Monolayers J. Immunol., February 15, 2000; 164(4): 1952 - 1960. [Abstract] [Full Text] [PDF]
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K. K. Jefferson, M. F. Smith Jr., and D. A. Bobak Roles of Intracellular Calcium and NF-{kappa}B in the Clostridium difficile Toxin A-Induced Up-Regulation and Secretion of IL-8 from Human Monocytes J. Immunol., November 15, 1999; 163(10): 5183 - 5191. [Abstract] [Full Text] [PDF]
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