IL-8 release and neutrophil activation by
Clostridium difficile toxin-exposed
human monocytes
Joanne K.
Linevsky1,2,
Charalabos
Pothoulakis3,
Sarah
Keates3,
Michel
Warny3,
Andrew C.
Keates3,
J. Thomas
Lamont3, and
Ciarán P.
Kelly3
1 Section of Gastroenterology,
Department of Veterans Affairs Medical Center, Boston
02130; 2 Evans Memorial
Department of Clinical Research, Boston University School of
Medicine, Boston 02118; and
3 Gastroenterology Division, Beth
Israel Deaconess Medical Center, Harvard Medical School, Boston,
Massachusetts 02215
 |
ABSTRACT |
Neutrophil infiltration is central to the
pathogenesis of Clostridium difficile
toxin A-induced enterocolitis. This study examines whether monocyte
activation by C. difficile toxins is instrumental in initiating neutrophil activation and recruitment. Human
monocytes were exposed to low concentrations of highly purified C. difficile toxins, and the
conditioned media were harvested for cytokine and functional assays.
Monocytes exposed to C. difficile toxin A (10
10 M) or toxin B
(10
12 M) released 100 and
20 times basal levels, respectively, of the neutrophil chemoattractant
interleukin-8 (IL-8). Reverse transcriptase-polymerase chain reaction
demonstrated a marked increase in IL-8 mRNA expression by monocytes 3 h
after toxin exposure. Conditioned media from toxin A- and toxin
B-treated monocytes stimulated neutrophil migration (324 and 245% of
control, respectively). This effect was completely blocked by IL-8
antiserum. These media also upregulated neutrophil CD11b/CD18 and
endothelial cell intercellular adhesion molecule-1 expression.
C. difficile toxins, at low
concentrations, potently activate monocytes to release factors,
including IL-8, that facilitate neutrophil extravasation and tissue
infiltration. Our findings indicate a major role for toxin-mediated
monocyte and macrophage activation in C. difficile colitis.
pseudomembranous colitis; intestinal inflammation; cytokines; adhesion molecules; interleukin-8
 |
INTRODUCTION |
OUR CURRENT UNDERSTANDING of the pathogenesis of
Clostridium difficile colitis is that
antibiotic therapy disturbs the normal colonic microflora to allow
colonization by toxigenic C. difficile (17). The organism releases two toxins, A and B. Toxin A, a 308-kDa
protein, is a potent inflammatory enterotoxin that, when injected into
rabbit ileal loops, elicits fluid secretion, increased mucosal
permeability, and a marked destructive inflammatory response (14, 17,
22, 24, 34). Toxin B, a 269-kDa protein, is a potent cytotoxin in vitro
but does not produce intestinal inflammation or alter intestinal
permeability in the rabbit ileal loop model (23, 33). However, more
recent studies indicate that toxin B may be even more harmful to human
colonic tissue than toxin A (27). The intracellular mechanism of action
of both toxin A and toxin B has recently been described. Both toxins
prevent ADP-ribosylation of the low-molecular-weight
guanosinetriphosphatase Rho, resulting in actin depolymerization and
cell death (8, 9, 13).
Neutrophil recruitment appears to be an essential step in the
pathogenesis of C. difficile
toxin-induced intestinal injury. Biopsy specimens from patients with
C. difficile colitis show striking
vascular congestion, neutrophil infiltration of the lamina propria, and
inflammatory pseudomembrane formation (22, 25). Systemic
polymorphonuclear activation is evidenced by the common finding of an
elevated peripheral blood neutrophil count with toxic granulations and
band forms, as well as the occasional finding of a "leukemoid"
reaction (20). Furthermore, we have previously shown that inhibition of
neutrophil recruitment using a blocking antibody to the CD18 leukocyte
adhesion molecule results in a marked reduction in fluid secretion,
epithelial injury, and mucosal inflammation in toxin A-exposed rabbit
intestinal loops (14). The mechanism by which neutrophils are activated
in C. difficile colitis has yet to be
elucidated. Toxin A directly stimulates human neutrophils as evidenced
by a rise in neutrophil cytosolic Ca2+ levels and stimulation of
neutrophil chemotaxis (14, 24). However, the concentrations of toxin A
needed to achieve these effects are relatively high
(10
7 M). In addition, toxin
B has no direct stimulatory effect on neutrophils (24). We therefore
sought an alternative mechanism by which C. difficile toxins, at low concentration, can effect neutrophil activation and tissue infiltration. The monocyte/macrophage was a clear candidate for an important role in this process.
In the normal colon, intestinal macrophages lie in close proximity to
the surface epithelial cells. These macrophages, which are members of
the mononuclear phagocytic system, are derived from monocytes in the
bone marrow. After entering the systemic circulation, monocytes gain
access to the intestinal tissue where they mature into tissue
macrophages. Both monocytes and macrophages have similar functions
including antigen presentation and phagocytosis (2). In addition, both
are key sources for an array of proinflammatory cytokines. Our
laboratory previously reported that C. difficile toxin A at a concentration of
10
9 M activates mouse
peritoneal macrophages to secrete interleukin (IL)-1 (21). In another
study, both toxins A and B stimulated human monocytes to release
IL-1
, tumor necrosis factor-
(TNF-
), and IL-6 (6). Monocytes
and macrophages are also a major source of IL-8, a low-molecular-mass
(10 kDa) protein that is a member of the chemokine family of
chemotactic cytokines (1). In addition to its chemoattractant
properties, IL-8 regulates neutrophil adhesion molecule expression and
directs neutrophil adhesion to the vascular endothelium, neutrophil
diapedesis, and tissue infiltration (12, 30).
In this study we demonstrate that human monocytes are activated by
C. difficile toxins A and B to release
IL-8 in addition to the proinflammatory cytokines IL-1
and TNF-
.
Much lower concentrations of toxin A
(10
10 M) are needed to
elicit neutrophil migration via monocyte-derived IL-8 as compared with
those needed for direct toxin-mediated neutrophil stimulation
(10
7 M). Toxin B, which has
no apparent direct effect on the neutrophil (24), stimulates monocyte
IL-8 release at even lower concentrations (10
12 M). Both toxin A and
toxin B also activate IL-8 production by macrophages. Cytokines
released by C. difficile toxin-exposed monocytes upregulate the expression of neutrophil and endothelial cell
adhesion receptors, thereby facilitating neutrophil extravasation. These findings support our hypothesis that macrophage activation is an
important step in the pathogenesis of C. difficile toxin-induced colitis.
 |
METHODS |
Toxin preparation.
Toxins A and B were purified, as previously described, from broth
culture supernatants of C. difficile
strain 10463 (14, 23, 31, 33). The enterotoxicity of purified toxin A
was confirmed in the ligated rabbit ileal loop assay (18, 14, 33), and
the cytotoxicity of both toxins was determined by cell rounding of
IMR-90 fibroblasts (18, 23). With the use of the E-toxate (Sigma) assay
system for lipopolysaccharide (LPS), the toxin preparations were tested
for and found to be free of LPS contamination.
Peripheral blood monocyte isolation.
Whole blood was obtained from healthy volunteers and peripheral blood
mononuclear cells isolated using lymphocyte separation media (Organon
Teknika, Durham, NC) as previously described (3, 16). The peripheral
blood mononuclear cells were washed three times with Hanks' balanced
salt solution (Cellgro, Herndon, VA) and plated on 24-well plastic
culture dishes at a density of 3 × 105 cells/well in 0.6 ml of R10
[RPM1 1640 medium (Cellgro) supplemented with heat-inactivated
10% fetal calf serum, 2 mM glutamine, 5 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 100 U/ml penicillin, and 100 µg/ml streptomycin, all
supplied by Sigma, St. Louis, MO]. After 90 min, nonadherent
lymphocytes were removed by washing. Monocyte purity, determined by
nonspecific esterase positivity, was >90%. After resting overnight,
the adherent monocytes were treated with varying doses of either toxin
A or toxin B. Bacterial endotoxin (10 ng/ml,
Escherichia coli 055:B5 LPS, Sigma)
was used as a positive control. Unless otherwise stated, monocytes were
treated for 3 h and washed, fresh medium was added, and the conditioned
media were harvested after an additional 21 h.
IL-8 measurement.
IL-8 protein levels in monocyte conditioned media were measured using a
double-ligand enzyme-linked immunosorbent assay (ELISA) (10, 15, 29).
Briefly, the wells of a 96-well Immulon II plate (Dyntatech, Chantilly,
VA) were coated with goat anti-human IL-8 (R & D Systems, Minneapolis,
MN) at a concentration of 5 µg/ml in carbonate coating buffer (pH
9.6) overnight and washed with phosphate-buffered saline (PBS)-0.05%
Tween 20, pH 7.5. Nonspecific binding was blocked with 2% bovine serum
albumin (BSA) in PBS-Tween 20 for 1 h at room temperature. Plates were
washed twice, and 100 µl of the recombinant human (rh)
IL-8 standard (R & D Systems) or samples were added to the wells and
incubated for 1 h at room temperature. Goat anti-human IL-8 antiserum
(R & D Systems), biotinylated using ImmunoPure (NHS-LC-Biotin; Pierce)
according to the manufacturer's directions, was used as the secondary
antibody. The plates were washed again, the biotinylated goat anti-IL-8
was added, and the plates were incubated for 1 h at room temperature.
The plates were washed, and biotinylated peroxidase streptavidin
complex (Amersham, Arlington Heights, IL) was added and incubated for 30 min at room temperature. After careful washing with PBS, 100 µl
tetramethylbenzidine substrate solution (Kirkegaard and Perry Labs,
Gaithersburg, MD) were added to each well, and the reaction was stopped
after 5 min with 100 µl of 1 M
o-phosphoric acid. The optical density
(OD) at 450 nm was then read using an automated microplate photometer
(Dynatech), and concentrations of IL-8 were determined by comparison
with the standard curve. IL-1
and TNF-
levels were also measured
in the monocyte media using commercially available ELISAs (R & D
Systems).
Monocyte total RNA extraction.
Peripheral blood mononuclear cells were isolated as above and plated on
150-cm2 coated polysterene culture
dishes at a density of 1 × 106 cells/ml in R10 media. After a
90-min incubation, the nonadherent cells were removed, and the adherent
monocytes were treated with toxin A
(10
9 M) for 3 or 6 h.
Untreated monocytes were used as control. The plates were then washed
with Hanks' balanced salt solution, and monocyte RNA was extracted
using the single-step method of Chomczynski and Sacchi (4; see also
Refs. 10, 11). Cells were homogenized with Tri Reagent LS (phenol and
guanidine thiocyanate in a mono-phase solution, Molecular Research
Center, Cincinnati, OH). Phase separation was performed using
chloroform and centrifugation at 10,000 g for 25 min. The aqueous phase was
removed, and RNA was precipitated with isopropanol.
Reverse transcription-polymerase chain reaction.
Total monocyte RNA (3 µg) was diluted to 10.5 µl with sterile
water, heated at 65°C for 10 min, and placed on ice for 5 min. After centrifugation at 12,000 g for 5 s, 4 µl of 5× avian myeloblastosis virus (AMV) reverse
transcriptase buffer (Promega, Madison, WI), 2 µl of a 10 mM mixture
of deoxyribonucleoside triphosphates (Pharmacia Biotech, Piscataway,
NJ), 20 U RNasin ribonuclease inhibitor (Promega), 100 pmol oligo(dT)
primer (Promega), and 20 U AMV reverse transcriptase (Promega) were
added. The mixture was incubated at 37°C for 90 min, and the
resulting complementary DNA (cDNA) was diluted to 200 µl with buffer
containing 10 mM tris(hydroxymethyl)aminomethane hydrochloride and 1 mM
EDTA (pH 6.5). Preparations of cDNA were stored until use at
20°C.
For subsequent amplification by polymerase chain reaction (PCR), 5 µl
monocyte cDNA were added to 45 µl of PCR mixture containing 5 µl
10× Taq polymerase buffer (Promega), 200 µM deoxyribonucleoside triphosphates, 1.5 mM MgCl2, 5 pmol human
-actin specific primers (Clontech, Palo Alto, CA) or 50 pmol human IL-8 specific primers (Stratagene, La Jolla, CA), and 1.25 U
Taq DNA polymerase (Promega) (11). Samples were amplified by initial
denaturation at 95°C for 3 min, then subjected to 35 cycles of
denaturation at 95°C for 1 min, annealing at 60°C for 1 min and
extension at 72°C for 1.5 min, followed by a final extension at
72°C for 5 min. PCR products (10 µl) were analyzed by
electrophoresis through 1% agarose gels containing 1 µg/ml ethidium
bromide, and DNA bands were visualized using an ultraviolet
transilluminator (Fisher Biotech, Pittsburgh, PA) at 312 nm.
Human neutrophil isolation.
Human neutrophils were isolated from heparinized blood as previously
described (24) using Ficoll-diatizoate density gradient centrifugation
(LSM, Organon Teknika), followed by dextran sedimentation (Pharmacia)
and hypotonic lysis of contaminating red blood cells (3). Neutrophil
suspensions prepared in this manner contained >98% granulocytes and
were >97% viable as determined by trypan blue exclusion.
Neutrophil migration assay.
The ability of conditioned medium from both toxin A- and toxin
B-exposed monocytes to induce neutrophil migration was measured using a
conventional migration assay (15, 24). Conditioned media from toxin A-
and toxin B-stimulated human monocytes were placed in the lower
chambers of a multiwell chemotaxis assembly (Neuro Probe, Cabin John,
MD) (2, 15, 24). Human neutrophils suspended in Dulbecco's PBS
(Cellgro) with 0.2% BSA at a concentration of 5 × 106 cells/ml were placed in the
upper chambers. The two chambers were separated by a 3-µm
nitrocellulose filter (Sartorius, Cherry Hill, NJ). After a 1-h
incubation at 37°C, the filters were removed, placed on glass
slides, fixed with ethanol, and stained with hematoxylin. Neutrophil
migration was quantified by counting the number of neutrophils
migrating a fixed distance into the filter (mean of 3 high-power fields
in each of duplicate filters). This distance was set at a point to
which 5-10 neutrophils per high-power field migrated in response
to R10 medium. Results were expressed as a mean percentage of basal
migration to R10 medium alone. rhIL-8 (100 ng/ml) was used as a
positive control. In some experiments, the conditioned media were
incubated with a blocking antibody to IL-8 (10 µg/ml, R & D Systems)
before performing the migration assay.
Flow cytometric analysis of neutrophil adhesion receptor
expression.
Human neutrophils were exposed for 30 min at 37°C to R10 medium,
R10 conditioned medium from untreated monocytes, and conditioned medium
from toxin A-treated monocytes (prepared as described above). The
neutrophils were then washed, suspended in PBS with 0.2% BSA and 0.1%
sodium azide, and incubated at 4°C for 25 min with one of the
following mouse monoclonal antibodies at a concentration of 5 µg/ml:
1) R15.7 (anti-CD18),
2) TS1.18 (anti-CD11b) [both of these antibodies were provided by Dr. R. Rothlein (Boerhringer Ingleheim Pharmaceuticals, Ridgefield, CT) and Dr. T. A. Springer (Center for Blood Research, Harvard Medical School, Boston, MA)], 3) DREG56 (anti-L-selectin; AMAC,
Westbrook, ME), or 4) DAK-GO5, a
mouse immunoglobulin G (IgG) negative control (Dako, Carpenteria, CA).
The cells were washed, labeled with fluorescein
isothiocyanate-conjugated F(ab')2 fragment of goat
anti-mouse IgG (Dako), fixed in Formalin, and analyzed using a FACScan
flow cytometer (Becton Dickinson, San Jose, CA) as previously described
(15).
Intercellular adhesion molecule-1 cell ELISA.
Surface expression of intercellular adhesion molecule-1 (ICAM-1) by
human endothelial cells (EndoPack-UV, Clonetics, San Diego, CA) was
measured by cell ELISA (16). Endothelial cells were grown to confluence
on a 96-well culture plate. Toxin-treated monocyte conditioned media
were mixed in equal parts with endothelial cell medium and incubated
with the endothelial cell monolayers for 24 h. The monolayers were then
fixed with 1% paraformaldehyde, washed with PBS, and nonspecific
binding blocked with 2% BSA in PBS for 1 h at 37°C. After the
cells were repeatedly washed, the primary antibody RR1/1, a mouse
IgG1 monoclonal directed against human ICAM-1 (provided by Dr. R. Rothlein) in a 1:200 dilution in 2%
BSA was added and incubated for 1 h at 37°C. The plates were
washed, and 100 µl horseradish peroxidase-conjugated goat anti-mouse
IgG (Pierce, Rockford, IL) in a 1:2,000 dilution in 2% BSA were added
to each well and incubated for 1 h at 37°C. After the cells were
carefully washed with PBS, 100 µl of tetramethylbenzidine substrate
solution (Kirkegaard and Perry) were added to each well, and the
reaction was stopped after 5 min with 100 µl of 1 M
o-phosphoric acid. Results are
expressed as an ELISA index (EI) derived as (OD 450 nm test
OD 450 nm background)/(OD 450 nm basal
OD 450 nm
background), where background refers to blank wells with no endothelial
cells and basal refers to unstimulated endothelial cells.
Monocyte and macrophage-differentiated THP-1 cells.
THP-1 cells, a human monocyte cell line (American Type Culture
Collection, Rockville, MD), were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 5 mM HEPES, 50 mM
-mercaptoethanol, 50 U/ml penicillin G, and 50 µg/ml streptomycin (GIBCO, Grand Island,
NY). For differentiation into macrophages, THP-1 cells (500,000/ml)
were seeded into 96-well plates (Primaria, Falcon, Lincoln Park, NJ)
and stimulated with 10 ng/ml phorbol 12-myristate 13-acetate (Sigma)
for 48 h. They were then further cultured in fresh medium for 4 days
(5, 26, 32). Monocyte and macrophage differentiated THP-1 cells were
stimulated for 3 h with varying doses of toxin A, toxin B, or
LPS. The conditioned media were then harvested, and IL-8 levels were
measured by ELISA.
Statistical analyses.
Statistical analyses were performed using SigmaStat (Jandel Scientific
Software, San Rafael, CA). Analysis of variance followed by the
Student-Newman-Keuls method were used for intergroup comparisons.
 |
RESULTS |
IL-8 release from C. difficile toxin-exposed monocytes.
Conditioned media from unstimulated monocytes contained low
concentrations of IL-8 (150 ± 13 pg/ml). As expected, monocytes treated with 10 ng/ml LPS produced greater amounts of IL-8 (2,820 ± 290 pg/ml). Conditioned media from monocytes exposed to toxin A at
concentrations of 10
11 to
10
8 M also contained
significantly greater amounts of IL-8 as compared with control (Fig.
1). Toxin B at low concentrations
(10
12 to
10
14 M) stimulated monocyte
IL-8 release, but at higher concentrations (10
11 and
10
10 M) caused a reduction
in IL-8 release (Fig. 1). Stimulation of monocyte IL-8 release by toxin
A was evident within 2 h of toxin exposure, peaked at 6 h, and
persisted for at least 24 h (Fig. 2). This
time course of IL-8 release paralleled that of LPS-stimulated monocytes.

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Fig. 1.
C. difficile toxins stimulate
interleukin (IL)-8 release from human monocytes. Isolated human
peripheral blood monocytes were exposed to C. difficile toxin A or toxin B at concentrations ranging
from 10 14 to
10 8 M. Conditioned media
from untreated monocytes served as control. After 3 h, toxins were
removed, cells were washed 3 times, and fresh media were added.
Monocyte conditioned media were harvested after an additional 21 h, and
IL-8 levels were measured by enzyme-linked immunosorbent assay (ELISA).
Results are expressed as means ± SE;
n > 4. ** P < 0.01 vs. control.
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Fig. 2.
Time course of IL-8 release from C. difficile toxin-exposed human monocytes. Human
monocytes were exposed to C. difficile
toxin A at 10 9 M. Conditioned media were removed, and fresh medium was added after 2, 4, 6, 8, and 24 h. IL-8 levels in monocyte conditioned media were measured
by ELISA. Unstimulated monocytes (control), toxin A-stimulated
monocytes, and monocytes stimulated by lipopolysaccharide (LPS) were
studied. Results are expressed as means ± SE;
n = 4. At all time points, values for
toxin A- and LPS-treated monocytes were significantly higher than
control (P < 0.01).
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Reverse transcriptase PCR analysis of IL-8 mRNA in human monocytes.
RNA from untreated monocytes (0 h) and monocytes treated with toxin A
for 3 and 6 h was reverse transcribed, and the IL-8 gene was amplified
by PCR. The results are shown in Fig. 3. We observed a substantial increase in IL-8 mRNA expression by monocytes exposed to toxin A for 3 h as compared with untreated monocytes. Monocyte IL-8 mRNA expression returned to baseline after 6 h of toxin A
exposure. Southern hybridization with an IL-8 cDNA probe confirmed the
identity of the IL-8 mRNA reverse transcriptase PCR product.

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Fig. 3.
C. difficile toxins stimulate
increased IL-8 mRNA levels in human monocytes. RNA from monocytes
exposed to C. difficile toxin A
(10 9 M) for 0, 3, or 6 h
was extracted, and cDNA was prepared by reverse transcription.
Amplification by polymerase chain reaction (PCR) was performed using
oligonucleotide primers for human IL-8 and human -actin. PCR
products were electrophoresed through a 1% agarose gel and visualized
with ethidium bromide.
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IL-1
and TNF-
release from C. difficile toxin-exposed monocytes.
Monocytes treated with toxin A produced significantly higher levels of
IL-1
and TNF-
as compared with untreated monocytes (Table
1). Peak levels of IL-1
and TNF-
were
obtained after exposure to toxin A at a concentration of
10
9 M for IL-1
release
and 10
10 M for TNF-
.
C. difficile toxin-exposed monocytes stimulate neutrophil migration.
Conditioned media from control monocytes increased neutrophil migration
only marginally (135% of basal migration, Fig.
4). Conditioned media from monocytes
exposed to toxin A or to toxin B caused a dramatic increase in
neutrophil migration (Fig. 4). The greatest effect was seen after
exposure of monocytes to toxin A at a concentration of
10
9 M (438% of basal
migration) or to toxin B at a concentration of
10
12 M (330% of basal
migration). Thus the concentrations of toxin A and toxin B that
resulted in maximal stimulation of neutrophil migration were the same
as those that induced maximal monocyte IL-8 release (Fig. 1). The
addition of a human IL-8 blocking antibody to the monocyte conditioned
media completely inhibited their ability to stimulate neutrophil
migration (Fig. 5).

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Fig. 4.
C. difficile toxin-exposed monocytes
stimulate human neutrophil migration. Human monocytes were exposed to
C. difficile toxins A or B at varying
concentrations, and their conditioned media were harvested after 24 h.
Ability of monocyte conditioned media to stimulate human neutrophil
migration was then determined using a multi-well chemotaxis assembly.
Data are expressed as a percentage of neutrophil migration to medium
alone (% basal migration) and are presented as means ± SE;
n = 4. * P < 0.05, ** P < 0.01 vs. control.
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Fig. 5.
A neutralizing antibody to IL-8 inhibits neutrophil migration induced
by C. difficile toxin-exposed
monocytes. Human monocytes were exposed to C. difficile toxin A
(10 9 M) or toxin B
(10 12 M) for 2 h. Cells
were washed, and fresh medium was added, which was harvested after
overnight incubation. Monocyte conditioned media (MCM) were then
incubated with a neutralizing antibody to IL-8 (solid bar) before
neutrophil migration assay was performed. Recombinant human IL-8
(rhIL-8; 100 ng/ml) was used as a positive control. Data are expressed
as a percentage of neutrophil migration to medium alone (% basal
migration) and presented as means ± SE;
n 4.
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C. difficile toxin-exposed monocytes regulate neutrophil adhesion
molecule expression.
Exposure of neutrophils to conditioned media from resting monocytes had
little effect on neutrophil surface expression of L-selectin, CD11b, or
CD18 as compared with control medium (Fig. 6,
A-C).
However, exposure to conditioned medium from monocytes treated with
toxin A (10
9 M) resulted in
marked shedding of neutrophil L-selectin and increased expression of
CD11b/CD18 adhesion receptors. These effects were similar to those
observed after exposure of neutrophils to rhIL-8 (positive control). To
be certain that neutrophil stimulation was not due to residual toxin A
in the conditioned media, we examined adhesion molecule expression by
neutrophils exposed directly to 10
9 M toxin A (the same
dose used to stimulate the monocytes in these and earlier experiments).
Toxin A at this dose has no direct effect on neutrophil adhesion
molecule expression as compared with control (data not shown).

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Fig. 6.
C. difficile toxin-exposed monocytes
regulate neutrophil adhesion molecule expression. Human neutrophils
were incubated for 30 min with R10 medium (control), with conditioned
medium from unstimulated monocytes (untreated MCM), or with conditioned
medium from monocytes that had previously been exposed to
C. difficile toxin A at
10 9 M (toxin A MCM). Flow
cytometric analysis was then performed to examine neutrophil surface
expression of adhesion molecules CD18
(A), CD11b
(B), and L-selectin
(C).
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C. difficile toxin-exposed monocytes upregulate endothelial cell
ICAM-1 expression.
Human endothelial cells exposed to toxin-treated monocyte conditioned
media demonstrated increased ICAM-1 expression. Upregulation of
endothelial ICAM-1 was dose dependent, and maximal effect was again
seen with media from monocytes exposed to
10
9 M toxin A (5.05 ± 1.93 EI) or 10
12 M toxin B
(3.46 ± 1.72 EI). Conditioned media from unstimulated monocytes had
little effect on endothelial cell ICAM-1 expression (1.24 ± 0.47 EI). Because IL-1 is known to upregulate ICAM-1 expression on
endothelial cells, we preincubated endothelial cells with a receptor
antagonist to IL-1 (rhIL-1ra, 25 µg/ml; R & D Systems) before
exposing them to the monocyte conditioned media. rhIL-1ra pretreatment
resulted in a 72% reduction in ICAM-1 upregulation using conditioned
media from monocytes exposed to
10
9 M toxin A (Fig.
7). rhIL-1ra had no significant effect on
ICAM-1 expression by control monocytes or by monocytes exposed to
conditioned medium from unstimulated monocytes.

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Fig. 7.
C. difficile toxin-exposed monocytes
upregulate endothelial cell intercellular adhesion molecule-1 (ICAM-1)
expression. Human endothelial cells were incubated for 24 h with
conditioned medium from unstimulated monocytes (control MCM), with
conditioned medium from monocytes that had previously been exposed to
C. difficile toxin A at
10 9 M (toxin A MCM), or
with conditioned medium from monocytes that had previously been exposed
to C. difficile toxin A at
10 9 M after pretreatment of
endothelial cells with rhIL-1 receptor antagonist (toxin A MCM + rhIL-1ra). Expression of ICAM-1 by endothelial cells was measured by
cell ELISA. Results are expressed as mean ELISA index ± SE,
n = 8. ** P < 0.01 vs. control MCM.
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IL-8 release from C. difficile toxin-exposed macrophages.
C. difficile toxin A, toxin B, and LPS
each stimulated IL-8 release from macrophage-differentiated THP-1 cells
(Fig. 8). Macrophages showed greater
sensitivity to each stimulus than monocytic THP-1 cells. Macrophages
were ~100-fold more sensitive to toxin A (Fig. 8A) and toxin B (Fig.
8B) and ~10-fold more sensitive to
LPS (Fig. 8C). The response of
macrophages was similar to that observed in peripheral blood monocytes
(Fig. 1) in a number of respects: toxin B stimulated IL-8 production at
lower doses than toxin A; the highest doses of toxin B were associated
with lesser IL-8 stimulation, and the greatest IL-8 production was seen
with high levels of toxin A (Fig. 1). The amount of IL-8 produced by
macrophages after maximal toxin stimulation was similar in degree to
maximal production in response to the potent macrophage activator LPS (Fig. 8).

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Fig. 8.
C. difficile toxins stimulate IL-8
release from macrophage-differentiated THP-1 cells. THP-1 cells, a
human monocyte cell line, were stimulated with phorbol 12-myristate
13-acetate and cultured for 6 days to induce macrophage
differentiation. Monocyte and macrophage-differentiated THP-1 cells
were then stimulated for 3 h with varying doses of C. difficile toxin A
(A), toxin B
(B), or LPS
(C). IL-8 levels in conditioned
media were measured by ELISA. Results are expressed as means ± SE;
n = 3. * P < 0.05, ** P < 0.01 vs. control.
|
|
 |
DISCUSSION |
The main finding of this study is that C. difficile toxins A and B potently stimulate monocytes
to release the neutrophil chemotactic factor IL-8 with maximal effects
at toxin concentrations of
10
10 M for toxin A and
10
12 M for toxin B. Significant monocyte stimulation continued to be evident at even lower
toxin concentrations (10
11
M for toxin A and 10
14 M
for toxin B). Toxin B at concentrations higher than
10
12 M reduced IL-8 release
to levels below those seen in control monocytes. One possible
explanation is that this reflects monocyte toxicity and death secondary
to the cytotoxic effects of higher doses of toxin B, as previously
reported (28). The concentration of toxin B needed to stimulate IL-8
release was 100-fold less than that of toxin A. This difference in
potency is similar to that reported in a recent study which found that
toxin B was 10 times more potent than toxin A in producing mucosal
damage in human colonic explants (27). Increased IL-8 mRNA levels and IL-8 protein release by monocytes occurred within 3 h of toxin exposure.
In C. difficile colitis, peripheral
blood monocytes are unlikely to be exposed directly to substantial
amounts of toxin A or toxin B. However, tissue macrophages may well
contact these toxins especially when colonic microulceration has
developed (25, 27). This led us to examine activation of
macrophage-differentiated THP-1 cells (5, 26, 32). Our studies confirm
that macrophages, like monocytes, are activated by both toxin A and
toxin B. Macrophage differentiation of THP-1 cells results in
heightened sensitivity to C. difficile
toxins. However, the absolute concentrations of toxin required to
activate THP-1 macrophages are somewhat higher than for nontransformed
human monocytes.
We also examined the functional importance of monocyte/macrophage
activation by demonstrating that both C. difficile toxins A and B activate human monocytes to
produce factors that promote both neutrophil migration and neutrophil
adhesion to the vascular endothelium. Conditioned media from monocytes
exposed to C. difficile toxin A or
toxin B potently stimulated neutrophil migration in comparison with
conditioned media from unstimulated monocytes. This effect is due
primarily to IL-8 release as evidenced by complete inhibition of
migration using a neutralizing antibody to IL-8. These data suggest
that IL-8 released by C. difficile
toxin-exposed macrophages in the lamina propria creates a chemotactic
gradient that induces neutrophil migration to the site of mucosal
inflammation in C. difficile colitis.
In addition to promoting neutrophil migration, the conditioned media of
C. difficile toxin-exposed monocytes
caused shedding of neutrophil L-selectin and increased the surface
expression of neutrophil CD11b/CD18. A variety of monocyte-derived
factors, including IL-8 and TNF-
, may be responsible for these
changes in neutrophil adhesion molecule expression. L-selectin mediates neutrophil rolling along the vascular wall while CD11b/CD18 (Mac-1) mediates firm adhesion of the neutrophil to the vascular endothelium (12). Shedding of L-selectin and upregulation of CD11b/CD18 marks a
critical step in neutrophil recruitment to sites of tissue injury,
since CD11b/CD18-dependent firm adhesion is a prerequisite to
neutrophil migration across the vascular endothelium. Again, toxin A
had no direct effect on neutrophil adhesion molecule expression.
Expression of ICAM-1, the endothelial cell ligand for neutrophil
CD11b/CD18, was upregulated on human endothelial cells after exposure
to conditioned media from C. difficile-toxin exposed monocytes. ICAM-1 upregulation
was largely, but not completely, inhibited in the presence of rhIL-1ra.
This is consistent with endothelial stimulation by IL-1
as well as
other monocyte-derived cytokines such as TNF-
. Direct exposure of
endothelial cells to toxin had no demonstrable effect on ICAM-1
expression (data not shown). Thus C. difficile toxins, acting through the
monocyte/macrophage, may activate both neutrophil and endothelial cell
adhesion receptors. We have previously shown that a blocking antibody
to CD18 markedly reduced neutrophil infiltration of toxin A-exposed
rabbit ileal loops (14). Inhibition of neutrophil recruitment in this
model was associated with a substantial reduction in
C. difficile toxin-induced intestinal
permeability, fluid secretion, and mucosal injury. In another study,
intravital video microscopy was used to examine the direct effects of
toxin A on the intestinal microvasculature (19). In that model,
monoclonal antibodies to CD11/CD18 and to ICAM-1 also inhibited toxin
A-induced leukocyte adhesion and extravasation. These studies
underscore the importance of neutrophil CD18 and endothelial cell
ICAM-1 interaction in the pathogenesis of C. difficile toxin-induced intestinal inflammation.
Monocyte/macrophage stimulation by C. difficile toxins requires these toxins to gain access
to the lamina propria of the colon. Both toxins A (308 kDa) and B (270 kDa) are very large molecules that would not easily cross the intact
intestinal epithelium. T84 colonic cell monolayers exposed to
C. difficile toxins A and B exhibit a
marked increase in permeability to mannitol (7). However, the increase
in monolayer permeability in this in vitro system is limited to
molecules with hydrodynamic radii <5.7 Å, significantly
smaller than either toxin A or toxin B. A more recent study examined
sheets of normal human colonic mucosa that were exposed to toxins A or
B. After 5 h, epithelial cell rounding and detachment from the basal
membrane were noted. Toxin B was more potent than toxin A in inducing
this colonocyte injury (27). Interestingly, undamaged epithelium was
observed immediately adjacent to severely damaged areas, consistent
with the classical in vivo findings of patchy pseudomembranes in human
C. difficile colitis (17, 25, 27). The
localized areas of injury and inflammation seen in vitro and in vivo
may result from cell rounding causing localized breaches in the colonic
epithelium through which tiny amounts of toxins A and B can pass. These
small amounts of toxin, although unable to directly activate
neutrophils, might activate tissue macrophages to produce IL-8 and
other proinflammatory cytokines. Once initiated, this inflammatory
cascade may result in a marked acute inflammatory cell infiltration,
further mucosal injury, and, ultimately, focal pseudomembrane
formation.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Research Grants DK-02128 and DK-34583-09. J. K. Linevsky held a Career Development Award from the Crohn's and Colitis Foundation of America and was an American Gastroenterological Association Senior Fellowship Award recipient.
 |
FOOTNOTES |
Address for reprint requests: J. K. Linevsky, Evans 201, Gastroenterology, Boston Medical Center, 88 East Newton St., Boston, MA
02118.
Received 26 December 1996; accepted in final form 3 September
1997.
 |
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