By
From the Immunopathology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114
The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-/
mice was explored by creating double mutant mice (TCR-
/
× immunoglobulin (Ig)µ
/
),
which lack B cells. TCR-
/
× Igµ
/
mice spontaneously developed colitis at an earlier age,
and the colitis was more severe than in TCR-
/
mice. Colitis was induced in recombination-activating gene-1 (RAG-1
/
) mice by the transfer of mesenteric lymph node (MLN) cells
from TCR-
/
× Igµ
/
mice. When purified B cells from TCR-
/
mice were mixed
with MLN cells before cell transfer, colitis did not develop in RAG-1
/
mice. Administration
of the purified Ig from TCR-
/
mice and a mixture of monoclonal autoAbs reactive with
colonic epithelial cells led to attenuation of colitis in TCR-
/
× Igµ
/
mice. Apoptotic
cells were increased in the colon, MLN, and spleen of TCR-
/
× Igµ
/
mice as compared
to Igµ
/
mice and TCR-
/
mice. Administration of the purified Ig from TCR-
/
mice
into TCR-
/
× Igµ
/
mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs
(autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.
Although autoantibodies (autoAbs) contribute to the
pathogenesis of certain autoimmune diseases such as
autoimmune hemolytic anemia and Graves' disease (1),
their role in disease such as ulcerative colitis (UC)1 is unknown (4). Recently, various animal models have been established to investigate the pathogenesis of human inflammatory bowel disease (IBD) (7). These animal models suggest the importance of CD4+ T cells or CD45RBhigh
CD4+ T cells and Th1 cytokines in the pathogenesis of
colitis (9). The spontaneous chronic colitis of IL-2- and
IL-10-deficient mice develops even when these mice are
made deficient in B cells by crossing them with Igµ TCR- The present study was designed to investigate the role of
B cells and autoAbs in the pathogenesis of colitis in TCR- Mice.
TCR- Flow Cytometry.
2 × 105 cells obtained from MLNs and
spleen were blocked by the incubation in FACS buffer (0.1% sodium azide and 0.2% BSA/PBS) containing 10% of normal rat
and hamster serum and 0.5 µg/2 × 105 cells CD16/CD32. After
washing with FACS buffer, cells were stained using anti-CD3 Histological Examination.
Specimens obtained from the distal,
middle, and proximal colon were fixed in 10% buffered formalin
and stained with hematoxylin and eosin. The severity of colitis
was determined according to the diagnostic criteria previously
described (17, 26).
Detection of Proliferative and Apoptotic Cells.
For labeling of the
proliferative cells, 5-bromo-2/
mice (15, 16).
/
mice also spontaneously develop chronic
colitis by 3-4 mo of age. The disease shares many features
with human UC (17) including restriction of the inflammation to the colon and a Th2-predominant cytokine profile (18). Furthermore, a negative association between incidence of appendectomy and development of UC in
human is supported by the lack of colitis in TCR-
/
mice after appendectomy (resection of cecal patch; reference 22). TCR-
/
mice harbor a unique population of
peripheral T cells (TCR-
+) that express TCR-
chains
without TCR-
or pre-T cell receptor
(pT
) chains on
the cell surface (18, 19, 23). The lack of regulatory
TCR-
+
+ T cells is associated with the presence of an
expanded population of B cells (80% of mesenteric LN
[MLN] cells are B cells [CD3
B220+ CD23+]) and increase
in production of autoAbs including anti-neutrophil cytoplasmic antibodies (ANCA) and antitropomyosin in TCR-
/
mice (22, 27, 28). These findings have raised the possibility that B cells, in particular autoAbs, may be involved
in the pathogenesis of colitis in TCR-
/
mice (7, 8, 17).
/
mice by creating double mutant (TCR-
/
× Igµ
/
) mice lacking B cells. The results suggest that although B cells are not required for the initiation of colitis,
B cells and Igs (autoAbs) can contribute by suppressing
colitis, presumably by affecting the clearance of apoptotic
cells and the related self Ags in TCR-
/
mice.
/
(23) and Igµ
/
(Igh 6 mutant) mice (29)
of C57BL/6 strain (H-2b) background were purchased from The
Jackson Laboratory (Bar Harbor, ME), crossed to generate the
double mutant (TCR-
/
× Igµ
/
) mice, and maintained under pathogen-free conditions at Massachusetts General Hospital
(Boston, MA). To distinguish heterozygous from homozygous mice,
pairs of three primers were used in PCR using tail DNA: KO1
(5
-TGCCTGTTCACCGACTTTGA), KO2 (5
-TGAACT-GGGGTAGGTGGCGT; reference 34), and pgk-neo (5
-CACCAAAGAACGGAGCCGGTT) for screening of C
locus, and
5
µM (5
-CTCTGTAAGGAGTCACCACC), 3
µM (5
-AAGCCTTCCTCCTCAGCATTC), and neoTK (5
-ATTCGGGAATGACAAGACGCTGG; reference 33) for screening of Cµ locus.
After screening by PCR, the nature of TCR-
/
× Igµ
/
mice was reconfirmed by immunophenotypic analysis of lymphocytes by FACScan® (Becton Dickinson, Mountain View, CA).
(145-2C11)-FITC (Boehringer Mannheim, Indianapolis, IN) or
B220 (RA3-6B2)-FITC and TCR-
(H57-597), TCR-
(GL3),
or Igµ-PE (PharMingen, San Diego, CA) at 4°C for 30 min. After
washing with FACS buffer, cells were analyzed on FACScan®.
-deoxyuridine (BrdU) (Sigma
Chemical Co., St. Louis, MO) was injected intraperitoneally (100 µg/g) 1 h before killing. In vivo BrdU-incorporated epithelial cells were detected by anti-BrdU mAb (Sera Lab, Crawley Down, England), followed by staining with avidin-biotinylated peroxidase complex method, and counted as previously described (22, 26).
20°C. After blocking endogenous peroxidase activity by 0.5% H2O2, the sections were rinsed with PBS
and immersed in terminal deoxynucleotidyl transferase (TdT)
buffer (30 mM Tris [pH 7.2], 140 mM sodium cacodylate, 1 mM
cobalt chloride). The sections were incubated with 2-4 mM biotinylated dUTP (Boehringer Mannheim) and 5-10 U TdT
(Promega, Madison, WI) in TdT buffer at 37°C for 2 h. After
terminating reactions by Tris-borate buffer, apoptotic cells were
detected by staining with avidin-biotinylated peroxidase complex
method. Apoptotic cells in the spleen were estimated by counting
the numbers of apoptotic cells in the entire frozen tissue sections
of spleen and expressing the counts as apoptotic cells per mm2.
Cell Transfer Studies.
MLN cells extracted from TCR-/
mice (8 or 20 wk of age) and TCR-
/
× Igµ
/
mice (8 wk
of age) were intraperitoneally transferred into RAG-1
/
mice
(5-6 wk of age), which were killed 8 wk after cell transfer. In
some experiments, B cells were partially depleted by the panning method using anti-Ig (30 µg/ml)-coating plates as previously described (18). Purified B cells from MLNs of TCR-
/
mice
were obtained by negative sorting using a mixture of biotinylated mAbs (anti-CD4 [RM4-5], CD5 [57-7.3], TCR-
[GL3], NK-1.1 [PK136], and Mac-1 [M1/70] from PharMingen [San Diego,
CA]), followed by incubation with streptavidin microbeads on
magnetic cell sorting system (Miltenyi Biotec Inc., Auburn, CA).
Administration of Ig or mAbs (AutoAbs) into TCR-/
× Igµ
/
Mice.
For Ig transfer, Ig was purified from sera pooled from 160 TCR-
+/
mice (6-15 wk of age) or 230 TCR-
/
mice (6-
12 wk of age) on a protein A affinity column, dialyzed, and concentrated. Monoclonal antibodies (autoAbs) capable of binding to
colonic epithelial cells were generated by the fusion of NS-1 cells
with B cells from MLNs of untreated TCR-
/
mice using polyethylene glycol (Sigma Chemical Co.) as previously described
(32). After screening by immunohistochemical staining and
ELISA using colonic epithelial cells of recombination-activating gene-1 (RAG-1
/
) mice (33), the positive clones were propagated and subcloned. These hybridoma cells were injected into
pristine-pretreated RAG-1
/
mice to obtain ascitic fluid containing mAb. After purification on a protein A affinity column,
five autoAbs (each 400 µg) reacting with colonic tissue were
cocktailed to form a combination of autoAbs. Seven weekly intraperitoneal injections of 2 mg of the purified Ig or mixture of
autoAbs were administrated into TCR-
/
× Igµ
/
mice starting at 12 d of age, and the mice were killed at 8 wk of age.
Detection of Circulating Self Ags.
To examine the presence of
circulating self Ags (colonic Ags), 200 µl of sera ( TCR-/
,
Igµ
/
, or TCR-
/
× Igµ
/
mice with or without Ig transfer) with CFA were injected into groups of five C57BL/6 mice
(6 wk of age). The sera obtained 14 d after immunization were
used for ELISA and immunohistochemical analysis of colonic tissue from RAG-1
/
mice (nonspecific binding of secondary Ab
to tissue is not present in RAG-1
/
mice due to lack of B cells
and Igs; reference 33). For ELISA, the purified colonic epithelial
cells (2 × 105/well) from RAG-1
/
mice were directly coated
on plates by centrifugation. After fixation with EtOH for 10 min,
the plates were blocked with 5% BSA and 2% rat serum/PBS, and
serial dilution of sera from the immunized C57BL/6 mice was
added and the antibody binding was detected by incubation with
alkaline-phosphatase rat anti-mouse Ig (PharMingen).
As in UC
patients, autoAbs such as ANCA and antitropomyosin are
frequently detectable in TCR-/
mice (16, 25, 26), and
B cells have been suspected to play a role in the pathogenesis of colitis in these mice (7, 8, 17). Therefore, to test the
role of B cells in the development of colitis in TCR-
/
mice, double mutant (TCR-
/
× Igµ
/
) mice were
generated by crossing TCR-
/
mice with Igµ
/
mice,
which lack B cells. All the mice were of inbred C57BL/6 strain. TCR-
/
× Igµ
/
mice were obtained from F3
parents and confirmed by PCR and FACScan® (Fig. 1). As
shown in Fig. 1, TCR-
/
× Igµ
/
mice, as well as
Igµ
/
mice lack mature B cells (B220+, sIgM+) and contain an increased percentage of T cells. The T cells consist
of TCR-
/
+ cells and the unique CD3+ TCR-
low cells
which express TCR-
chains in the absence of TCR-
chains on the cell surface (23, 24).
Fig. 2 shows the gross appearance of the distal part of colons from TCR-/
and TCR-
/
× Igµ
/
mice at
8 wk of age. The colons from TCR-
/
mice have a normal beaded appearance due to the presence of firm stools in
the lumen. In contrast, the colons from TCR-
/
× Igµ
/
mice have thickened wall with presence of loose
stools. Fig. 3 shows the severity of colitis in TCR-
/
,
Igµ
/
, and TCR-
/
× Igµ
/
mice maintained under
specific pathogen-free conditions. Igµ
/
mice did not develop colitis. In TCR-
/
mice, ~70% of mice developed colitis by 20 wk of age, whereas only 17% of mice
showed evidence of colitis by 12 wk of age. In contrast, all
the TCR-
/
× Igµ
/
mice developed a more severe
colitis by 8 wk of age, suggesting that the disease in TCR-
/
× Igµ
/
mice develops faster than in TCR-
/
mice. Since TCR-
/
× Igµ
/
mice are more immunocompromised than TCR-
/
mice, it is possible that
the severe colitis in these mice may be related to the presence of pathogens. However, enteric pathogenic organisms
were not detected in the TCR-
/
and TCR-
/
× Igµ
/
mice maintained under pathogen-free conditions as
confirmed by the studies performed at The Charles River
Laboratories (Wilmington, MA). We also orally administered (three times) cecal contents from TCR-
/
× Igµ
/
mice with colitis into immunodeficient RAG-1
/
and SCID mice to investigate the possibility that an unknown pathogen may be present in TCR-
/
× Igµ
/
mice. However, no colitis was recognized in these RAG-1
/
and SCID mice 8 wk after oral administration (data
not shown). These findings taken together indicate that,
like the other murine models of human IBD (15, 16), B
cells are not necessary for the development of spontaneous
colitis in TCR-
/
mice. However, unlike other models,
mature B cells or their products may have a regulatory role
in the pathogenesis of this colitis in TCR-
/
mice.
Cell Transfer to RAG-1
To further investigate
the role of B cells in colitis, we transferred lymphocytes
from TCR-/
and TCR-
/
× Igµ
/
mice to
RAG-1
/
mice that lack T and B cells (33; Table 1). The
transfer of MLN cells from TCR-
/
mice of 8 or 20 wk
of age did not induce colitis in RAG-1
/
mice within an
8 wk period of observation. Since 80% of cells in MLN of
TCR-
/
mice contain B cells (18), cell transfer studies
were performed after B cells were depleted by panning.
RAG-1
/
mice reconstituted with B cell-reduced (B220+,
10-15%) population from TCR-
/
mice also did not
show evidence of colitis. In contrast, 82% of RAG-1
/
mice reconstituted with MLN cells from TCR-
/
× Igµ
/
mice developed colitis. However, when MLN cells
from TCR-
/
× Igµ
/
mice were mixed with equal
numbers of purified MLN B cells (B220+, >98%) from
TCR-
/
mice before cell transfer, no colitis was detected in the reconstituted RAG-1
/
mice. Since our previous studies have indicated that increased colonic epithelial cell proliferation is a sensitive index of development of
colitis in TCR-
/
mice (22, 26), the results of cell transfer studies were confirmed by in vivo BrdU incorporation
to detect the colonic epithelial cell proliferation. Proliferation index of colonic epithelium as detected by BrdU incorporation was markedly higher in RAG-1
/
mice reconstituted with MLN cells of TCR-
/
× Igµ
/
mice
as compared with mice reconstituted with MLN cells from TCR-
/
mice. These findings support a suppressive role
of B cells in the development of colitis.
|
B cells
possess many immunological functions such as secretion of
Ig, antigen presentation, and cytokine production. In
TCR-/
mice, ANCA and autoAbs against tropomyosin
(a constituent of colonic epithelial cells), small nuclear ribonucleoproteins, and DNA have been frequently detected
(26, 27, 34). To define how B cells alter the pathogenesis
of colitis, we passively transferred Ig into TCR-
/
× Igµ
/
mice. Injection of purified Ig from TCR-
/
mice clearly decreased the severity of colitis in TCR-
/
× Igµ
/
mice (Fig. 4). TCR-
/
× Igµ
/
mice injected
with Ig from wild-type mice (TCR-
+/
mice) also showed
an improvement of disease; however, the severity of colitis
in these mice seemed to be greater than that in TCR-
/
× Igµ
/
mice injected with Ig from TCR-
/
mice. It is
possible that the suppression of colitis is due to autoAbs
present in TCR-
/
mice. To confirm our hypothesis,
we generated five autoAb-secreting hybridomas by using B
cells of MLNs from unimmunized TCR-
/
mice. These
autoAbs showed strong reactivity against colonic tissue by
immunohistochemical studies and ELISA (data not shown). The generated autoAbs were intraperitoneally injected into
TCR-
/
× Igµ
/
mice. The injection of a mixture of
autoAbs generated by these hybridomas also attenuated the
severity of colitis in TCR-
/
× Igµ
/
mice (Fig. 4).
These findings strongly suggest that autoAbs can contribute
to suppression of colitis.
Increase of Apoptotic Cells in TCR-
Apoptotic bodies comprise the major source of autoAgs
and provide powerful immunogens for autoreactive T cells
(35, 36). Translocation of intracytoplasmic autoantigens to
cell surface during apoptosis (37) indicates that autoAbs
(ANCA) can bind intracytoplasmic Ags in the apoptotic
process. It has been postulated that rapid clearance of apoptotic bodies by macrophages can prevent tissue damage
caused by the harmful exposure to self Ags (38, 39). Therefore, to investigate the suppressive role of autoAbs in spontaneous colitis, apoptotic cells were enumerated by TUNEL assay (Fig. 5). TUNEL assay revealed marked increase in
the number of apoptotic cells in the colon (epithelial cells
and lamina propria cells), MLN, and spleen of TCR-/
× Igµ
/
mice compared to TCR-
/
and Igµ
/
mice.
However, when TCR-
/
× Igµ
/
mice received passively transferred Ig from TCR-
/
mice, the number of
detectable apoptotic cells strikingly decreased. We also examined the expression of Fas, Fas ligand, bcl-2, and IL-1
converting enzymes (ICE) in the colon by immunohistochemical analysis, FACScan®, and/or reverse transcriptase
PCR. There was no detectable difference in the expression
of the molecules involved in apoptosis between the TCR-
/
× Igµ
/
mice and TCR-
/
mice (data not shown).
This suggests that the increase in the number of apoptotic
cells in TCR-
/
× Igµ
/
mice is caused by alteration
in the clearance of apoptotic cells rather than due to increase in apoptosis.
Increase of Circulating Self Ags in TCR-
Since apoptotic cells are the major source of self
Ags (35, 36), the presence of circulating colonic Ags (self
Ags) was examined. C57BL/6 mice were immunized with
sera from TCR-/
, Igµ
/
, or TCR-
/
× Igµ
/
mice (8 wk of age), and the reactivity of sera from the immunized C57BL/6 mice (14 d after immunization) to colonic epithelial Ags was analyzed by ELISA. The sera from
C57BL/6 mice immunized with sera of TCR-
/
× Igµ
/
mice showed significantly higher (P <0.001) reactivity to colonic Ags compared to the other groups including the sera from the C57BL/6 mice immunized with sera
from Ig-transferred TCR-
/
× Igµ
/
mice (Fig. 6 A).
Immunohistochemical analysis confirmed these results; the
sera from C57BL/6 mice immunized with sera from TCR-
/
× Igµ
/
mice, but not from other groups, strongly
reacted with colonic epithelial cells. The reactivity was mostly
associated with the nucleus of the cells (Fig. 6, B and C).
These findings indicate that in TCR-
/
× Igµ
/
mice,
there is an increase of circulating colon-associated self Ags
as compared to TCR-
+/
, Igµ
/
, and TCR-
/
mice,
and the transfer of Ig into TCR-
/
× Igµ
/
mice leads
to marked decrease of circulating self Ags. Furthermore, these findings support the hypothesis that failure of normal
clearance mechanisms for apoptotic cells by lack of autoAbs
leads to an increase of circulating self Ags. The increased
circulating self Ags may activate self-reactive T cells and provoke organ-specific autoimmune diseases (40) such as IBD.
The increase of apoptotic cells shown in lamina propria
cells of colon as well as spleen and MLNs in TCR-
/
× Igµ
/
mice is likely to reflect the activation-induced cell
death (41) of effector cells caused by harmful exposure to
the increased local and circulating self Ags.
In the organ-specific autoimmune disease model of experimental autoimmune encephalomyelitis (EAE), the data
indicates that lack of mature B cells acting as secondary
APCs may delay the recovery of the disease (42). The administration of Ig suppressed the severity of colitis, but did
not completely prevent the development of colitis in
TCR-/
× Igµ
/
mice. These findings suggest that B
cells play important functions in the complex immunological network of autoimmune diseases and in the pathogenesis of colitis in TCR-
/
mice. Since normal mice also
produce natural autoAbs (43, 44), it is possible that these
Abs may also contribute to the regulation of the immunological homeostasis and suppress the development of autoimmune disease such as IBD.
Address correspondence to Dr. Atul K. Bhan, Immunopathology Unit-Cox5, Massachusetts General Hospital, 100 Blossom St., Boston, MA 02114. Phone: 617-726-2588; FAX: 617-726-2365; E-mail: bhan{at}helix.mgh.harvard.edu
Received for publication 25 July 1997.
1 Abbreviations used in this paper: BrdU, 5-bromo-2We are grateful to Dr. Susumu Tonegawa (Massachusetts Institute of Technology, Boston, MA) for reviewing the manuscript. TCR-/
mice were originally developed in Dr. S. Tonegawa's laboratory. We also
thank Dr. M. Haramaki, Mr. D. Dombkowski, and Ms. I. Olszak for technical assistance and Miss C.A. Nason for preparation of the manuscript.
This work was supported by National Institutes of Health grants (DK47677, to A.K. Bhan) and the Massachusetts General Hospital/New England Regional Primate Research Center for the Study of Inflammatory Bowel Disease (DK43551).
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