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,
Departments of
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Pediatrics and
Microbiology and Immunology and
The Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, TX 77555
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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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and mediate cell-mediated immunity
(1). Because the relative proportions of Th1 and Th2 cells
have such an impact on the host response, the factors controlling T
cell differentiation are of interest.
IL-10 is a potent suppressor of macrophage function and leads to an
inhibition of Th1-type cytokine production from T cells
(2). This immunoregulatory effect is associated with
inhibiting the expression of class II MHC (3), reducing
the production of proinflammatory cytokines such as IL-1, IL-6, and
TNF-
(4), and down-regulating the expression of
costimulatory molecules such as B7 (5) or ICAM-1
(6) on macrophages. IL-10 also plays a role in the
maintenance of anergy in T cells activated by a primary mixed
lymphocyte reaction (7). The failure to develop tolerance
to otherwise benign Ags that persist in the lumen may contribute to
chronic inflammatory bowel disease, such as Crohn’s disease or
ulcerative colitis, in humans (8). In fact, tolerance
against autologous intestinal flora is broken in lamina propria
lymphocytes isolated from active lesions of inflammatory bowel disease
patients (9) or in mononuclear cells from spleen or
intestine from mice with experimental colitis (10, 11).
Interestingly, tolerance toward autologous flora was restored by
treatment with IL-10 or Abs to IL-12 in the murine model
(10).
Recent advances in genetic engineering have revealed several experimental models of colitis. For example, spontaneous colitis develops in mice in which there is a disruption of genes encoding IL-2 (12), TCR ß-chain (13), TGF-ß (14), or IL-10 (15). Moreover, the intestinal flora that is present in wild-type mice without colitis is sufficient to trigger enteric disease in the animals with these genetic disruptions (16, 17, 18, 19). The role for flora as a trigger of colitis was also established in SCID mice reconstituted with CD45RBhigh CD4+ lymphocytes (20). Furthermore, disease in these animals was prevented when they were treated with exogenous IL-10 (21) or the transfer of T cells that express high levels of IL-10 (22, 23). Because colitis is also induced after the transfer of CD4+ T cells from IL-10-deficient mice to recombinase-activating gene-2-deficient mouse recipients exposed to a normal flora (24), it is reasonable to suggest that IL-10 plays a key role in controlling the excessive immune responses to luminal flora.
The present study investigated the hypothesis that potentially destructive autoreactive responses can be induced by microbial Ags and that these responses are inhibited by IL-10. The results suggest that highly immunogenic stimuli, such as staphylococcal enterotoxin B (SEB),3 are capable of inducing autoreactive T cells that express a repertoire that recognizes self Ags, proliferate in response to syngeneic cells, and are capable of injuring syngeneic cells through cell-mediated immunity. In addition, these responses are regulated by IL-10. The implications for the roles of these responses in immune-mediated disease of the intestine are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-10-/- 129/SvEv mice (H-2b, I-E-) were provided by DNAX Research Institute (Palo Alto, CA) and were bred in the Animal Resource Center of the University of Texas Medical Branch (Galveston, TX). Wild-type 129/SvEv mice were purchased from Taconic Farms (Germantown, NY) and were bred under the same conditions. C57BL/10 IL-10-/- mice (H-2b, I-E-, Mls-1b) and wild-type controls, CBA/J mice (H-2k, Mls-1a), as well as C3H/HeJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The animal research committee of the University of Texas Medical Branch approved all procedures used in these studies.
Cytokines and Abs
Recombinant murine IL-10 was provided by Schering-Plough
(Kenilworth, NJ). Hybridomas producing anti-mouse CD8 (53-6.72,
TIB-105) or anti-mouse I-Ak (10.2.16, TIB-93)
were purchased from American Type Culture Collection (Manassas, VA) and
grown according to the instructions provided. Supernatants were used as
a source of Ab. Anti-mouse I-Ek,
I-Ab Ab (M5/114) were produced from ascites and
were concentrated using ammonium sulfite sedimentation by Dr. Victor
Reyes (University of Texas Medical Branch). Anti-IL-10R Ab (1B1.2) was
provided by Dr. K. W. Moore (DNAX, Palo Alto, CA). The activity of
rIL-10 and the neutralizing activity of this Ab were confirmed as
previously described (25) using the IL-10-dependent mouse
mast cell line (MC/9, American Type Culture Collection CRL 8306), in
which 0.1 µg/ml Ab inhibited the proliferation of 2 x
104 MC/9 cells in the presence of 1 ng/ml mouse
rIL-10 and 5 pg/ml mouse rIL-3 (data not shown). Isotype controls for
mouse IgG (ChromPure Mouse IgG) and rat IgG (ChromPure Rat IgG) were
purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
PE- or FITC-conjugated anti-mouse CD3, CD4, or CD8 was purchased
from Caltag (Burlingame, CA). FITC-conjugated anti-mouse Vß3,
Vß6, Vß7, Vß8.1, Vß8.2, biotin-conjugated anti-mouse
TCR-ß, PE-conjugated anti-mouse CD8, biotinylated-human IL-10
(26), and isotype controls for the corresponding Abs were
obtained from PharMingen (San Diego, CA). Streptavidin-PE was purchased
from Sigma (St. Louis, MO).
Cell isolation
Single-cell preparations were made by mincing spleen and lysing RBC in ammoniun chloride (0.15 M), potassium carborate (1 nM), and EDTA (0.1 nM) buffer (pH 7.3) lysing buffer; using standard techniques (27). Colonic mucosal T cells were isolated using a modification of a previously described procedure (28). Briefly, large intestines from five C3H/HeJ mice were isolated and flushed, lymphoid nodules were removed, and tissue was opened longitudinally in HBSS (Life Technologies, Grand Island, NY). Tissue was cut into 2- to 5-mm segments and stirred at 37°C for 45 min in DMEM containing 50 U/ml of type VIII collagenase (Sigma), 0.5 mg/ml of dispase grade II (Roche, Indianapolis, IN), 0.01% gentamicin (Life Technologies), and 5% FCS. After collecting the cell suspension in the supernatant, the enzymatic treatment was repeated again. Pooled supernatants were filtered through nylon wool, and enriched lymphocytes were centrifuged through a discontinuous 44/70% Percoll (Pharmacia, Alameda, CA) gradient for 18 min at 800 x g at 4°C. Cells at the interface between the 70 and 44% layers were collected and washed. Approximately 95% of the lymphocytes were viable, as measured by trypan blue exclusion.
T cell stimulation
T cells prepared from spleen or colon (2.5 x 106 cells/ml) were stimulated with 10 µg/ml SEB (Sigma) in RPMI 1640 (Life Technologies) containing 10% FCS (HyClone, Logan, UT) as a primary stimulation. After 72 h of incubation the cells were washed three times to remove the residual SEB and resuspended in 10 µg/ml SEB in RPMI 1640. After 4 days of resting, cells were washed once, and 2 x 105 viable responder cells were cultured with 2.5 x 105 syngeneic feeder cells in 96-well flat-bottom plates. Feeder cells were prepared by incubating fresh splenocytes with 100 µg/ml of mitomycin C (Sigma) at 37°C for 30 min and washing three times. In some experiments recombinant mouse IL-10 and/or anti-IL-10R Ab (1B1.2) was added in either the priming or secondary stimulation. T cells were also activated with anti-CD-3 or PMA (5 ng/ml) and ionomycin (500 ng/ml; Sigma) as previously described (29) to compare their autoreactivity with that of SEB-primed cells. To detect proliferative responses, responder cells were incubated for 72 h, and [3H]thymidine (1 µCi/well; ICN, Irvine, CA) was added for the last 16 h. The cells were harvested using an automatic cell harvester (Skatron, Sterling, VA), and [3H]thymidine incorporation was determined by liquid scintillation counting using a Liquid Scintillator (Beckman, Fullerton, CA). All experiments were performed in triplicate.
Purification of T cells
In some experiments MHC class II+ cells and/or CD8+ cells were depleted to obtain highly enriched CD4+ T cells (30). Spleen cells or mesenteric lymph node cells (1 x 108) were incubated in DMEM containing 10% FCS with M5/114 (anti-MHC class II) and/or 53-6.72 (anti-CD8) Abs for 30 min on ice. After being washed three times, cells were incubated at 4°C with sheep anti-rat IgG Ab-coated magnetic beads (Dynabeads M-450; Dynal, Oslo, Norway) in DMEM at one bead per cell for 20 min while rotating. Magnetic bead-binding cells were removed using a magnetic separator (PerSeptive Biosystems, Framingham, MA). Flow cytometric analysis always revealed the residual MHC class II+ or CD8+ cells to be <1%. The recovered cells were resuspended in RPMI and used for culture.
Flow cytometry of surface Ag expression
One million cells were incubated in PBS-0.1% azide containing fluorescence- or biotin-conjugated Abs on ice for 30 min. When using biotin-conjugated Abs, cells were washed once and incubated with streptavidin-PE for 20 min on ice. After being washed three times, cells were fixed in 1% paraformaldehyde and analyzed by FACScan (Becton Dickinson, San Jose, CA).
Cytokine assay
The IL-2 concentration of culture supernatant was detected by bioassay using the CTLL-2 hybridoma (27). Cells (5000/50 µl medium) were incubated with 50 µl of samples or standards (recombinant human IL-2, Genzyme, Cambridge, MA) in triplicate for 24 h. [3H]thymidine (1 µCi/well) was added during the last 8 h of incubation, and [3H]thymidine incorporation was detected as described above. Specific IL-2 activity (units per milliliter) was calculated according to the standard curve of the identical assay.
Cytotoxicity assay
The cytotoxic activity of SEB-primed effector cells (splenocytes or colonic mucosal lymphocytes) was detected by the JAM assay (31) using Mode-K cells (duodenal epithelial cell line from C3H/HeJ origin (32)) as target cells. Effector cells from C3H/HeJ mice were stimulated with 10 µg/ml of SEB for 18 h, washed three times, and used as effector cells. In some experiments cells were pretreated with recombinant mouse IL-10 and/or anti-IL-10R Ab (1B1.2) for 2 h before addition of SEB. Mode-K cells were labeled with 10 µCi/ml of [3H]thymidine for 4 h, washed three times, and used as target cells. Labeled Mode-K cells (1 x 104) were added to each well of a 96-well flat-bottom plate and incubated with increasing numbers of effector cells up to a 40:1 E:T cell ratio. After 24 h cells were treated with trypsin/EDTA for 3 min and harvested onto a glass-fiber filter. The amount of tritium, which was retained only in the live cells, was counted by beta counter. Data are expressed as the percent survival and are calculated by dividing the counts per minute of samples with that of Mode-K cells only (average of 18 wells). In some experiments, supernatant from SEB-primed responder cells was checked for cytotoxicity. Culture inserts (0.4 mm pore size; Transwell, Corning Costar, Cambridge, MA) were also used between effector and target cells.
Statistical analysis
Paired Student’s t test (two tailed) was performed to compare the mean values of two corresponding datasets.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether activation of splenic T cells by a microbial
Ag could induce autoreactive responses, splenocytes from IL-10 KO
(C57BL/10) or wild-type mice were primed with SEB in vitro (Fig. 1
). The primary proliferative response to
10 µg/ml of SEB of splenocytes from IL-10 KO mice was comparable to
that of wild-type splenocytes. SEB-primed splenocytes from C57BL/10
mice showed a significant proliferative response after secondary
culture with mitomycin C-treated syngeneic feeder cells. The magnitude
of the secondary response in IL-10-deficient mice of the same strain
was comparable to that of wild-type mice. In contrast, medium-primed
splenocytes from wild-type mice did not respond to the secondary
stimulation with the syngeneic feeders, whereas those from
IL-10-deficient mice underwent a significant proliferation in response
to the syngeneic feeders, albeit less than that observed after priming
with SEB. Interestingly, 67.6 ± 5.2 U of IL-2 were produced in
the supernatant of SEB-primed splenocytes from IL-10-deficient mice,
which was higher than that in wild-type (46.1 ± 3.1). Adding
recombinant mouse IL-10 (10–100 ng/ml) to the culture suppressed the
IL-2 production of both IL-10 KO and wild-type splenocytes, whereas the
constitutive proliferative response observed in IL-10 KO mice was not
affected. These data indicate that priming with SEB induced
autoreactive proliferative responses, and IL-10-deficient cells tend to
be autoreactive without priming (Fig. 1).
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We examined the possibility that IL-10 regulates the induction of
the autoreactive response by SEB in several strains of mice.
Splenocytes from three different strains of mice were primed with SEB
in the presence or the absence of recombinant mouse IL-10, and
secondary autoreactive responses to the syngeneic feeder cells were
detected. Although the magnitude of the secondary response varied among
strains, IL-10 in the primary culture significantly inhibited the
autoreactive response in the 129 and CBA/J mice (Fig. 2A). We also examined whether
IL-10 inhibited the autoreactive response of SEB-primed cells (Fig. 2B). Adding IL-10 to the secondary culture of SEB-primed
cells suppressed the autoreactive response in all strains. This
inhibition was confirmed to be IL-10 specific, since the effect of
IL-10 was completely abrogated by addition of anti-IL-10R Ab, but
not by isotype control, in C57BL/10 and 129/SvEv strain. Interestingly,
this Ab was not effective in the CBA/J strain, suggesting heterogeneity
of the structure of IL-10R in CBA/J mice.
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C),
or mRNA, detected by RT-PCR (data not shown). Binding of IL-10 did not
change with stimulation. Polyclonal stimuli induce autoreactivity
As SEB, which stimulates about 20% of T cells, induced
autoreactivity, we examined the hypothesis that polyclonal stimuli in
general have a potential to induce autoreactivity. Splenocytes from
CBA/J mice were primed with SEB as well as anti-CD3 or PMA plus
ionomycin, and secondary responses to the feeder cells were detected.
All these polyclonal stimuli induced the secondary autoreactive
responses, and these responses were inhibited by IL-10 in the secondary
cultures (Fig. 3). Ag-specific
stimulation with OVA did not induce the secondary response (data not
shown).
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To determine the expansion of autoreactive T cells, we employed the model involving endogenous viral superantigen in mice. Mice that have the integration of Mtv-7 genome derived from mouse mammary tumor virus are designated Mls-1a (33, 34). Mls-1a mice, such as CBA/J, have only a marginal number of T cells bearing TCR Vß6, -7, and -8.1 chains, because these populations are depleted in the thymus due to the endogenous viral superantigen Mls-1a (33, 35). Expansion of these T cell populations directly indicates the expansion of autoreactive T cells in Mls-1a mice.
SEB-primed autoreactive T cell lines from CBA/J splenocytes were
established by growing cells with feeder cells in medium containing
IL-2. After several weeks of culture, TCR Vß-chain usage was
determined by flow cytometric analysis. More than 95% of the cells
expressed CD4 and TCR /ß (P. Ernst et al., unpublished
observations). Although freshly isolated splenocytes from CBA/J mice
contained only a marginal number of Vß7+ T
cells (2% of CD3+ cells), this population
increased to 12% in the T cells lines. T cells recognizing the
endogenous viral superantigen and bearing the Vß6 TCR were also
expanded in the T cell lines (Fig. 4).
Splenocytes from C3H/HeJ mice (Mls-1b) also had
more Vß6+ or Vß7+ T
cells after expansion in response to SEB.
Vß8.1+ T cells are depleted in
Mls-1a strains; however, we detected 9% of Vß8
cells in the freshly isolated splenocytes from both CBA/J and C3H/HeJ
strains, because the Ab also recognizes Vß8.2+
cells. Expansion of this population was also observed in the T cell
lines. In other experiments anti-Mls-1a Ab (33)
partially inhibited the secondary response of SEB-primed CBA/J cells,
suggesting that part of the autoreactive response was directed toward
the Mls-1a Ag itself (data not shown). These data
provide direct evidence that priming with superantigen abrogated self
tolerance and expanded autoreactive T cells in vitro.
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Because Th cells are capable of transferring disease in several
models of colitis, the role of CD4+ Th cells in
the secondary autoreactive response was determined. CD8-depleted cells
showed a comparable level of secondary response as the splenocyte
preparation (Fig. 5A).
Moreover, addition of anti-MHC class II Ab to the secondary
culture, but not an isotype control Ab, significantly blocked the
autoreactive response in all three strains examined (Fig. 5B). These observations suggest that
CD4+ T cells mediated the autoreactive response.
Adding IL-10 in the primary or secondary culture of CD8-depleted cells
suppressed the secondary response, indicating that the regulatory
effect of IL-10 was independent of the presence of
CD8+ T cells (Fig. 5A).
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To address the possibility that autoreactive T cells may be able
to contribute to epithelial cell damage and inflammation in the
digestive tract, Mode-K cells were used as a target for cell-mediated
immunity. Splenocytes from C3H/HeJ mice (syngeneic to Mode-K) were
stimulated with SEB for 18 h and checked for a cytotoxic effect by
JAM assay. Fig. 6A shows that
SEB-stimulated splenocytes as well as CD8-depleted cells mediated
cytotoxic activity against the Mode-K target cells. Supernatant from
SEB-stimulated splenocytes did not kill the target (data not shown).
Furthermore, the cytotoxic effect was abolished when a culture insert
was used to separate effector and target cells, indicating that this
cytotoxicity was facilitated by direct contact with the target
cells.
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B, SEB-activated colonic
mucosal T cells kill the syngeneic epithelial cell target. Moreover,
this cytotoxicity was inhibited by the addition of IL-10, and the Ab
recognizing the IL-10R (1B1.2) blocked the inhibition. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2
(42). Based on the results presented in this report, any
stimulation that activates a relatively large subset of T cells
generates autoreactive T cells that proliferate when re-exposed to self
Ags expressing class II MHC. Moreover, these autoreactive cells are
cytolytic to epithelial cells in vitro.
The autoreactive cells induced by SEB included
CD4+ T cells, because most of the primed cells
contained CD4+ TCRß+
phenotypes detected by FACS analysis. Depletion of
CD8+ cells before priming did not attenuate the
autoreactivity. Moreover, this autoreactivity was mediated by MHC class
II molecules on the feeder cells, because this reaction was inhibited
by anti-MHC class II Abs. These findings support the idea that the
autoreactive cells are CD4+ T cells. As activated
T cells may express only modest amounts of class II MHC, the T cells
are not capable of long term expansion in the absence of feeder cells.
Moreover, because SEB induces the expression of FasL on T cells, it is
likely that, in time, activation will lead to a negative selection of
the T cells as they die due to Fas/FasL interactions (43).
The magnitude of the proliferative responses varied among strains of
mice, but they were present in all strains tested. In addition,
autoreactive T cells could be generated from spleen or colon, which
supports our previous preliminary studies suggesting that these
responses occur in gut-associated lymphoid tissue, including the
mesenteric lymph nodes or intestinal Peyer’s patches
(44).
CD4+ autoreactive T cells prepared from the spleens of IL-10-deficient mice displayed a level of spontaneous autoreactivity without prior priming with SEB. Despite this intrinsic autoreactivity, stimulation with SEB did not increase the response to levels greater than those observed in control mice. This somewhat unanticipated finding may reflect a ceiling for autoreactivity that is defined by the number of autoreactive precursors and the magnitude of their expansion. It should be remembered that normally, autoreactive cells are almost nonexistent, so any increase may be quite significant. This is supported by the observation that adoptive transfer of these T cells into T cell-deficient mice can induce colitis in the recipient (24). These observations suggest that IL-10 confers a protective effect against the activation of autoreactive T cells induced by normal flora. Although there was no difference between IL-10-deficient and wild-type mice in the level of secondary autoreactive responses after priming with SEB, exogenous IL-10 suppressed the autoreactivity, including both proliferative and cytolytic effects, when delivered during the priming or secondary stimulation. IL-10R were expressed on both T cells and cells of other lineages, so the inhibitory activity of IL-10 may have been mediated at different levels.
The specificity of the effect of IL-10 was confirmed by blocking the IL-10R with anti-IL-10R Ab. This Ab was effective in the 129/SvEv and C57BL/10 strains, but not in the CBA/J strain. Interestingly, IL-10 inhibited IL-2 production in the C57BL/10 and 129SvEv strains after stimulation with SEB, but not in CBA/J mice (data not shown). Heterogeneity of cell surface molecule among mouse strains has been reported for CD8 (45), IL-4R (46), or FasL (47), which affects not only the recognition by mAbs but also the function of the molecules. Our observations suggest that there is heterogeneity in the structure and function of the IL-10R; however, further experimentation is necessary to precisely evaluate the basis for the inability of the Ab to inhibit IL-10 responses in CBA/J mice.
Regulation of autoreactivity in the priming phase by IL-10 is consistent with the finding that supplementation of IL-10 prevented the development of colitis in IL-10-deficient mice (48) or in the CD45RBhigh transfer model (22, 23). IL-10 also suppressed autoreactivity in the secondary culture. Endogenous IL-10 produced from feeder cells seemed to be insufficient to inhibit the autoreactivity, because secondary proliferative responses were similar whether the feeder cells were obtained from IL-10-deficient or control mice.
Although proliferation in response to syngeneic feeders provides evidence that SEB can abrogate tolerance, it is possible that the proliferative response was not directed to self Ags, but, rather, to residual SEB or some factor in the growth medium. The possibility that residual SEB in the primed cells was restimulating the responder cells was discounted for several reasons: 1) the responder cells contained only a marginal number of APC, which might carry over SEB into the secondary stimulation, because SEB induced only a limited proliferation to the responder cells without adding fresh feeder cells; 2) responder cells could be maintained for up to 12 wk after repeated washing and restimulation with syngeneic feeder cells, suggesting that little residual SEB was carried over; and 3) the autologous response was significantly inhibited by IL-10, but IL-10 did not inhibit the proliferative response caused by SEB. The fact that T cells from IL-10-deficient mice were capable of modest proliferative responses upon stimulation with syngeneic Ags without priming suggests that the responses were indeed autoreactive. Although it is possible that xenogenic proteins in the growth medium, such as those provided by FCS, may be presented by feeders, small amounts of proliferation were still observed when cultures were maintained in mouse serum (data not shown). However, the most direct evidence for the expansion of autoreactive cells was the expression of autoreactive T cell repertoires.
To address the affect of SEB on T cells that bear autoreactive repertoires, the well-characterized murine model of TCR repertoires to endogenous viral superantigens was employed. Mls-1 has been known to act as a minor lymphocyte-stimulating Ag. Subsequently, the Mls-1 Ag was shown to be encoded by the mtv-7 gene, which is a murine mammary tumor viral gene that has integrated into their genome of some strains of mice (34). These mice are referred to as Mls-1a mice, while mice lacking this Ag are Mls-1b. It is known that specific T cells in Mls-1b mice bearing specific TCR ß-chains (i.e., Vß6 and Vß7) proliferate in response to this Ag. Because the Mls-1 Ag is expressed on host cells, these T cells are deleted in Mls-1a mice. The results described in this report show that SEB could expand the number of T cells from Mls-1a mice that bear this autoreactive repertoire. Although most of these autoreactive T cells are deleted during T cell development, this process is imperfect, leading to the escape of potentially autoreactive clones. The intestinal intraepithelial lymphocyte compartment is particularly enriched with T cells expressing these autoreactive TCRs (49, 50). Because Mls-1 Ag can be expressed on intestinal epithelial cells (51), autoreactivity of T cells recognizing this Ag must be prevented by a state of anergy. However, cytokines, such as IL-2, can abrogate anergy in autoreactive IEL (29, 52). Zinkernagel and colleagues (53) have also shown that transgenic T cells expressing a receptor recognizing a transgenic viral peptide that is expressed on pancreatic ß islet cells remain anergic. However, upon stimulation with virus infection, the anergic T cells react to viral Ag and induce diabetes. Thus, presentation of a microbial Ag in an immunogenic fashion can induce autoreactivity that can contribute to disease. This may be due to antigenic mimicry between the pathogen and the host Ag or the activation of autoreactive T cells due to the burst of cytokines, such as IL-2, that are released during the infection.
Having established that autoreactive T cells can expand after exposure
to SEB, we showed that these cells were capable of inducing damage to
host cells. As described in Fig. 6, T cells from the spleen or colon
that were activated with SEB could induce death and DNA degradation in
Mode-K cells. Because SEB induces the expression of FasL on T cells
(43), it is possible that apoptosis of epithelial cells
was mediated by interactions between Fas and FasL. This model is
supported by the results of other studies documenting the expression of
Fas on epithelial cells and FasL by intestinal T cells in graft-vs-host
disease (54, 55). Previous reports have suggested that
changes in epithelial cell structure and function can occur after
exposing tissue to SEB (41, 56). The ability of SEB to
induce cytokines (42) as well as apoptosis in epithelial
cells following exposure to SEB may contribute to the T cell-mediated
pathology.
As described above, colitis in many animal models is dependent on normal flora, and T cells recognizing luminal microbial Ags become activated during colitis. Moreover, adoptive transfer of T cells from colitic mice will lead to colitis in T cell-deficient recipients. These studies suggest that microbial triggers activate a deleterious T cell response; however, a role for autoreactive T cells has not been implicated previously. In this report we show that a microbial superantigen, SEB, is capable of inducing autoreactive T cells in systemic and colonic tissue and that this response is prevented by IL-10. Additional studies are required to determine the relative contribution of this process to chronic inflammatory diseases in the digestive tract.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter B. Ernst, Children’s Hospital, Room 2.300, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0366. E-mail address:
3 Abbreviations used in this paper: SEB, staphylococcal enterotoxin B; KO, knockout; IEL, intestinal intraepithelial epithelial lymphocytes; FasL, Fas ligand.
Received for publication June 28, 1999. Accepted for publication January 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß-deficient mice fail to develop colitis in the absence of a microbial environment. Am. J. Pathol. 150:91.[Abstract]
ß TCR. Int. Immunol. 6:1467. CD8+ intraepithelial T cell receptor /ß+ lymphocytes reveals a major extrathymic pathway of T cell differentiation. J. Exp. Med. 173:483./ß in the intestinal epithelium: evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes. J. Exp. Med. 176:187.
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cells in the intestine. J. Exp. Med. 177:1755.This article has been cited by other articles:
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  P. Zhou, C. Streutker, R. Borojevic, Y. Wang, and K. Croitoru IL-10 modulates intestinal damage and epithelial cell apoptosis in T cell-mediated enteropathy Am J Physiol Gastrointest Liver Physiol, September 1, 2004; 287(3): G599 - G604. [Abstract] [Full Text] [PDF]
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 P. Brest, B. Mograbi, V. Hofman, A. Loubat, B. Rossi, P. Auberger, and P. Hofman Rho GTPase Is Activated by Cytotoxic Necrotizing Factor 1 in Peripheral Blood T Lymphocytes: Potential Cytotoxicity for Intestinal Epithelial Cells Infect. Immun., March 1, 2003; 71(3): 1161 - 1169. [Abstract] [Full Text] [PDF]
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) or C3H/HeJ (Mls-1b; 
) mice
were established and maintained in growth medium supplemented with IL-2
and feeder cells. Cells were double stained with CD3 and TCR
Vß-specific Abs and were analyzed by FACS. The percentage of specific
Vß-chain usage in CD3+ cells was calculated. Freshly
isolated splenic T cells (left end of each line)
recognizing SEB (Vß3, Vß8) in both strains of mice as well as Vß7
and Vß6 TCR+ in C3H/HeJ mice underwent expansion
(right end of each line). Vß7 and Vß6
TCR+ cells in freshly isolated splenic T cells from CBA/J
were depleted due to the expression of the endogenous viral
superantigen. However, the percentage of T cells bearing these
Vß-chains increased to 14 and 6%, respectively, after stimulation.
This observation provided direct evidence that priming with SEB
expanded autoreactive T cell populations.
