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Division of Gastroenterology and Hepatology and
Department of Pathology, University of Alabama, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA and
protein. Pathogenic Bir T cell lines expressed CD40 ligand (CD40L) when
cultured with Ag-pulsed APCs in vitro. Production of IL-12 was also
increased in such cultures, an effect that was Ag- and T cell-dependent
and required costimulation by CD40, but not by B7. The two Bir T cell
lines that did not induce lesions after transfer failed to
significantly express CD40L or increase IL-12 when cultured with
Ag-pulsed APCs. Administration of anti-CD40L blocked disease
expression induced by pathogenic T cells. We conclude that interactions
in the colon mucosa between CD40L-expressing Bir Th1 cells with APCs
endogenously loaded with commensal bacterial Ags are critical for
sustained increases in local IL-12 production and progression to
colitis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(2, 3, 4). Although
the deficiency of such molecules is global, inflammatory disease has
been localized mainly to the colon, suggesting a role for the enteric
bacterial flora in driving disease. Indeed, in multiple instances
disease is abolished under germfree conditions (2, 5, 6, 7, 8)
or is ameliorated by antibiotics (9) or by limited
bacterial colonization (10). The effector cell in most of
these new animal models is the TCRß+,
CD4+ T cell (11, 12, 13, 14). In most cases
these T cells produce high levels of IFN-. IL-12 has been shown in
some studies to play an important role in the initiation and
progression of disease (15, 16, 17, 18, 19). The exact mechanisms by
which the flora triggers pathogenic responses by
CD4+ T cells remains unknown in most models,
i.e., whether by nonspecific or Ag-specific stimulation. Some insights into the last question have come from studies in the C3H/HeJBir mouse, a new substrain that is highly susceptible to colitis (20). Under certain housing conditions, these mice can develop a spontaneous colitis that resolves over time. These mice have been previously shown to have increased Ab reactivity to commensal bacteria in the gut as well as increased CD4+ Th1 reactivity to enteric bacterial Ags (21, 22). CD4+ T cells from C3H/HeJBir mice stimulated with enteric bacteria-pulsed APCs induced colitis when transferred into histocompatible scid recipients. In this instance the stimulatory material in the enteric bacterial preparations was found to be conventional protein Ags of the commensal flora (22).
There are several mechanisms that direct the phenotype development of
responding CD4+ T cells toward IL-2- and
IFN--producing Th1 cells or toward IL-4- and IL-10-producing Th2
cells. For example, low Ag concentration (23) and the
presence of IL-4 (24) promote the development of Th2
cells. In contrast, the presence of IL-12, a heterodimeric cytokine
produced by APCs, has been shown to greatly enhance Th1 cell
development and IFN- production (25, 26, 27, 28). An important
role of IL-12 in experimental colitis has been demonstrated, e.g.,
treatment with anti-IL-12 mAb inhibited colitis in a
trinitrobenzenesulfonic acid (TNBS)-induced model (16), in
IL-10-/- mice, and in a
CD4+ CD45RBhigh T cell
scid transfer model (18, 19). Transfer of bone
marrow cells from STAT-4-deficient mice, in which T cells do not
respond to IL-12, into Tg
26 mice induced
milder disease compared with transfer of bone marrow cells from normal
mice (18), but the precise mechanisms that regulate the
production of IL-12 in enteric bacterial Ag-driven responses, and thus
drive disease development, are not yet known. Several recent studies
have demonstrated that the interaction of CD40 ligand
(CD40L)3 on activated
T cells with CD40 on APC leads to the secretion of IL-12 by APC,
including monocytes, dendritic cells, and macrophages, and that such
interactions are critical for IL-12 production in Ag-driven responses
(29, 30, 31).
For these reasons, the role of IL-12 production in the C3H/HeJBir
transfer model and the effects of CD40L-CD40 interactions were given
particular emphasis in this study. To this end, eight enteric bacterial
Ag-specific Bir T cell lines from colitic C3H/HeJBir mice have been
established. Six of the T cell lines expressed CD40L, stimulated IL-12
production by cecal bacterial Ag (CBA)-pulsed APCs in vitro, and
induced colitis when transferred into scid recipients,
lesions marked by increased IL-12 and IFN- production. The two Bir T
cell lines that did not express CD40L and did not stimulate IL-12
production did not induce colitis in scid recipients despite
their production of Th1 cytokines in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C3H/HeJBir, C3H/HeJ, and C3H/HeSnJ-Pkrdcscid/Pkrdcscid (scid) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in the University of Alabama animal facility. All studies were approved by the animal care and use committee of the University of Alabama at Birmingham.
Reagents and Abs
RPMI 1640, FBS, 2-ME, HEPES, L-glutamine, and sodium
pyruvate were purchased from Life Technologies (Long Island, NY). The
multiprobe RNase protection assay kit was purchased from PharMingen
(San Diego, CA). mAbs to CD3, CD4, CD8, ICAM-1,
ß7 integrin, CD40L, B7.1, B7.2, CD44,
L-selectin, TCRß, TCRVß2, Vß3, Vß6, Vß7, Vß8, Vß9,
Vß10, Vß13, and Vß14 were purchased from PharMingen.
Anti-I-Ab and anti-I-Ak
were purchased from American Type Culture Collection (Manassas, VA).
Hamster anti-mouse CD40L mAb was purified on a protein A affinity
column from culture supernatants of the hybridoma line MR1, which was a
gift from Dr. Brian Kelsoe (National Institutes of Health, Bethesda,
MD). Control hamster IgG of the same isotype was obtained from
PharMingen.
Preparation of enteric bacterial Ags
Enteric bacterial Ags were prepared as previously described (21). Briefly, C3H/HeJ or C3H/HeSnJ scid mice were sacrificed, and their ceca were removed. The ceca were opened and placed in 1 ml of PBS. Cecal bacteria were expelled by mixing with a vortex, and residual cecal tissue was removed. After addition of DNase (10 µg/ml), 1 ml of this bacterial suspension was added to 1 ml of glass beads. The cells were disrupted at 5000 rpm in a MiniBead Beater (BioSpec Products, Bartlesville, OK) for 3 min and then placed on ice. The glass beads and unlysed cells were removed by centrifugation at 5000 x g for 5 min. The lysates were filter sterilized through a 0.2-µm pore size syringe filter.
Establishment and maintenance of enteric bacterial Ag-specific CD4+ T cells
C3H/HeJBir spleen and mesenteric lymph nodes were removed from groups of four or five mice and placed into cell suspension by straining through a small mesh sieve as described previously (22). After two washes, the cells were passed through a nylon wool column to obtain T cells as previously described (32). The column-passed cells were washed twice and treated with anti-CD8 mAb (TIB 211, American Type Culture Collection) supernatant (1 ml of supernatant/1 x 107 cells) for 30 min on ice. After washing three times, magnetic beads coated with anti-rat IgM were added to the cells (BioMag, Cambridge, MA) and incubated for 30 min on ice. After passing through a magnet, CD4+ T cells were collected and reconstituted at 4 x 106 cells/ml in complete RPMI medium containing 10% FCS for use in cell culture.
APCs from the spleens of C3H/HeJ mice were prepared and treated with 200 µg/ml of enteric bacterial Ag at 2 x 107 cells/5 ml in a 15-ml tube overnight at 37°C. After washing twice, the cells were reconstituted at 4 x 106 cells/ml in complete medium containing RPMI 1640 10% FCS, 2 mM L-glutamine, 0.05 mM 2-ME, 100 U/ml penicillin and 10 µg/ml streptomycin for use in cell culture. These APCs were irradiated with 3000 rad before addition to T cell cultures.
T cells (4 x 106/well) and CBA-pulsed APCs (4 x 106/well) were cultured in 24-well plates. T cells were restimulated with freshly prepared Ag-pulsed APCs at intervals of 10–14 days. The resulting T cell lines were designated by the prefix Bir followed by a number denoting the order of their derivation.
Assay of Ag-specific proliferation of T cells
T cell lines generated as described above were cultured at 2 x 105 cells/well in triplicate in the presence of 2 x 105 Ag-pulsed APC/well in 96-well flat-bottom tissue culture plates (Falcon 3072, Lincoln Park, NJ) at 37°C in 5% CO2 humidified air. After 3 days of culture, 0.5 µCi of [3H]thymidine (New England Nuclear, Boston, MA) was added to each culture for the last 16–20 h of the incubation period. The cells were harvested on glass-fiber filters on a PHD cell harvester (Cambridge Technology, Watertown, MA). Proliferation was measured as the incorporation of [3H]thymidine into cell DNA, as measured by beta scintillation counting (Beckman, Palo Alto, CA) of the harvested samples. Data were expressed as the mean counts per minute ± SD.
Ex vivo organ culture
Ex vivo organ fragment cultures of colon and small intestine were performed by removing and longitudinally opening small and large intestines. After washing with cold RPMI containing 20 µg/ml of gentamicin three times, three 3-mm circular full thickness pieces of small intestine and colon were obtained using a 3-mm dermal punch (Baker-Cummins, Miami, FL). Each fragment was then placed in 0.5 ml of complete medium in separate wells of a 48-well plate and incubated for 24 h at 37°C in 5% CO2 humid air. Culture supernatant from each biopsy was collected and stored at -20°C before analysis for cytokine content.
Cytokine assays
T cell lines were cultured with Ag-pulsed APCs as described
above. Culture supernatants were collected at different times and
pooled for assay. The supernatants collected after 24 h of culture
were used for IL-2 assay, and the supernatants collected at 48–72 h of
culture were used for IL-4, IL-10, and IFN- assays. For IL-12
production, C3H/HeSnJ scid spleen APC were cultured with T
cell lines under different conditions as described in text, and the
supernatant was collected at 48 h of culture.
The cytokine content in supernatants was determined by ELISA. Briefly,
96-well polystyrene microtiter plates (Dynatech, Rockville, MD) were
coated with primary anti-cytokine capture mAb (2 µg/ml)
overnight. After an incubation with sample supernatant or cytokine
standards, the wells were incubated with biotinylated detection
anti-cytokine mAb, followed by an incubation with HRP-avidin.
Finally, 50 µl of tetramethylbenzidine was added to each well, and
the color development after incubation at room temperature was measured
at OD450 in an EL 312e ELISA reader (Bio-Tek
Instruments, Winooski, VT). A standard curve was constructed for each
assay, and the values of the samples were interpolated using the
Deltasoft III program (BioMetallics, Princeton, NJ). All mAb pairs were
purchased from PharMingen and are listed by capture/biotinylated
detection mAb as follows: IL-2, JES6-1A12/JES6–5H4; IL-4,
BVD4-1D11/BVD6-24G2; IL-10, JES5-2A5/SXC-1; IFN-, R4-6A2/XMG1.2; and
IL-12, C17.8/C15.6.
Flow cytometric analysis
Flow cytometric analysis of T cell lines was conducted as described previously (33). In brief, T cell lines were harvested from plates 8–10 days after restimulation, and dead cells were removed by centrifugation over Ficoll. After washing by PBS with 0.1% sodium azide plus 2% heat-inactivated newborn calf serum, the cells were incubated with various FITC- or PE-conjugated mAbs, washed, and fixed in 1% buffered paraformaldehyde. 104 stained cells were analyzed using a FACStar flow cytometer (Becton Dickinson, Mountain View, CA). An FITC- or PE-labeled mAb of the same isotype but irrelevant specificity was used as a negative control in all experiments.
Immunoscope analysis
Details of the method have been published (34, 35).
Briefly, total RNA was isolated from viable cells of the Bir cell lines
using RNeasy miniprep columns according to the manufacturer’s
instructions (Qiagen, Santa Clarita, CA). cDNA was synthesized from 10
µg of total RNA using (dT)15 primer and 10 U of
avian myeloblastosis virus (AMV) reverse transcriptase (Roche,
Indianapolis, IN) in the presence of 25 U of RNasin (Promega, Madison,
WI). The TCRBC- and TCRBV-specific primers used in the PCR were
previously described (36). All PCRs were performed using
25 U/ml Taq polymerase, dNTPs (0.2 mM each), primers (0.5
µM each), and 
10 µg of reverse transcribed RNA in a 25-µl
final volume. Forty cycles of PCR amplification were performed, each
consisting of 45 s at 94°C, 45 s at 60°C, and 45 s
at 72°C, following a hot start of 30 s at 94°C. An aliquot of
the resulting PCR products was subject to a run-off reaction in which
one to five cycles of further amplification were performed under
similar conditions as the PCR, but substituting a single TCRBC
dye-labeled primer at 0.1 µM (34). The run-off products
were loaded on a 6% polyacrylamide sequencing gel and electrophoresed
on an ABI-373 DNA sequencer (Perkin-Elmer, Emeryville, CA) together
with size standards. The data were analyzed with Immunoscope software
developed by C. Pannetier (35).
Cell transfer and evaluation of colitis
For control CD4+ T cell transfers, 2 x 106 freshly prepared or anti-CD3-activated CD4+ T cells from spleen and mesenteric lymph nodes of C3H/HeJ or C3H/HeJBir mice were transferred i.v. into C3H/HeSnJ scid recipients. For CD4+ Bir T cell line transfers, the T cell lines were harvested 5 days after restimulation with CBA-pulsed APCs, and dead cells were removed by centrifugation over Lymphocyte-M (Cedarlane, Hornby, Canada). Then, 2 x 106 viable T cells were transferred i.v. into C3H/HeSnJ scid recipients. Three months later, the recipients were killed, and the cecum and the proximal, middle, and distal portions of the colon were fixed in formalin. If animals looked ill and had lost >20% of initial body weight, they were killed at that point. In that instance one mouse from the control group was also sacrificed at the same time to make the comparison equivalent. Fixed tissues were embedded in paraffin, and sections were stained with hematoxylin and eosin for histologic examination. All slides were read by an experienced pathologist (A.L.) without knowledge of their origin.
In certain experiments pathogenic Bir T cells were transferred into C3H/HeSnJ scid recipients. The recipients received 200 µg of either anti-mouse CD40L mAb or control IgG at the time of cell transfer and then weekly until sacrifice.
RNase protection assay (RPA)
RPA was conducted using the RiboQuant multiprobe template set according to the instructions provided by PharMingen. Briefly, RNA was isolated from tissues using the guanidinium isothiocyanate method (37). Next, 10 µg of RNA from the colon tissues of scid recipient mice was dissolved in hybridization buffer, after which 2 µl of a 32P-labeled RNA probe set was added. The solution was overlaid with mineral oil and incubated at 56°C for 12–16 h. Unhybridized probes and RNA were digested with an RNase mixture containing RNase A and RNase T1 in 100 µl of digestion buffer for 45 min at 30°C. The enzymes were inactivated by proteinase K for 15 min at 37°C. Digested samples containing nondegraded probe-target mRNA hybrids were extracted with phenol-chloroform, precipitated with ethanol, dissolved in loading buffer, and electrophoresed on a 5% polyacrylamide-urea sequencing gel. The wet gel was placed on photographic film overnight at -80°C, then the film was developed.
Statistics
The results were expressed as the mean ± SD. The significance of the difference between means was determined by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies indicated that C3H/HeJBir mice, a substrain
of C3H/HeJ that can develop colitis spontaneously, have a strong
CD4+ T cell response to Ags of the commensal
bacterial flora (22). To further characterize such enteric
bacterial Ag-specific T cells, T cell lines specific for enteric
bacterial Ags were established by repeated stimulation of purified
CD4+ T cells from spleen and mesenteric lymph
nodes of C3H/HeJBir mice with APCs pulsed with lysates of enteric
bacteria obtained from the cecum. Eight T cell lines were generated at
different times over a 1-yr interval, and each was maintained in
continuous culture with CBA-pulsed APCs for over 6 mo. To determine the
Ag specificity of CBA-specific Bir T cell lines, we assessed their
ability to proliferate when stimulated with CBA, food, and epithelial
or unrelated Ags. None responded to CBA in the absence of APC or in the
presence of unpulsed APC. All strongly responded to CBA-pulsed APCs,
but not to APCs pulsed with epithelial cell, food, or unrelated protein
Ags such as OVA (Table I
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Phenotypic characterization of Bir T cell lines
To define the phenotype of the CBA-specific T cell lines, Bir T
cells were harvested 8–10 days after restimulation with CBA-pulsed
APCs, stained with mAb to various surface membrane molecules, and
analyzed by flow cytometry. All T cell lines were
CD4+, TCRß+ and
expressed high levels of CD44 and ICAM-1 and low levels of L-selectin
and CD69. They also expressed CD25, the high affinity IL-2R.
Interestingly, even after long-term maintenance in culture, a
significant fraction of the cells expressed high levels of CD45RB Ag.
ß7 integrin was expressed by 30–60% of cells
in the different T cell lines, compatible with cells able to home to
mucosal sites. Fig. 1 shows the flow
cytometric profile of Bir 3 T cells as a representative example.
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To measure the cytokine production of Bir T cell lines stimulated
with CBA-pulsed APCs, culture supernatants were collected at different
times after Ag restimulation, and IL-2, IL-4, IL-10, and IFN-
production was measured by cytokine-specific ELISA (see Materials
and Methods). As shown in Table II,
although different T cell lines produced varying amounts of cytokines
in response to CBA stimulation, all T cell lines produced relatively
high levels of IL-2 (97–222 U/ml) and IFN- (82–282 U/ml) and
relatively small amounts of IL-4 (11–32 U/ml) and IL-10 (11–31 U/ml),
indicating that all eight Bir T cell lines were Th1 predominant.
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To determine the TCR Vß usage by the Bir T cell lines, cells
were stained with different anti-TCR Vß Abs and analyzed by flow
cytometry. In addition, TCR repertoire was assessed by Immunoscope, a
PCR-based CDR3 length display analysis. At the time of these analyses,
the cell lines had been in culture for at least 6 mo. Compared with
fresh splenic CD4+ T cells, Bir T cell lines used
a restricted set of TCR Vß families (Fig. 2). Six Bir T cell lines (Bir 3, Bir 4,
Bir 5, Bir 6, Bir 7, and Bir 9) used predominantly TCR Vß6, -8, and
-10. Bir 1 and Bir 2 lines used mostly TCR Vß10 (>95% of the total
cells). These lines thus use the same TCR Vßs that are predominant in
the C3H/HeJBir colon lamina propria (Fig. 2). Using Immunoscope, fresh
splenic CD4+ T cells demonstrated a polyclonal
TCRVß CDR3 pattern as expected (Fig. 3). In contrast, the Bir 3 and Bir 4 cell
lines had a pauciclonal TCRVß CDR3 phenotype (Fig. 3) despite being
stimulated with an unselected mixture of thousands of enteric bacterial
Ags. Other Bir lines had a pauciclonal phenotype as well, consistent
with a response to a restricted number of immunodominant Ags.
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To investigate the ability of these CBA-specific-Bir T cell lines
to induce colitis, 2 x 106 cells from
different Bir T cell lines were separately transferred into groups of
four histocompatible C3H/HeSnJ scid recipients 5 days after
restimulation with CBA-pulsed APCs. Three months after cell transfer,
the recipients were sacrificed, and the histopathology of cecum and
proximal, middle, and distal portions of colon and small intestine was
examined. All recipients of six T cell lines, Bir 3, Bir 4, Bir 5, Bir
6, Bir 7, and Bir 9, developed colitis (Table II
and Fig. 4). The lesions in the recipients were
focal and localized to the cecum and proximal colon, similar to the
pattern seen in the donor C3H/HeJBir mice. The small intestines of
these recipients were not inflamed. Transfer of Bir 1 and Bir 2 cells
did not induce colitis, although both lines reconstituted gut lymphoid
tissue based on histological analysis. Transfer to scid mice
of either fresh C3H/HeJBir CD4+ T cells or
C3H/HeJBir CD4+ T cells activated in vitro with
anti-CD3 mAb did not result in colitis in any recipient (Table II
and data not shown). Cotransfer of Bir 1 and Bir 2 with pathogenic Bir
T cell lines did not inhibit disease caused by the latter (data not
shown).
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expression in colon lesions
To investigate cytokine production in the colonic lesions of Bir T
cell recipients, cytokine mRNA expression was measured by a multiprobe
RPA. Representative data are shown in Fig. 5. IL-12p40 and IFN- mRNA expression
was increased, as were IL-1, IL-1ß, and IL-6 mRNA in the colons of
mice receiving pathogenic Bir T cell lines. In contrast, none of these
cytokine mRNA was increased in the colon of the recipients of Bir 1 T
cells, Bir 2 T cells, and either fresh or anti-CD3-activated
C3H/HeJBir CD4+ T cells, none of which developed
colitis (Fig. 5 and data not shown).
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levels were measured by ELISA. Colon from Bir 3 T cell recipients
produced high levels of IL-12p40. In contrast, colon from recipients of
anti-CD3-activated CD4+ T cells did not
produce detectable IL-12p40, nor did colon from scid mice
that received no T cells (Fig. 6 and data
not shown). Colon of Bir 3 T cell recipients, but not that of
recipients of anti-CD3-activated CD4+ T cells
or of naive scid mice, also produced detectable IFN-
(Fig. 6 and data not shown). No small intestinal cultures for any group
produced detectable IL-12 or IFN- after overnight culture.
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To investigate the mechanism of the increased IL-12 production
driven by Bir T cells, IL-12p40 production by APCs interacting with
pathogenic Bir T cell lines was assessed in vitro. C3H/HeSnJ
scid spleen APCs were incubated overnight with or without
CBA, then cultured with various Bir T cells. Culture supernatants were
collected after 48 h, and IL-12p40 production was measured by
ELISA. As shown in Fig. 7A,
high levels of IL-12p40 were produced in cocultures containing
CBA-pulsed APCs, and each of the six pathogenic Bir T cell lines (Bir
3, Bir 4, Bir 5, Bir 6, Bir 7, and Bir 9). No detectable IL-12p40 was
produced by APCs in the absence of Bir T cells regardless of whether
these APCs were pulsed with CBA (Fig. 7B). Notably, APCs did
not produce detectable IL-12p40 in the presence of Bir T cells and the
absence of CBA (Fig. 7B), indicating that APC IL-12p40
production is both Ag and T cell dependent. In contrast, CBA-pulsed
APCs cocultured with nonpathogenic Bir 1 or Bir 2 T cell lines produced
very low levels of IL-12p40 compared with the pathogenic Bir T cell
lines under the same conditions (Fig. 7A).
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To determine whether CD40-CD40L interactions between CBA-pulsed
APC and Bir T cells were involved in APC IL-12p40 production,
anti-CD40L mAb MR1 was added to the cultures. As shown in Fig. 7B, addition of anti-CD40L inhibited IL-12p40 production
by 80–90%. Anti-CD40L also inhibited IFN- production by Bir 3 T
cells (data not shown). To examine whether the anti-CD40L mAb
inhibition of IL-12p40 production was mediated by decreased IFN-
production by Bir T cells, anti-IFN- mAb was added to cultures
of Bir 3 T cells and CBA-pulsed APCs. Addition of anti-IFN- mAb
inhibited IL-12p40 production by about 30–40% (data not shown), but
never reached the degree of inhibition seen with anti-CD40L,
indicating that CD40-CD40L interactions were directly involved in the
induction of IL-12p40 production. Addition of anti-B7.1 (CD80) and
anti-B7.2 (CD86) mAbs, which block B7-CD28 costimulation, had no
effect on IL-12 production, but did inhibit the Bir 3 T cell
proliferation stimulated by CBA-pulsed APCs (Fig. 7B).
Interestingly, addition of anti-CD40L had no effect on Bir 3 T cell
proliferation (Fig. 7B).
Because the nonpathogenic Bir 1 and Bir 2 T cell lines stimulated only
low levels of IL-12p40 in vitro, we examined their expression of CD40L
after CBA-pulsed/APC activation using flow cytometry. Eight hours after
Ag stimulation, 31% of Bir 3 T cells expressed CD40L on the surface
(Fig. 8A), whereas only 2–3%
of the nonpathogenic Bir 1 or Bir 2 T cells expressed CD40L (Fig. 8B). To determine whether the low level of CD40L expression
on Bir 1 and Bir 2 T cells was due to differences in the kinetics of
expression, CD40L expression on Bir 1 and Bir 2 T cells was measured at
4, 8, 12, 24, and 48 h after Ag stimulation. Bir 1 and Bir 2 T
cells expressed low levels of CD40L at all these time points; these
cell lines were also deficient in CD40L expression after activation by
CD3 mAb (data not shown).
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To directly test the role of CD40L-CD40 interactions in the
pathogenesis of colitis in vivo, anti-CD40L was administered to
C3H/HeSnJ scid recipients of a pathogenic Bir T cell line. A control
group received polyclonal IgG. All four recipients in the control group
developed colitis at 10 wk after transfer (Fig. 9). None of the four recipients of
anti-CD40L mAb developed colitis, although
CD3+ T cells were clearly present in the colon
based on immunohistochemical stain.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in
vitro upon Ag stimulation. Transfer of C3H/HeJBir
CD4+ T cells after activation by enteric
bacterial Ag, but not after activation by anti-CD3, into
histocompatible scid mice resulted in colitis in the
majority of the recipients (22). This result prompted
derivation of multiple C3H/HeJBir CD4+ T cell
lines reactive to enteric bacterial Ags, so that their properties and
mechanism of disease induction could be studied. These Bir T cell lines
are specific for enteric bacterial Ags in a MHC class II-dependent
manner. Although generated at different times from different donors,
the cell surface phenotype of these T cell lines was remarkably
uniform: CD4+, CD8-,
TCRß+, L-selectinlow,
CD44high, CD69low. There
was a variable expression of CD25, but 30–80% of the cells in various
lines are positive for the high affinity IL-2R. Similarly, all these
cell lines expressed the ß7 integrin, which
forms part of the homing receptor for mucosal trafficking. There is
also a variable expression of CD45RB, with each line containing cells
expressing both high and low levels of this marker. Flow cytometry was
used to determine the TCR usage by the different lines. From this
analysis it is clear that there is a predominant expression of Vß6,
-8, -10, and -14 (Fig. 2), which are the same TCRBVs predominant in the
colon lamina propria of C3H/HeJBir mice. All Bir T cell lines maintain
a Th1-predominant cytokine phenotype in long-term cultures in vitro,
similar to the cytokine phenotype identified previously in freshly
obtained C3H/HeJBir CD4+ T cells activated by
CBA-pulsed APCs (22).
In a previous study of the Ab response to enteric bacterial Ags of the
C3H/HeJBir mouse, we found the response to be highly selective,
targeting a very small number of the large numbers of protein Ags
present in the enteric bacteria (21). Because the Bir T
cell lines are stimulated by freshly obtained enteric bacterial Ags,
which contain a myriad of different proteins, a polyclonal T cell
response would be expected. We used the Immunoscope technique, which
provides a more sensitive analysis of the TCR repertoire than is
possible by flow cytometry. All the Bir T cell lines were pauciclonal
rather than polyclonal. Most of the TCR peaks were individual to the
different cell lines (compare Bir 3 and Bir 4 in Fig. 4); however,
there were some TCR that were shared among lines, suggesting the
existence of public specificities in the T cell antibacterial response
(38). The most dramatic example of such a public
specificity came from analysis of the Bir 1 and Bir 2 cell lines, which
both use a Vß10, Jß2.7 chain with a CDR3 length of eight residues.
Sequencing of the CDR3 region of these two lines has demonstrated that
they use the same TCR ß-chain even though they were generated from
different mice at different times. This fits the definition of a public
specificity TCR response and is consistent with a T cell response to
the same immunodominant enteric bacterial Ag. The pauciclonal Bir T
cell response is consistent with the highly selected Ab response
identified in the previous study (21). Indeed, the same
immunodominant Ags are probably triggering both B cells and T cells,
because depletion of CBA preparations by immunoprecipitation with serum
Ab reduces the CD4+ T cell response by 70% (data
not shown).
The transfer of six of the Bir T cell lines to histocompatible
scid mice induced colitis in all recipients (Table II). The
resulting colitis was focal and transmural, similar to that previously
shown after transfer of C3H/HeJBir CD4+ T cells
activated by CBA-pulsed APCs in short term cultures. The reasons for
the focality of lesions are not yet known, but may relate to the
pauciclonal TCR used by the lines and/or the presence of relevant
bacterial Ags in high concentrations in certain niches in the colon.
Most lesions are found in the cecum and proximal colon, which is where
the bulk of the bacterial Ag is present. There were two notable
exceptions to the uniform induction of colitis after transfer of these
Th1-predominant cell lines. Transfer of the Bir 1 or Bir 2 cell lines
did not cause colitis in scid recipients, although they
clearly repopulated the colon lamina propria as seen on histology.
These lines did not inhibit colitis when cotransferred with one of the
pathogenic Bir T cell lines. As mentioned above, the Bir 1 and Bir 2
cell lines were nearly clonal and shared the same TCR ß-chain, but
otherwise they were indistinguishable from the pathogenic cell lines,
e.g., producing substantial amounts of IFN- in vitro. Although these
two lines had a much more limited TCRBV expression, the Ags triggering
them were presumably immunodominant, and thus the reasons for their
lack of pathogenicity remained puzzling.
Analysis of the local cytokine production in the colon of
scid recipients of pathogenic Bir cell lines demonstrated a
pattern consistent with a Th1-mediated disease. Both IFN- and IL-12
were increased, as measured by mRNA expression determined by RPA and by
cytokine protein levels found in ex vivo organ explant cultures of
affected colon. In addition, IL-1ß, IL-1, and IL-6 were elevated.
IL-12 has been implicated as playing a central role in colitis
induction and progression in several other animal models, including
trinitrobenzenesulfonic acid-ethanol-induced colitis (16),
IL-2-deficient mice (17), IL-10-deficient mice
(19), and bone marrow-reconstituted
Tg26 mice (18). Recent data
indicate that IL-12 may play a role in progression of disease
independently of IFN- by mechanisms that remain obscure
(19). The results from the present study demonstrate an
important role for IL-12 in the C3H/HeJBir mouse as well. Recent
reports indicate that IL-12 production by APCs requires CD40-CD40L
interactions (29, 30, 31). Accordingly, we tested the
interactions between pathogenic Bir cell lines with enteric bacterial
Ag-pulsed APCs in vitro, specifically asking what costimulatory signals
were required in this interaction. Interestingly, different
costimulatory signals were involved in IL-12 production compared with T
cell expansion. Blocking CD40-CD40L interaction substantially inhibited
IL-12 production by APCs, but had no effect on Bir
CD4+ T cell proliferation, whereas blocking
CD28-B7 interactions had no effect on IL-12 production, but
substantially inhibited Bir CD4+ T cell
proliferation. In regard to the latter, anti-B7.1 had more effect
than did anti-B7.2, but the combination of the two was consistently
superior to either alone, suggesting a role for both B7.1 and B7.2 in
the expansion of pathogenic Bir cell lines both in vitro and in
vivo.
Although these studies were performed in vitro, the local increase in
IL-12 mRNA and production in the colon of scid recipients of
pathogenic Bir T cell lines argues strongly that the same interactions
occur in vivo between transferred Bir CD4+ T
cells and local APCs in the colon mucosa. Because no IL-12 or IFN-
is produced in the absence of Ag, i.e., in cultures of
CD4+ T cells with naive APCs, it follows that
disease following transfer with pathogenic Bir T cells requires
interaction with APCs in the colon that are primed naturally with
enteric bacterial Ags (39, 40). Further support for this
idea comes from additional studies performed on the nonpathogenic, but
Th1-predominant, Bir 1 and Bir 2 cell lines. Analysis of the
interactions of these cell lines with Ag-pulsed APCs in vitro revealed
that these lines only weakly expressed CD40L and stimulated little or
no IL-12 production by APCs. The former was not due to insufficient
activation by Ag, in that the same result occurred after anti-CD3
activation of the Bir 1 and Bir 2 cell lines. Moreover, the Bir 1 and
Bir 2 T cell proliferative responses to enteric Ag-pulsed APCs are
comparable to those of the pathogenic T cell lines. The Bir 1 and Bir 2
T cell lines also fail to increase IL-12 or IFN- mRNA in the colon
after transfer to scid recipients. The inability to
up-regulate CD40L by the Bir 1 and Bir 2 cell lines appears to explain
their lack of pathogenicity in vivo. If so, then blocking CD40L-CD40
interactions between a pathogenic Bir T cell line and endogenous APCs
in vivo should prevent the development of colitis. Indeed,
anti-CD40L treatment of recipients of the pathogenic Bir 3 cell
line did prevent lesions from developing. These results indicate that
CD40L-up-regulation and stimulation of IL-12 production in the mucosa
are pivotal events in the pathogenesis of colitis mediated by
bacteria-reactive CD4+ T cells and support
previous evidence of this in a hapten-mediated system
(41).
These studies clearly demonstrate that CD4+ T cells directed at commensal bacterial Ags can cause chronic inflammatory bowel disease. Significantly, these CD4+ Bir T cell lines are derived from C3H/HeJBir mice at 3 mo of age, at a time when the mice themselves have recovered from colitis. The reason for the lack of disease despite the presence of potentially pathogenic T cells in vivo is probably due to the activity of regulatory T cells that prevent expression of the pathogenic T cell program in the bowel. The identity of such regulatory T cells is unclear, but candidates include Th2, Th3, and Tr1 cells. The lack of colonic disease in normal mice despite the huge antigenic load in the colon indicates a tight regulation of the mucosal immune response to these Ags (1). Perturbation of this regulation is the likely explanation for the colitis developing in a number of gene-targeted knockout mice. Indeed, a state of tolerance to Ags of the enteric bacterial flora may exist in normal hosts (42, 43), but how such tolerance to living organisms is induced and maintained remains unknown. However, the present studies give support to this idea by demonstrating the detrimental consequences of nontolerized, unregulated CD4+ effector T cells in the colon mucosa.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Charles O. Elson, Division of Gastroenterology and Hepatology, University of Alabama, 633 Zeigler Research Building, 703 South 19th Street, Birmingham, AL 35294-0007.
3 Abbreviations used in this paper: CD40L, CD40 ligand; CBA, cecal bacterial Ag; RPA, RNase protection assay.
Received for publication August 4, 1999. Accepted for publication June 5, 2000.
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