Intestinal Disease Research Program, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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The immunomodulatory properties of bacterial
superantigens (SAgs) have been defined, yet comparatively little is
known of how SAgs may affect enteric physiology.
Staphylococcus aureus enterotoxin B
(SEB) was used to examine the ability of SAgs to alter epithelial ion
transport. BALB/c mice, severe combined immunodeficient (SCID, lack T
cells) mice, or SCID mice reconstituted with lymphocytes or
CD4+ T cells received SEB
intraperitoneally, and jejunal segments were examined in Ussing
chambers; controls received saline only. Baseline short-circuit current
(Isc, indicates
net ion transport) and
Isc responses
evoked by electrical nerve stimulation, histamine, carbachol, or
forskolin were recorded. Serum levels of interleukin-2 (IL-2) and
interferon- (IFN-
) were measured. SEB-treated BALB/c mice showed elevated serum IL-2 and IFN-
levels, and jejunal segments displayed a time- and dose-dependent increase in baseline Isc compared with
controls. Conversely, evoked ion secretion was selectively reduced in
jejunum from SEB-treated mice. Elevated cytokine levels and changes in
jejunal Isc were
not observed in SEB-treated SCID mice. In contrast, SCID mice
reconstituted with T cells were responsive to SEB challenge as shown by
increased cytokine production and altered jejunal
Isc responses
that were similar to those observed in jejunum from SEB-treated BALB/c
mice. We conclude that exposure to a model bacterial SAg causes
distinct changes in epithelial physiology and that these events can be mediated by CD4+ T cells.
Staphylococcus aureus enterotoxin B; intestine
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INTRODUCTION |
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SUPERANTIGENS (SAgs) are low molecular weight peptides
that are produced by a variety of bacteria (that can occur commonly or
sporadically in the gut), viruses, and possibly some protozoan parasites (10). Studies with Staphylococcus
aureus enterotoxins and other bacterial SAgs have shown
that these peptides are potent stimulants of specific T cell subsets
(17). Unlike conventional antigens, SAgs bypass the classical route of
antigen processing and presentation and cross-link outside domains of
major histocompatibility class II (MHC II) molecules with the variable
portion of -chain (V
) of the T cell receptor, binding beyond the
antigen-specific site. This initial activation
(proliferation, cytokine production, and increased cytotoxic activity)
can be followed by a period of anergy or depletion of the appropriate
V
+ T cells (10, 14, 17). Thus
SAgs can activate up to 25% of the host's T cells and therefore are
potentially important and environmentally relevant inflammatory
stimuli. In this context SAgs have been implicated in the
pathophysiology of inflammatory and autoimmune disorders, such as
rheumatoid arthritis, multisystem vasculitis (Kawasaki disease), and
diabetes (8, 38). However, whereas the immunomodulatory properties of
SAgs are being precisely defined, there is a dearth of information on
the physiological consequences of exposure to SAgs, particularly in
terms of gut function.
There is increasing evidence implicating bacteria in the
pathophysiology of enteric secretory and inflammatory disorders (42). Recently it has been postulated that bacterial SAgs could be involved in inflammatory bowel disease (19). This hypothesis is not
unprecedented. In the mid-1960s it was shown that dogs and rhesus
monkeys treated with an S. aureus
extract or partially purified S. aureus enterotoxin B (SEB) developed diarrhea and
intestinal changes typified by loss of epithelial microvilli and
mitochondrial destruction, a mucopurulent exudent, and varying degrees
of lymphocytic infiltration (20, 33, 46). Many of these histological
changes were apparent 2-8 h after treatment. The mechanism(s) of
these changes in the gut remains undefined. In hindsight, it appears
that these changes in gut morphology are temporally consistent with
SEB-induced increased levels of circulating cytokines, such as tumor
necrosis factor- (TNF-
) and interferon-
(IFN-
) (34). It has
also been shown that mice display a significant weight loss in response
to SEB (27) and that treatment with the related enterotoxin,
staphylococcal enterotoxin A, results in a rapid recruitment of
lymphocytes to the epithelial compartment in the duodenum of rats
(4). Moreover, we (32) and others (13) have recently found
that functionally intact SEB can cross confluent monolayers of human
epithelial cell lines (i.e., T84 and Caco-2) in vitro and murine
intestine in vivo. In the former study we also reported that immune
activation elicited by SEB resulted in increased epithelial
permeability and diminished the secretory responsiveness of T84
monolayers (32).
Extrapolating from these studies, the present study is the first in a series of investigations to delineate the enteric consequences of exposure to SAgs, where epithelial electrolyte ion transport was used as an index of gut function. Our findings indicate that mice treated with SEB develop a self-limiting enteropathy characterized by a rapid onset of dramatic irregularities in epithelial ion transport and that these events can be mediated by CD4+ helper T cells. We speculate that given the correct environmental conditions and genetic background, exposure to SAgs could result in severe intestinal electrolyte abnormalities and so initiate or contribute to enteric secretory functional disorders. Furthermore, we contend that this model can be used to examine not only the effects of bacterial SAgs per se but also the role of T cells and T cell subsets in the immunophysiological regulation of gut function.
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MATERIALS AND METHODS |
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Animals and Experimental Treatment
BALB/c mice. Male mice (7-9 wk old) were purchased from Charles River animal suppliers (St. Constant, Quebec, Canada) and housed under conventional conditions for 1-2 wk before treatment with a single intraperitoneal injection of 1-100 µg SEB (Sigma Chemical, St. Louis, MO). With use of the limulus amoebocyte lysate assay (Sigma Chemical) our SEB stocks were found to be free of lipopolysaccharide contamination. After treatment (2-144 h) mice were killed by cervical dislocation. Time-matched PBS (vehicle for SEB administration)-treated mice served as controls.
SCID/beige mice.
Mice were obtained from the breeding colony at McMaster University. To
check for the presence of T cells 1)
segments of gut and spleen were Formalin fixed and proceeded to wax,
and 3-µm sections were immunostained with polyclonal anti-CD3
antibodies (Dako Diagnostics, Mississauga, ON, Canada) and
2) splenocytes (106/ml) were stimulated in vitro
for 48 h with 1 µg/ml SEB or concanavalin A (Con A; Sigma Chemical),
and proliferation was assessed by
[3H]thymidine
incorporation and interleukin-2 (IL-2) measured in the culture medium
by ELISA (PharMingen, Mississauga, ON, Canada). Severe combined
immunodeficient (SCID) mice (8-12 wk old) were injected with SEB
(5 µg ip) and examined 4 or 48 h later. In additional studies, SCID
mice were reconstituted with a mixed lymphocyte population or purified
CD4+ T cells. Briefly, spleens and
mesenteric lymph nodes from normal BALB/c mice (not treated with SEB)
were mechanically dispersed into a single cell suspension, and the
erythrocytes were lysed by a 2-min incubation in ammonium chloride
buffer. After centrifugation, the immune cells were resuspended in
sterile PBS and 30 × 106
cells/0.2 ml PBS were administered intravenously to SCID mice (21). In
other preparations the mixed spleen and lymph node cells were
resuspended in RPMI medium (containing 10% FCS and antibiotics; GIBCO
BRL, Burlington, Canada) and incubated for 2 h at 37°C on sterile
petri dishes to allow the adherence of monocytes/macrophages. The
nonadherent lymphocytes were recovered, and
CD4+ T cells were positively
selected using magnetic cell sorting (MACs; Miltenyi Biotec, Auburn,
CA); 107 lymphocytes were
incubated with 10 µl of colloidal superparamagnetic microbeads
conjugated to rat anti-mouse CD4 antibodies (L3T4, Miltenyi Biotec) for
20 min at 4°C, and the cell suspension was passed through an
LS+ separation column mounted in a
Midi-MACs magnet. After a buffer wash (PBS, 2 mM EDTA, and
0.5% wt/vol BSA), the column was removed from the magnet and the
CD4+ T cells were eluted with cold
buffer. The positively selected CD4+ cells were resuspended in
sterile PBS, and 15 × 106
cells/0.2 ml PBS were administered intravenously to SCID mice (samples
of immune cells were analyzed by fluorescent-activated cell sorting and
monoclonal antibodies to murine CD3, CD4, and V8). Four weeks later
the reconstituted SCID mice were challenged with SEB (5 µg) and
jejunal segments examined in Ussing chambers 4 h later. We concentrated
on 4 h post-SEB (5 µg) treatment because altered ion transport was
consistently observed in the intestine from SEB-treated BALB/c mice at
this time.
Immune Activation
Splenocyte proliferation. Splenocytes were isolated and resuspended in RPMI medium, and 105 cells were added to each well of 96-well sterile culture plates ± 100 ng/well of SEB or Con A (as a positive control). Forty-eight hours later each well was pulsed with 1 µCi of [3H]thymidine (DuPont-NEN, Wilmington, DE) in 50 µl of fresh medium, and after an 18-h incubation at 37°C, cells were harvested onto glass fiber filters and radioactivity was determined in a scintillation counter (Becton-Dickinson, Mississauga, ON, Canada). The results are expressed as the stimulation index (SI), obtained by dividing the radioactivity count (counts per minute) from stimulated splenocytes by that from nonstimulated splenocytes from the same mouse.
Cytokine production.
At autopsy blood samples were collected and serum was stored at
70°C before measurement of IL-2 and IFN-
levels.
Cytokines were measured in duplicate in three serial dilutions by
sandwich ELISA, using paired antibodies from PharMingen and following
the instructions of the manufacturer. Also, lamina propria lymphocytes (LPLs) were isolated (using standard techniques) from control mice or
mice treated with SEB (100 µg) 4 h previously and incubated at
37°C with or without SEB treatment (1 µg
SEB/106 LPLs). Twenty-four hours
later cell-free supernatants were collected and assayed for IL-2 and
IFN-
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Intestinal Ion Transport
A 12-cm portion of jejunum was excised beginning at the ligament of Treitz and was divided into four segments. Each piece of tissue was mounted in an Ussing chamber (exposed surface area 0.6 cm2) and bathed in 10 ml of warm (37°C), oxygenated Krebs buffer (in mM): 115.0 NaCl, 8.0 KCl, 1.25 CaCl2, 1.2 MgCl2, 2.0 KH2PO4, and 25.0 NaHCO3; pH 7.35 ± 0.02 (28). Some experiments were conducted in a modified buffer in which chloride ions were replaced with isethionate and acetate ions (43). Glucose (10 mM) was added to the buffer on the serosal side of the tissue, and this was osmotically balanced by 10 mM mannitol in the luminal buffer. The buffers were maintained at 37°C by a surrounding heated water jacket and circulated by a CO2-O2 gas lift. Tissues were short-circuited at zero volts using an automated voltage clamp (WPI Instruments, Narco Scientific, Mississauga, ON, Canada), and the short-circuit current (Isc, in µA/cm2) was continuously monitored as an indication of net ion transport. The circuit was opened at intervals to obtain potential difference values (mV), and tissue conductance (G in mS/cm2; indicates barrier to passive ion flow) was calculated according to Ohm's law. After a 15-min equilibration period, baseline Isc and G were recorded.Stimulated Ion Transport
Enteric nerves in each preparation were activated by an electrical transmural nerve stimulation (ETS, at 10 Hz, 10 mA, 0.5 ms for a total time of 5 s), and the peak in Isc was recorded. Subsequently, histamine (10MPO Activity
Mucosal scrapings of intestinal tissue were snap frozen in liquid N2 and stored atData Presentation and Analysis
Examination of all PBS-treated control mice showed no significant differences in any of the parameters examined in this study, and therefore, for illustrative purposes, they have been grouped together as a single control group (in some figures the control animals time-matched to the 4-h and 48-h post-SEB-treated mice are shown as individual groups). All data are expressed as means ± SE; n values are the number of mice in each experiment, in which 2-4 intestinal segments were used in the ion transport studies. Data were compared using one-way ANOVA followed by post hoc comparisons with the Newman-Keuls test, where P < 0.05 was accepted as the level of statistically significant difference. Statistical analysis compared data from test mice with time-matched controls. ![]() |
RESULTS |
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BALB/c Mice
After SEB treatment some mice displayed obvious piloerection and hunched posture, and when the mice were killed portions of the intestine were filled with fluid. Similar events have been reported after in vivo T cell activation via anti-CD3 antibodies (11).SCIDs and SCID Reconstitution
Immunohistochemical staining of sections of jejunum or spleen for CD3 positivity confirmed the general absence of T cells in the SCID mice used in this study. Corroborating this observation, splenocytes from SCID mice failed to proliferate or synthesize IL-2 in response to in vitro SEB or Con A challenge (Table 1). Fluorescence-activated cell-sorting analysis revealed that the mixed splenocyte-mesenteric lymph node population used to repopulate SCID mice contained 34.5 ± 10.5% CD3+ T cells, of which 9 ± 1% were V
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Immune Activation
Splenocyte proliferation. BALB/C MICE. Splenocytes isolated from SEB-treated mice and challenged with SEB in an in vitro proliferation assay showed a significant increase in [3H]thymidine incorporation compared with spleen cells from control mice. The calculated SI for controls was 3.8 ± 0.7, whereas the SI of splenocytes from mice treated with SEB 2-48 h previously ranged from 6.9 to 12.2 (data not shown, Table 1). In contrast, splenocytes from saline-treated and SEB-treated mice displayed similar proliferative responses to in vitro exposure to Con A (Table 1).
SCID MICE. There was no increase in the SI in response to SEB or Con A in splenocytes from SCID mice with or without SEB treatment (Table 1). Splenocytes from reconstituted SCID mice did show a slightly enhanced immune responsiveness evoked by in vivo SEB treatment, such that in vitro proliferation to Con A was increased 3.5-fold (n = 4).Cytokine production.
BALB/C MICE.
Four hours after SEB (5 µg) treatment, serum levels of IL-2 and
IFN- were increased (Fig. 1); by 48 h
posttreatment the levels of IL-2 and IFN-
had returned to control
values. Mice treated with high-dose SEB (100 µg) also displayed
increased serum levels of IL-2 (60.2 ± 11.1 ng/ml) and IFN-
(6.7 ± 1.7 ng/ml) 4 h after treatment that remained elevated at 48 h
posttreatment (48.4 ± 13.8 and 3.8 ± 1.8 ng/ml for IL-2 and
IFN-
, respectively). LPLs isolated from mice treated 4 h earlier
with SEB showed increased spontaneous and stimulated IL-2 and IFN-
production in culture, compared with LPLs from control mice (Table
2).
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Intestinal Ion Transport
Baseline Isc and ion
conductance (G).
BALB/C MICE.
Assessment of jejunal segments from mice treated with 5 µg of SEB
revealed a time-dependent increase in baseline
Isc that was
significantly different from controls at 12 h posttreatment, peaked at
48 h posttreatment, and had returned to control levels at 144 h
posttreatment (Table 3). Dose responses
were examined at 4 and 48 h after SEB treatment
(Fig. 2), and the data revealed that 5
µg of SEB caused an increase in baseline
Isc. The use of
chloride ion substitution buffers eliminated the increase in Isc observed in
tissues from SEB-treated mice (Fig. 3),
implicating active anion secretion as the cause of the elevated
Isc. Calculation of tissue ionic conductance (G)
showed that this was significantly reduced 2 and 4 h after
administration of 5 µg of SEB intraperitoneally (Table 3). Likewise,
treatment with higher doses of SEB led to a drop in
G [21.3 ± 1.5, 14.6 ± 4.9 (P < 0.05), and 13.7 ± 3.4 (P < 0.05)
mS/cm2 for jejunum from control
mice and those treated with 100 µg SEB and examined 4 and 48 h later,
respectively (n = 4)].
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Stimulated Ion Secretion
ETS. BALB/C MICE. Jejunum from mice treated 2 or 4 h previously with low-dose SEB (5 µg) displayed a dramatically reduced (<50% of control values) secretory response to nerve stimulation (Fig. 4A). When mice were treated with higher doses of SEB the reduced responsiveness to electrical nerve stimulation was apparent 48 h after intraperitoneal administration of the SAg (Fig. 4B).
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Ca2+-evoked
active Cl secretion.
BALB/C MICE.
Jejunal tissues from SEB-treated mice displayed a diminished
Isc response when
CCh was added to the serosal side of the tissue (Fig.
6A). This response was time
dependent with the
Isc being significantly attenuated 2 and 4 h after SEB treatment. The reduced response to CCh was also observed in jejunal segments examined 4 h
after mice had been given a single intraperitoneal injection of 1, 50, or 100 µg SEB. In addition, higher doses of SEB prolonged the
abnormal secretory nature of the epithelium, with diminished responses
to CCh being evident 48 h after treatment (Fig.
6B). In contrast to the SEB-evoked
alteration in the ability of the epithelium to respond to CCh, the
secretory response to exogenous histamine at a maximal dose
(10
4 M) was not
consistently altered at any time point (2-144 h posttreatment), irrespective of the administered dose (1-100 µg/mouse) of SEB. For instance, in jejunal segments from control mice, histamine caused a
transient increase in
Isc of 32.0 ± 3.5 µA/cm2 compared with 28.3 ± 5.3 and 30.1 ± 5.2 µA/cm2 in jejunal tissues from
mice treated with 5 µg SEB 4 and 48 h previously.
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cAMP-evoked active Cl secretion.
BALB/C MICE.
Addition of forskolin to jejunal segments resulted in significantly
reduced responses in tissues from SEB-treated mice
(Fig. 9). Reduced jejunal responsiveness to
forskolin (as indicated by smaller
Isc) was
evident 2 h after a single intraperitoneal injection of 5 µg SEB.
Unlike the abnormal secretory responses to ETS and CCh, the diminished
ability of the tissue to respond to forskolin was protracted and was
still apparent 24 h after treatment with this low dose of SEB (data not
shown). The diminished secretory response to forskolin occurred
independent of whether the tissue had been previously exposed to CCh or
histamine (Fig. 9). Also jejunal segments from mice treated with higher
doses of SEB (50 or 100 µg) displayed a prolongation of the secretory abnormality, with
Isc responses to
forskolin being significantly reduced 48 h after treatment compared
with intestine from time-matched control mice (Fig. 9). Because
Ca2+ and cAMP-mediated
Isc responses can
be linked (see Ref. 18), we also examined the
Isc response
to forskolin in tissue that had not been previously exposed to
histamine or CCh. Under these in vitro conditions, jejunal tissues from
SEB-treated (5 µg) mice still displayed a ~60% reduction in the
response to forskolin [49.9 ± 18.9 µA/cm2 compared with 115.3 ± 18.2 µA/cm2 in control tissues
(n = 4 mice)].
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MPO Activity
There was no significant difference in MPO levels in jejunal homogenates from saline-treated control mice and SEB (5 µg)-treated mice autopsied 4-48 h later. MPO levels in jejunal homogenates from saline-treated control mice and mice examined 4 and 48 h after SEB treatment were 0.21 ± 0.05, 0.17 ± 0.04, and 0.14 ± 0.03 U/ml, respectively (n = 6). ![]() |
DISCUSSION |
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The primary role of the immune response is the recognition and
destruction and/or elimination of antigens and
pathogens. However, it is also clear that inappropriate
or exaggerated immune reactions can result in tissue damage and
pathology (24). For instance, mice that lack the regulatory cytokine
IL-10 develop a spontaneous enterocolitis, the severity of which is
dramatically reduced when the animals are maintained under germ-free
conditions (22). Indeed, microflora and/or bacterial products
have been repeatedly implicated in enteric disease (42). Recently
reported examples (7, 48) of this show that
Helicobacter hepaticus and
Cryptosporidium parvum infections
enhance the gut inflammation that develops in SCID mice reconstituted
with CD4+,
CD45RBhigh T cells and in mice
lacking the -chain of the T cell receptor, respectively.
Extrapolating from these observations, we hypothesized that exposure to
a potent T cell activator, that is bacterial SAgs, could result in
altered intestinal function. To test this hypothesis, we treated mice
with SEB, as a model bacterial SAg, and subsequently examined
epithelial ion transport as our key index of gut function. To
summarize, we found that 1) exposure to SEB resulted in a time- and dose-dependent disruption of jejunal tonic and stimulated ion transport,
2) these events were not observed in
mice devoid of T cells (i.e., SCID mice), and
3) abnormal ion transport events in
response to SEB-immune activation could be reproduced in SCID mice by
repopulating these animals with
CD4+ T cells. Thus we have shown
that bacterial SAgs can perturb enteric ion transport and that T cells
are important in this enteropathy.
In defining the immunomodulatory properties of bacterial SAgs, many
studies have shown that T cells isolated from SEB-treated mice are
hyperresponsive to in vitro SEB stimulation (6, 23). Therefore, before
examining any intestinal physiological implications of exposure to SEB
it was important to confirm that the SEB did affect the immune system
in the treated mice. In accordance with the recognized
immunostimulatory nature of SAg, we observed that splenocytes from
SEB-treated mice showed an enhanced proliferative response (i.e.,
increased
[3H]thymidine
incorporation) to SEB in vitro compared with splenocytes from control
mice. Confirmatory evidence of SEB causing an immune activation was
provided by the increased levels of IL-2 and IFN- in the serum of
SEB mice compared with controls. In addition, LPLs from SEB-treated
BALB/c mice produced greater amounts of IL-2 and IFN-
on secondary
challenge with SEB in vitro; this is indicative of previous in vivo
exposure (14). Having shown that SEB did have an immune effect, we
proceeded to consider the enteric physiological ramifications of
exposure to this model SAg.
Functional assessment of jejunal tissue revealed that baseline
Isc was
significantly increased by ~40% 12-48 h after SEB treatment. Subsequent studies showed that this increase in tonic ion transport was
most likely due to heightened anion secretion because it was not
observed in tissues bathed in
Cl-free buffer. Enhanced
luminally directed anion secretion would create a driving force for
water movement and so could initiate or contribute to a diarrheal
response (39). Indeed, macroscopic examination often revealed an
increased fluid content in the gut lumen of SEB-treated mice. Thus
should SEB gain access to the mucosa it could elicit an immune cascade
that could result in increased movement of water into the gut lumen. In
this context, SEB has been found to cross monolayers of confluent human
epithelial cells in vitro and murine intestine in vivo after oral
administration (13, 32). In addition, the barrier function of rat
jejunal epithelium is disrupted after exposure to a mild stress regimen (43). If the same is true of human intestine, then SAgs in the gut
lumen could gain access to the mucosa after stressful life events and
therefore evoke or augment enteric secretory or inflammatory disorders.
This scenario may be particularly relevant in situations in which there
are increased numbers of T cells and/or increased MHC II
expression in the intestine (see Ref. 24).
In contrast to the increase in baseline
Isc, jejunal
segments from SEB-treated mice consistently displayed diminished
Isc responses to
electrical nerve stimulation and exogenous CCh and forskolin.
Diminished Isc
responses to these three prosecretory stimuli were evident in jejunal
tissue from mice treated 2 or 4 h previously with 5 µg of SEB, and
these abnormalities were prolonged in mice given higher doses of SEB
(i.e., 50 or 100 µg). Although the exact mechanism and physiological
importance of this diminished ion transport responsiveness are unknown,
it is clear that exposure to SEB results in dramatic, but reversible,
disruption of normal intestinal ion transport. Histamine, similar to
CCh, evokes active Cl
secretion using intracellular Ca2+
as a second messenger (3). The changes in
Isc evoked by
maximal doses of exogenous histamine were not significantly different in tissues excised from saline-treated controls and SEB-treated mice at
any time point. This differential responsiveness of jejunal tissue from
SEB-treated mice to secretagogues that utilize intracellular Ca2+ as a second messenger implies
specific changes in the transporting enterocyte, rather than a general
loss of epithelial secretory capacity. This postulate is supported by
studies with other models of gut inflammation (18, 28, 39) or
examination of resected inflamed human tissue (16, 40), where
diminished secretory responses and differential responses to known
secretagogues have been reported. In one such model where colitis was
chemically induced in rats, a loss of
Na+ and
Cl
absorption was
correlated with epithelial destruction (5). Epithelial viability was
not examined in the present study, but we have presented preliminary
data showing altered enteric morphology in response to SEB (5a). It is
still unclear as to how these changes in villus and crypt structure
directly impact on the ion transport abnormalities. In addition we
report that SEB treatment results in ~50% drop in the magnitude of
stimulated ion transport events, and this compares favorably with other
enteropathies (e.g., mitomycin-induced colitis, parasitized rats) in
which increases in
Isc evoked by nerve stimulation, substance P, prostaglandin
E2, or serotonin were reduced by
30-75% compared with responses in tissues from control animals.
Voluminous literature has accumulated illustrating the range of cells
(immune, neural, and stromal) and messenger molecules that can regulate
(enhance and downregulate) intestinal electrolyte transport (39).
Because the T cell has been identified as an important cell in the
mediation of SAg effects (23, 27, 29), we questioned the role of T
cells in the current model of SAg-induced enteropathy. The SCID mouse
(derived from a congenic partner of BALB/c) lacks mature functional T
cells but does contain natural killer cells and cells of the monocytic
lineage are reportedly normal (1). After confirming the general absence
of T cells in the SCID mice used in this study (immunocytochemically
and functionally), we observed that SEB treatment of these animals had
minimal or no effect on jejunal ion transport. These data suggested a
role for T cells in the SAg-induced epithelial abnormalities. To
further test this postulate, SCID mice were reconstituted with a mixed
population of spleen and/or mesenteric lymph node cells or
purified CD4+ T cells. SEB
challenge of these mice resulted in increased serum IL-2 and IFN-
levels, and although CD3+ T cells
were present in the gut of these mice, we do not suggest that these
cells were the source of the IL-2, but rather that T cell
reconstitution conferred the ability to respond to SEB (also supported
by the enhanced responsiveness of LPLs from SEB-treated mice to in
vitro SEB challenge; Table 2). Furthermore, on challenge with SEB (5 µg ip) reconstituted SCID mice displayed very similar, and on
occasions more severe, alterations in ion transport compared with those
observed in SEB-treated normal BALB/c mice. These data demonstrate a
central role for CD4+ T cells in
the mediation of SAg-induced epithelial ion transport abnormalities.
Similarly, CD4+ cells have been
implicated in other models of enteric dysfunction (9, 45). However, our
data do not negate a putative role for
CD8+ T cells in SAg-induced
changes in gut function in normal mice. CD8+ cells do respond to SEB (10,
17, 23), and the role of these cells in SAg-induced enteropathy needs
to be tested. A role for non-T cells in SAg-induced changes in the gut
must also be considered (note small drop in
Isc to CCh in
the jejunum of SEB-treated SCID mice). In this context, there is
fragmentary evidence showing effects of SAg on MHC
II+ monocytes and fibroblasts
(35), mast cells (44), and dendritic cells (36). Thus, whereas we have
shown an unequivocal role for CD4+
T cells in SEB-induced changes in epithelial ion transport, we do not
dismiss the putative involvement of other cells, possibly working in
concert with T cells, in the modulation of epithelial ion transport in
response to SEB. Additionally, the selective administration of T cell
cytokines (see below) to SCID mice may be of use in more fully
elucidating T cell modulation of epithelial physiology.
The onset of the epithelial electrolyte transport abnormalities in this
model of SAg enteropathy is fairly rapid. These findings complement
those of Neumann et al. (37), who showed that SEB could evoke rapid
(within 6 h) pathology in the lungs of mice, and earlier studies
documenting enteric morphological and ultrastructural changes in
response to SEB exposure (20, 33, 46). Although studies are in progress
to more fully define the mechanism(s) underlying this irregular enteric
ion transport, clues are available from the literature and our
investigations with an in vitro model of SEB-induced epithelial (T84)
transport and barrier dysfunction (32). For instance, levels of IL-2,
IFN-, and TNF-
are elevated in murine serum 2-8 h after
challenge with SEB and the related SAg, staphylococcal enterotoxin A
(12, 34). These changes in cytokine levels are temporally consistent
with the altered ion transport observed in the current study. Also,
data from a variety of model systems show that IFN-
and TNF-
can
directly or indirectly affect epithelial electrolyte transport (15, 26, 30, 41). Corroborating these findings we have recently described the
ability of SEB-activated peripheral blood mononuclear cells to increase
the permeability characteristics and reduce the secretory responses to
CCh and forskolin in a model epithelium, namely monolayers of the human
colonic T84 cell line (32). The epithelial ion transport dysfunction in
this in vitro model is very similar to that observed in jejunal tissue
excised from SEB-treated mice; reduced T84 secretory responsiveness is
apparent 3-6 h after coculture with SEB-activated immune cells.
Furthermore, neutralizing antibodies to IFN-
and TNF-
were found
to wholly or partially inhibit the epithelial abnormalities in the
coculture model. Collectively these studies implicate IFN-
and
TNF-
as likely candidates in the mediation of the intestinal changes
elicited in response to SEB. Further research efforts are required to
confirm or refute this postulate.
A neutrophilic infiltrate is a common feature of acute inflammation.
Neutrophils can contribute to tissue damage, and in vitro studies have
documented the ability of these cells to evoke active Cl secretion (25). In the
present study, an elevation in MPO levels (as an indicator of
granulocyte infiltration) was not observed. Thus we have shown that T
cell activation, as evidenced by increased IL-2, can result in
significant changes in epithelial ion transport in the absence of a
neutrophilic infiltrate.
In summary, this study describes an enteropathy evoked by a model
bacterial superantigen (SEB) that is characterized by alterations in
electrolyte transport and occurs in the absence of any significant neutrophilic infiltrate. CD4+ T
cells have been identified as an integral component in the mediation of
altered epithelial ion transport in response to SEB, although the
precise mechanisms (i.e., mediators, cell-cell signaling) underlying
these events remain to be elucidated. We speculate that given suitable
environmental conditions (e.g., T cell V expression, altered barrier
function of the epithelium, etc.) bacterial SAgs could contribute to
the initiation or exaggeration of intestinal secretory abnormalities
and may, in some patients with chronic or low-grade inflammation, lead
to relapses in active disease.
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ACKNOWLEDGEMENTS |
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This study was supported by a Crohn's and Colitis Foundation of Canada operating grant to D. M. McKay.
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FOOTNOTES |
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Address for reprint requests: D. M. McKay, Intestinal Disease Research Program, HSC-3N5, Dept. of Pathology, McMaster Univ., 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5.
Received 14 October 1997; accepted in final form 9 March 1998.
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