- Email: [email protected]
CD4+ T-cell population mediates development of inflammatory bowel disease in T-cell receptor alpha chain-deficient mice
GASTROENTEROLOGY 1997;112:1876–1886
CD4/ T-cell Population Mediates Development of Inflammatory Bowel Disease in T-cell Receptor a Chain– Deficient Mice ICHIRO TAKAHASHI,*,‡ HIROSHI KIYONO,‡ and SHIGEYUKI HAMADA* *Department of Oral Microbiology, Faculty of Dentistry, and ‡Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita-Osaka, Japan
Background & Aims: Increase of T cells expressing CD4 and T-cell receptor (TCR) a0b/ (bdim) was observed in the mucosal and peripheral lymphoid tissues of TCR a0/0 mice with inflammatory bowel disease (IBD). The aim of this study was to characterize the CD4/ TCR a0b/ T cells. Methods: Cytokine production, TCR Vb usage, and helper function for Peyer’s patch B cells by the CD4/ TCR a0b/ T cells were assessed. Results: The CD4/ TCR a0b/ T cells purified from mesenteric lymph nodes and lamina propria of the intestine of IBD mice exclusively produced interleukin 4, used selected subsets (Vb6, Vb8, Vb14, and Vb15) of TCR, and massively proliferated after stimulation with staphylococcal enterotoxin B. Addition of the CD4/ TCR a0b/ T cells to Peyer’s patch B-cell cultures markedly enhanced immunoglobulin (Ig) A, IgG, and IgM antibody responses. Furthermore, depletion of the TCR a0b/ T cells with monoclonal antibody against TCR b chain completely suppressed the onset of IBD and polyclonal B-cell activation in the TCR a0/0 mice. Conclusions: These findings suggest the CD4/ TCR a0b/ T cells–mediated development of IBD in TCR a0/0 mice.
I
n the last 3 years, a number of murine models of inflammatory bowel disease (IBD) have been developed.1,2 These IBD mice showed the common feature of disrupting a T cell–dependent regulatory system that includes alterations in T-cell subpopulations and T-cell selection3 – 8 as well as those with targeted disruption of cytokine genes9 – 13 and cytokine receptor genes.14,15 Results obtained from these experimental IBD models indicate that either destruction of homeostasis in regulatory T cells or emergence of the forbidden T cells plays a crucial role in the development of intestinal immune disorders. It has also been shown that ubiquitous nonpathogenic microorganisms in the gut flora worsen IBD, whereas IBD is much milder in symptoms and slower in progress, or even completely absent, in germfree mice.10 Accumulated results from a number of these different experimental models of IBD indicated that a subpopulation of CD4/ T cells and their cytokines play a critical / 5e1d$$0011
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role for the induction of IBD. For example, either Thelper type 1 (Th1) or Th2 subset supports the development of IBD. Th2 cells and their cytokines, particularly interleukin (IL)-4, have been suggested to enhance the development of IBD.7,16,17 IL-2 gene–disrupted mice that harbored abnormal B-cell responses, including production of autoantibodies and anti–gut constituent antibodies, developed IBD.10 On the other hand, it was shown that CD4//CD45RBhigh T-cell transfer model of colitis in mice with severe combined immunodeficiency disease resembles a dysregulated Th1 response.4,18 In this study, we investigated the cellular and molecular characteristics of CD4/ T-cell receptor (TCR) a0b/ (bdim) T cells in mucosal and systemic compartments of TCR a0/0 mice with IBD. We show that CD4/ TCR bdim T cells become increasingly detectable with age in the mucosal (Peyer’s patches, intestinal lamina propria, and mesenteric lymph nodes) and peripheral lymphoid organs (spleen) of TCR a0/0 mice. The increase of the aberrant CD4/ TCR bdim T cells accompanied the elevated immunoglobulin (Ig) secretion, anti–self constituent and food antibodies, including those of IgE isotype (i.e., disruption of naturally induced peripheral tolerance), and development of ulcerative colitis in the TCR a–deficient mice. These aberrant T cells selectively produced IL-4, used limited Vb genes, and strongly responded on stimulation with staphylococcal enterotoxin B (SEB). They could also support the development of germinal centers of Peyer’s patches and spleen and provide helper function for B-cell differentiation to IgM-, IgG-, and IgA-producing cells. Erroneous selection and expansion of the CD4/ TCR bdim T cells in extrathymic lymphoid tissue may be responsible for the aberrant imAbbreviations used in this paper: CCD, charge-coupled device; ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme-linked immunospot; IFN-g, interferon gamma; IL, interleukin; MAb, monoclonal antibody; PBS-T, PBS containing 0.05% Tween 20; RT-PCR, reverse-transcription polymerase chain reaction; SEB, staphylococcal enterotoxin B; TCR, T-cell receptor; Th, T helper. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
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mune responses occurring in TCR a0/0 mice that may account for the development of IBD.
phosphatase buffer (100 mmol/L Tris-HCl [pH 9.5] containing 100 mmol/L NaCl and 5 mmol/L MgCl2 ).
Cytokine-Specific ELISPOT
Materials and Methods Mice TCR a– and TCR d–deficient (TCR a0/0 and TCR d ) mice with a background of (129 1 C57BL/6) were obtained from the Jackson Laboratory (Bar Harbor, ME). C57BL/ 6 mice were purchased from Charles River Japan (Kanagawa, Japan). TCR a0/0 and TCR d0/0 mice were originally generated by a targeted disruption of TCR genes in embryonic stem cells.19 The mice were housed in two rooms in the Experimental Animal Facility at the Research Institute for Microbial Diseases at Osaka University. All mice were kept on a 12hour light-dark cycle, housed under specific pathogen–free conditions, and received sterilized food (Certified diet MF; Oriental Yeast Co., Osaka, Japan) and autoclaved distilled water ad libitum. The cages were changed every 5 days, and the mice were handled with sterilized gloves. 0/0
Reagents SEB was purchased from Sigma Chemical Co. (St. Louis, MO). Purified a-casein from bovine milk was obtained from Wako Pure Chemicals (Osaka, Japan). Myelin basic protein from bovine brain was purchased from Sigma. Protein extracts from soy bean and wheat, the main constituents of the sterilized food, were provided by Oriental Yeast Co.
Isolation of Lymphoid Cells From MucosaAssociated Tissues and Spleen The spleen and mesenteric lymph nodes were aseptically removed, and single cell suspensions were prepared by a standard mechanical disruption procedure as described previously.20,21 Single-cell suspensions of Peyer’s patches and the lamina propria lymphocytes were prepared by an enzymatic dissociation method using Dispase (Boehringer Mannheim Biochemicals, Indianapolis, IN) as described previously.20,21 In these procedures, the cell viability of Peyer’s patches and spleen were ú98% and that of lamina propria lymphocytes were ú95% viable. Antigen-reactive and total IgA, IgG, and IgM antibodyforming cells were evaluated in mononuclear cells isolated from mucosa-associated and peripheral lymphoid tissues by a modified enzyme-linked immunospot (ELISPOT) assay as described previously.20,22 Nitrocellulose microtiter plates (Millipore Co., Bedford, MA) were coated with 100 mL of anti-mouse IgA, IgE, IgG, or IgM (Southern Biotechnology Associates, Birmingham, AL) for total Ig antibody-forming cells or various antigens (e.g., protein extracts from soybean and wheat) for antigen-reactive antibody-forming cells at a concentration of 5 mg/mL in phosphate-buffered saline (PBS). For the detection of isotype and antigen-reactive antibody-producing cells, alkaline phosphatase–conjugated anti-mouse IgA, IgG, or IgM (1 mg/mL; Southern Biotechnology Associates) was added and then visualized with the substrate, 5-bromo-4-chloro-3-indolyl phosphate (Wako)/nitroblue tetrazolium (Wako) in alkaline
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For the assessment of cytokine production by CD4/ TCR b T cells at the single-cell level, cytokine-specific ELISPOT assay was used. Purified CD4/ TCR bdim T cells were sorted by FACStar Plus (Becton Dickinson, Palo Alto, CA) from the TCR a0/0 mice with IBD. Cells producing IL-2, interferon (IFN)g, IL-4, IL-6, and IL-10 were detected by the cytokine-specific ELISPOT assay as described previously.23,24 In brief, 96-well Multiscreen HA plates equipped with 0.45-mm cellulose ester membrane (Millipore) were coated with the rat monoclonal antibodies (MAbs) anti-mouse IL-2 (clone no. JES6-1A12), IL-4 (BVD4-1D11), IL-6 (MP5-20F3), IL-10 (JES5-2A5), or IFN-g (R4-6A2) (20 mg/mL) at 47C for 16 hours. Then the plates were washed three times with PBS, followed by preincubation with RPMI 1640 (Sigma) containing 10% fetal calf serum for 1 hour at 377C. Thereafter, different concentrations of purified CD4/ TCR bdim T cells (105 –103 cells in 100 mL of RPMI 1640 supplemented with insulin-transferrin-selenium-S [Life Technologies, Gaithersburg, MD]) were added to the plates and incubated at 377C overnight in a humidified incubator at 5% CO2 . As positive controls for cytokine-specific ELISPOT assay, a Th clone (D10.G4.1) specific for conalbumin25 and SEB-stimulated Peyer’s patch CD4/ T cells from TCR a/// mice were used. The plates were then washed with PBS followed by PBS containing 0.05% Tween 20 (PBS-T) (Wako). Subsequently, biotinylated rat antimouse IL-2 (JES6-5H4), IL-4 (BVD6-24G2), IL-6 (MP5-32C11), IL-10 (SXC-1), or IFN-g (XMG1.2) MAbs (5 mg/mL) were added, and the incubation was performed at 47C overnight. After washing with PBS-T, streptavidin-peroxidase (1:2000 dilution; Zymed Laboratories, San Francisco, CA) was added for 1 hour at 377C. The plates were washed again in PBS-T and the substrate solution containing 3-amino-9-ethylcarbazole (Aldrich Chemical Co., Milwaukee, WI) and H2O2 was added. Cytokine-specific spots were quantitated by using an Olympus SZH stereo-zoom microscope (Olympus, Tokyo, Japan). dim
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Enzyme-Linked Immunosorbent Assay Antibody concentrations in sera from TCR a0/0 mice with or without IBD were determined by enzyme-linked immunosorbent assay (ELISA).26 ELISA plates (Nunc, Naperville, IL) were coated with 100 mL of 5 mg/mL of goat anti-mouse IgM, IgG, or IgA or rat anti-mouse IgE (Southern Biotechnology Associates) in PBS. Serial twofold dilutions of sera or fecal extracts from 5 individual mice were added (100 mL/well). Fecal extracts were prepared by mixing fresh feces with PBS containing 0.5% sodium azide (100 mg wet weight of feces per milliliter), and the centrifuged supernatant was used as fecal extract for ELISA.27 After 2-hour incubation at 377C, unbound antibodies were removed and alkaline phosphatase– conjugated goat or rat anti-mouse m, g, a, and e heavy chain (Southern Biotechnology Associates) was added. The plates were incubated at 377C for 2 hours and developed with pnitrophenyl phosphate (1 mg/mL; Wako) in 10 mmol/L di-
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ethanolamine buffer (pH 9.6). Antibody concentrations were calculated from the standard curve using purified mouse IgA, IgG, IgM, or IgE (Southern Biotechnology Associates).
In Vitro T-cell Proliferative Responses Mesenteric lymph nodes and spleen were aseptically removed, and single-cell suspensions of the tissues were prepared by mechanical disruption, washed extensively, and resuspended in RPMI 1640 (Sigma) supplemented with sodium bicarbonate, HEPES (15 mmol/L), L-glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (100 mg/mL), and gentamicin (100 mg/mL). The CD3/ T cells were enriched by negative panning on Petri dishes coated with goat anti-mouse IgG F(ab*)2 (20 mg/mL; Jackson ImmunoResearch Laboratories, West Grove, PA). CD4/ T cells were further purified by positive panning on a Petri dish coated with anti-CD4 MAb.27 The MAb specific for CD4 (clone GK1.5, American Type Culture Collection, Rockville, MD) was purified from ascites through a protein G column (Pharmacia LKB, Uppsala, Sweden). The purified CD4/ T cell population was ú95% CD4/ CD80 as determined by flow cytometry.27 The CD4/ T cells (1 1 105/well) were cultured in flat-bottom 96-well microculture plates (Corning, Corning, NY) in the presence of mitomycin C–treated feeder spleen cells (1 1 104 cells/well) for 3 days.26 SEB-specific CD4/ T-cell proliferative responses were assessed by addition of 0.5 mCi of [3H]thymidine (ICN, Costa Mesa, CA) 6 hours before the cell collection. The level of proliferation was determined by liquid scintillation counting. As a control, CD4/ T cells were cultured with feeder cells in the absence of SEB.
Reverse-Transcription Polymerase Chain Reaction Reverse-transcription polymerase chain reaction (RTPCR) analysis was performed to detect cytokine and TCR Vb messenger RNA (mRNA) expression according to the method previously described.23,27 To isolate RNA from the fluorescenceactivated cell sorter (FACS) purified CD4/ T cells, TRIzol reagent (Life Technologies) was used. The RNA was reverse-transcribed into complementary DNA (cDNA) by use of 10 mL of an RT mixture containing 10 ng of total cellular RNA, 11 PCR buffer composed of 10 mmol/L Tris-HCl (pH 8.3) and 50 mmol/L KCl, 5 mmol/L MgCl2 , 1 mmol/L each deoxynucleoside triphosphate, 10 U of RNase inhibitor (Promega, Madison, WI), 0.1 mg of oligo(dT16), and 2 U of Superscript II reverse transcriptase (Life Technologies). The mixture was incubated at 427C for 30 minutes, heated to 957C for 5 minutes, and chilled quickly on ice. The PCR mixture for TCR Vb–specific mRNA analysis consisted of 11 PCR buffer, 2 mmol/L MgCl2 , 50 mmol/L deoxynucleoside triphosphate, 0.4 mmol/L Vb-specific sense primer28 and Cb antisense primers, and 1.25 U of AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Branchburg, NJ) in a total volume of 50 mL. PCR amplification was performed in sequential 21–33 cycles at 947C for 1 minute and 607C for 1 minute with a 727C extension step for 7 minutes after the last cycle with GeneAmp PCR System 9600 (Perkin-Elmer Cetus). One fifth of the amplified products
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were separated by electrophoresis in 2% agarose gel, and amplified products were visualized with ethidium bromide (1 mg/mL). The intensity of ethdium bromide fluorescence of each TCR Vb–Cb bands was measured by the charge-coupled device (CCD) imaging system (Densitograph AE 6900-F; Atto Co., Tokyo, Japan) as previously described.29 To normalize the condition of gel staining and CCD imaging, a constant amount of control DNA (b-actin cDNA) was electrophoresed in every time. Quantitation of PCR products was performed by a formula described by Chelly et al.30 Briefly, the extent of amplification (Y) is given by the formula: Y Å A(1 / R)n, where A is the initial amount of the original template (cDNA), R is the efficiency, and n is the number of cycles. It is possible to estimate Y by measuring the intensity of ethidium bromide fluorescence of amplified products by CCD imaging system, and R can be deduced from the slope of the semi-log plot. Thus, the proportion of a certain Vb gene expressed to total Vb genes expressed was calculated: %Vbx Å A(Vbx) 1 100/ [A(Vb1) / A(Vb2) / rrrrr / A(Vb19)].
Helper Assay To examine helper functions of CD4/ TCR bdim T cells in intestinal lamina propria, Peyer’s patch B cells were prepared by the treatment of lymphocytes with two cycles of anti-Thy 1.2 MAb (Cedarlane, Ontario, Canada) and low-toxic guinea pig complement (Cedarlane), followed by fractionation on discontinuous gradient of Percoll (Pharmacia LKB) as described previously23 and resuspended in complete RPMI 1640 consisting of sodium bicarbonate, L-glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (100 mg/mL), and 10% fetal calf serum. Purified CD4/ TCR bdim T cells (105 cells per well) were added to individual B-cell cultures (2 1 106 cells per well). In some experiments, 100 mg/mL of MAb anti– IL-4 (BVD4-1D11) was added to these cultures. As controls, CD4/ T cells purified from intestinal lamina propria of TCR a/// mice were added to Peyer’s patch B-cell cultures. These cultures were incubated for 4 days at 377C in humidified atmosphere of 5% CO2 . Nonadherent, viable B cells were fractionated by discontinuous Percoll density gradient centrifugation, and the number and isotype of Ig-producing cells were determined by ELISPOT assay.
Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Western Immunoblotting Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed as described previously.31 Purified casein and myelin basic protein (1 mg each) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred electrophoretically to a nitrocellulose sheet. The sheet was then treated either with a 1:40 dilution of sera from the mice with or without IBD for 2 hours at 257C. The antibody bound to the immobilized replica proteins was incubated with a 1:1000 dilution of alkaline phosphatase– conjugated rat anti-mouse e heavy chain (Southern Biotechnology Associates) for 2 hours at 257C. The sheet was then washed
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with PBS-T and developed with the substrate 5-bromo-4chloro-3-indolyl phosphate (Wako)/nitroblue tetrazolium (Wako) in alkaline phosphatase buffer (100 mmol/L Tris-HCl [pH 9.5] containing 100 mmol/L NaCl and 5 mmol/L MgCl2 ).
Flow Cytometric Analysis and Cell Sorting Immunofluorescent analysis was performed using a FACScan flow cytometry (Becton Dickinson). Cells stained with single-color reagent were used to set the appropriate compensation levels, and at least 10,000 events were analyzed. Cell sorting was performed on a FACStar (Becton Dickinson). The following MAb from Pharmingen (San Diego, CA) were used: anti-CD4 (RM4-5), anti-CD5 (53-7.3), anti-TCRb (H57-597), anti-TCR Vb4 (KT4), anti-TCR Vb5.1, 5.2 (MR9-4), anti-TCR Vb6 (RR4-7), anti-TCR Vb7 (TR310), anti-TCR Vb8.1, 8.2 (MR5-2), anti-TCR Vb9 (MR 10-2), anti-TCR Vb10b (B21.5), anti-TCR Vb11 (RR3-15), antiTCR Vb12 (MR11-1), anti-TCR Vb13 (MR12-3), anti-TCR Vb14 (14-2), and anti-TCRd (GL3). For two-color flow cytometry, 1 1 106 cells in 20 mL PBS containing 2% fetal calf serum and 0.02% sodium azide were first incubated with antiFc receptor MAb (2.4G2) to prevent nonspecific staining and then stained with appropriate fluorescein isothiocyanate–conjugated MAb and phycoerythrin-conjugated MAb or biotinylated MAb followed by streptavidin-phycoerythrin.21 All MAbs were used at the saturating concentrations. Flow cytometric analysis was performed with a FACScan flow cytometer using Lysis II software (Becton Dickinson). Results are shown as loglog dot plots. For cell sorting, mononuclear cells were stained simultaneously for CD4 (RM4-5) and ab TCR (H57-597) and were positively selected with a FACStar (Becton Dickinson).
Treatment With Anti-ab TCR (Clone H57597) MAb Young healthy TCR a0/0 mice (4–5 weeks of age) were treated with hamster MAb anti-TCRb (H57-597) purified by protein G–Sepharose CL-4B (Pharmacia LKB) as described previously.26 Mice were injected once a week with the MAb (1 mg in a volume of 200 mL) intraperitoneally for 12 weeks according to the protocol described previously.32 Control mice were injected with hamster IgG (Pharmingen).
had extended to the ileum, colon, and rectum with marked hyperplasia, dilatation, and thickening of the colon wall compared with the TCR a0/0 mice without IBD. Histological examination showed marked hyperplasia, elongation, and distortion of crypts, with areas of microabscesses, reduced numbers of goblet cells, and infiltration of inflammatory lymphocytes within the lamina propria of the colon wall (data not shown). Enlargement of Peripheral Lymphoid Tissues Associated With Enhanced B-cell Responses TCR a–deficient mice between 4 and 6 months of age with IBD were found to have developed massive enlargement of Peyer’s patch, mesentric lymph nodes, and spleen. Histological examination of Peyer’s patch revealed a marked expansion of lymphoid tissues with abnormally developed germinal centers (data not shown). There were striking elevations of serum and fecal antibodies in the TCR a0/0 mice with IBD (Table 1). The level of IgA in fecal sample of the mice with IBD was comparable to those of the normal C57BL/6 (TCR a///) mice. In contrast, extremely lower levels of IgA were seen in fecal samples obtained from the TCR a0/0 mice without IBD. A similar pattern was also observed with serum IgA (Table 1). The levels of serum IgG and IgM in the TCR a0/0 mice with IBD were also dramatically increased to the levels of the TCR a/// mice. Furthermore, elevated IgE antibodies were seen in the serum of the TCR a0/0 mice with IBD but not the TCR a0/0 mice without IBD and the TCR a/// mice (Table 1). Concerning antibody-producing cells in lamina propria of the small and large intestines, Peyer’s patch, mesenteric lymph nodes, and spleen from the TCR a0/0 mice with
Table 1. Comparison of Ig Levels in Feces and Serum of TCR a0/0 Mice With and Without IBD TCR a0/0 mice
Statistical Method Significant differences between mean values were determined by Student’s t test. P values of õ0.05 were considered statistically significant.
Sample Feces (ng/100 mg)
Results Morphometric Assessment of the Large Intestine of TCR a0/0 Mice
Serum (mg/mL)
Starting at 4–6 months of age, approximately 60% of the TCR a0/0 mice were found to have developed IBD, characterized by anorectal prolapse, diarrhea, and wasting syndrome such as weight loss and hunched posture. The necropsy revealed that there was bowel inflammation, which / 5e1d$$0011
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Isotype
With IBD
IgA IgE IgG IgM
10,000 { 86 0a 640 { 45 310 { 20
IgA IgE IgG IgM
164 { 11 2.5 { 0.2 640 { 30 320 { 12
Without IBD
TCR a///
1240 { 48 9800 { 24 0 0 644 { 65 84 { 7 0 0 5.0 { 1 0 5.1 { 0.4 5.2 { 0.2
204 { 5 0 820 { 60 200 { 16
NOTE. Data represent mean { SD from 5 mice per group of three separate experiments. a Zero indicated that antibody was undetectable.
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Figure 1. Correlation of IBD induction in the TCR a0/0 mice with selected increases in IgA(j), IgG- (h), and IgM-producing ( ) cells. Mononuclear cells isolated from spleen, mesenteric lymph nodes, Peyer’s patch, and lamina propria of the small intestine of TCR a0/0 mice with or without IBD were examined by isotype-specific ELISPOT.
IBD, increase in IgA antibody–producing cells was mainly found in the lamina propria followed by mesenteric lymph nodes and Peyer’s patch, while IgG-producing cells were predominantly found in mesenteric lymph nodes followed by spleen and lamina propria (Figure 1). Although the frequency of IgM-producing cells was lower compared with the other two isotypes, these isotype antibody-producing cells were noted in lamina propria, mesenteric lymph nodes, and spleen. The number of total Ig-producing cells in lamina propria of the small intestine were similar to those in the large intestine. Increase of CD4/ TCR bdim T Cells in TCR a0/0 Mice With IBD A unique subset of T cells that could be co-stained with MAb anti-CD4 (RM4-5) and MAb H57-597 spe-
cific for pan TCRb became increasingly detectable in the mucosa-associated and peripheral lymphoid tissues in TCR a–deficient mice with IBD (Figure 2). The intensity of staining with MAb H57-597 was weak, and these T-cell populations (approximately 6%–9% of lymphocyte population) were termed CD4/ TCR bdim T cells. These cells had developed in both mucosa-associated lymphoid tissues (e.g., intestinal epithelium, lamina propria, and mesenteric lymph nodes) and the spleen of the TCR a0/0 mice with IBD. The Peyer’s patch of the mice also contained a similar level (6% of lymphocyte population) of CD4/ TCR bdim T cells. In contrast, only a few cells of this subset were seen in the TCR a0/0 mice without IBD (0%–0.8%). Furthermore, these T cells were undetectable in the normal TCR a/// mice (data not shown).
Figure 2. Flow cytometric analysis of lymphocytes isolated from spleen, mesenteric lymph nodes (MLN), and lamina propria (LP) in the large intestine and intestinal intraepithelial lymphocytes (IEL) in the TCR a0/0 mice with IBD (left panels ) and the TCR a0/0 mice without IBD (right panels ). Cell suspensions were stained with MAbs specific for fluorescein isothiocyanate–TCR b and phycoerythrin-CD4.
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Table 2. Numbers of Th1- and Th2-Type Cytokine-Producing CD4/ TCR bdim T Cells Isolated From MucosaAssociated Tissues of TCR a0/0 Mice With IBD Numbers of cytokine-producing cells per 106 cells
Cytokine
LPL
MLN
PP
IFN-g IL-2 IL-4 IL-6 IL-10
0 0 18,300 { 1700 0 0
0 0 520 { 140 0 0
0 0 1300 { 50 0 0
D10.G4.1 (Th2 clone)
2450 6780 25,620 5170
0 { { { {
210 540 1740 420
NOTE. The results are representative of five separate experiments. LPL, lamina propria lymphocytes; MLN, mesenteric lymph nodes; PP, Peyer’s patches.
Histochemical examination of colon and spleen also revealed TCR bdim cells. TCR bdim T cells were observed in the parafollicular region (i.e., T-cell area) of spleen (data not shown). The CD4/ TCR bdim T cells in spleen might support the development of germinal centers of these lymphoid tissues. Focal clusters of the TCR bdim T cells were found in the lamina propria of the colon, suggesting that clonal expansion of the TCR bdim T cells was induced by prolonged exposure to luminal food and bacterial antigens (data not shown). Cytokine Production by CD4/ TCR bdim T Cells The CD4/ TCR bdim T cells isolated from mucosa-associated lymphoid tissues of the TCR a0/0 mice with IBD prominently contained IL-4–producing cells (Table 2). However, none of the IL-2, IFN-g, IL-6, and IL-10 cytokine-producing cells were detected in the FACS-purified CD4/ TCR bdim T cells. To confirm valid-
Figure 4. RT-PCR analysis of TCR Vb repertoire usage of CD4/ TCRbdim cells from intestinal lamina propria of TCR a0/0 mice with IBD. The percentages of each TCR Vb mRNA expression were calculated from PCR products as described in Materials and Methods. The percentage of T cells was the mean of five separate experiments { SD.
ity of our ELISPOT assay, a Th clone (D10.G4.1) was used as a positive control; the Th clone produced spots in IL-2–, IL-4–, IL-6–, and IL-10–specific cytokine assay (Table 2). SEB-stimulated Peyer’s patch CD4/ T cells from TCR a/// mice could form spots specific for IFN-g (1530 { 210/104 cells), IL-2 (980 { 57/104 cells), and IL-4 (680 { 72/104 cells). Furthermore, cytokinespecific RT-PCR analysis revealed the expression of IL4–specific message in RNA extracted from the purified CD4/ TCR bdim T cells (data not shown). This finding is the first direct demonstration that the CD4/ TCR bdim T cells predominantly produce IL-4 in the TCR a0/0 mice with IBD. Responses of the CD4/ TCR bdim T Cells to SEB TCR Vb usage of these CD4/ TCR bdim T cells was then examined by flow cytometry and Vb family-
Figure 3. Flow cytometric analysis of TCR Vb repertoire of CD4/ TCR bdim cells from the lamina propria of intestine of TCR a0/0 mice with IBD. Cell suspensions were stained with MAbs specific for fluorescein isothiocyanate–TCR Vb and phycoerythrin-CD4. The percentages of T cells bearing each TCR Vb were calculated as 100 1 (% Vbx)/%(Vb4 / Vb5 / Vb6 / Vb7 / Vb8 / Vb9 / Vb10 / Vb11 / Vb12 / Vb13 / Vb14). The percentage of T cells was the mean of five separate experiments { SD.
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Figure 5. T-cell proliferative responses to SEB in CD4/ T-cell cultures prepared from the TCR a–deficient mice with IBD and from the TCR a/// mice. [3H]thymidine uptake in CD4/ T-cell cultures in the absence of SEB was 1780 { 145 for mesenteric lymph nodes (MLN) and 2300 { 230 for spleen (SP) in diseased TCR a0/0 mice and 1035 { 61 for mesenteric lymph nodes and 1473 { 85 in TCR a/// mice, respectively. The results presented are representative of five separate experiments. Data are expressed as mean values { SD for triplicate cultures.
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Figure 6. Comparison of antibody responses reactive with soybean and wheat in TCR a0/0 mice with IBD and TCR a/// mice. Mononuclear cells isolated from spleen and lamina propria of both TCR a0/0 mice with IBD and TCR a/// mice were subjected to the soybeanand wheat-reactive ELISPOT assay. Results are expressed as mean values { SD.
specific RT-PCR. The flow cytometric analysis revealed that the CD4/ TCR bdim T cells used TCR Vb8 most frequently, followed by TCR Vb14 and TCR Vb6 (Figure 3). To examine the potential expression of TCR Vb for which antibodies were not available to us, analysis of TCR Vb gene usage by the CD4/ TCR bdim T cells was also conducted by RT-PCR amplification using a panel of TCR Vb–specific oligonucleotide primers (Vb1–Vb19). The RT-PCR analysis revealed Vb6, Vb11, Vb14, and Vb15 mRNA were frequently expressed in the infiltrating CD4/ TCR bdim T cells from lamina propria of the
intestine, while Vb8 message was most frequently detected (Figure 4). Because of the latter finding, it was important to examine whether these CD4/ TCR bdim T cells from the IBD mice proliferated in response to SEB. It was found that these cells massively proliferated in response to SEB (0–10 mg per well) in a dose-dependent manner (Figure 5). The intensity of the [3H]thymidine uptake by the CD4/ T cells from the diseased mice was well over the level of CD4/ T cells from TCR a/// mice. However, CD3/ T cells isolated from the nondiseased TCR a0/0 mice did not respond to SEB (data not shown).
Figure 7. Immunoblots showing reactivity of serum IgE toward (A ) milk casein (food antigen) and (B ) purified myelin basic protein (self antigen). One microgram of purified proteins was analyzed by 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose sheets by electroblotting. The proteins on the sheet allowed to react with serum IgE from the TCR a0/0 mice with IBD, the TCR a0/0 mice without IBD, or the TCR d0/0 mice were detected by alkaline phosphatase–conjugated rat anti-mouse IgE. Molecular sizes are indicated on the left.
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Figure 8. Assessment of helper activity for Peyer’s patch B cells by CD4/ TCR bdim T cells isolated from lamina propria of colon of the TCR a0/0 mice. The CD4/ TCR bdim T cells (1 1 105 cells per well) purified by flow cytometry were cultured with Peyer’s patch B cells (2 1 106 cells per well) for 4 days. Nonadherent cells were collected and analyzed by the isotype-specific ELISPOT assay. To some of the wells, 100 mg per well of anti–IL-4 MAb was added. As controls, CD4/ T cells isolated from TCR a/// mice were added to the culture. Numbers of antibody-forming cells was determined in triplicate cultures and is expressed as the mean value { SD. Numbers of antibodyforming cells in cultures containing Peyer’s patch B cells only were IgA (j), 65 { 4.0/105 antibody-forming cells; IgG (h), 40 { 3.5/105 antibody-forming cells; and IgM ( ), 28 { 2.5/105 antibody-forming cells. The results are representative of five separate experiments.
Unique B-cell Responses in TCR a Mice With IBD
Effect of CD4/ TCR bdim T-cell Depletion on the Development of IBD in TCR a0/0 Mice
0/0
The role of CD4/ TCR bdim T cells for B-cell responses in the TCRa0/0 mice with regard to the development of IBD was examined. The TCR a0/0 mice with IBD were found to show marked B-cell responses reactive with food proteins such as soybean and wheat in the lamina propria of the intestine, mesenteric lymph nodes, and spleen (Figure 6). In particular, lamina propria lymphocytes of the intestine from the mice with IBD contained high numbers of plasma cells that produced IgA antibody reactive with soybean and wheat. It was also interesting to note that high numbers of soybean-reactive IgG-producing cells were detected in the intestinal lamina propria of the TCR a0/0 mice with IBD. In contrast, low numbers of antibody-producing cells reactive with food antigen were seen in the lamina propria and mesenteric lymph nodes of the TCR a/// (C57BL/6) mice. In regard to the systemic lymphoid tissues of the TCR a0/0 mice with IBD, our results revealed the presence of antibody-producing cells reactive with food antigen in spleen (Figure 6). In contrast, spleen of TCR a/// mice did not contain these antibody-producing cells. Furthermore, serum IgE from the TCR a0/0 mice with IBD was found to react strongly with milk casein and myelin-basic protein (Figure 7). Neither the TCR a0/0 mice without IBD nor the TCR a/// mice developed IgE responses against milk casein and myelin-basic protein in serum. Based on the above results, we examined whether the CD4/ TCR bdim T cells found in the TCR a0/0 mice with IBD were capable of providing helper function for / 5e1d$$0011
Peyer’s patch B cells. When the FACS-purified CD4/ TCR bdim T cells were added to Peyer’s patch B-cell cultures without any stimulants, IgA-, IgG-, and IgMproducing cells were prominently demonstrated (Figure 8). The levels of helper function of the CD4/ TCR bdim T cells were comparable to those of Peyer’s patch CD4/ T cells from intestinal lamina propria of TCR a/// mice. Because the CD4/ TCR bdim T cells prominently contained IL-4–producing cells (Table 2), it was important to investigate whether IL-4 was involved in the helper function. When anti–IL4 MAb were added to the Peyer’s patch B-cell cultures in the presence of the CD4/ TCR bdim T cells, induction of IgA- and IgM-producing cells was markedly suppressed, while IgG-producing cells were only partially inhibited by this MAb treatment (Figure 8). These results clearly suggested that the CD4/ TCR bdim T cells exclusively producing IL-4 supported Peyer’s patch B cells to synthesize IgA, IgG, and IgM antibody.
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Young TCR a0/0 mice without IBD were treated with weekly injection of anti-TCRb MAb or hamster IgG (1 mg/mouse). Onset of IBD and appearance of CD4/ TCR bdim T cells were then assessed. Monthly histopathological assessment of TCR a0/0 mice revealed that there were no signs of IBD in the anti-TCRb MAb– treated mice up to 6 months of age (Table 3). In addition, flow cytometric analysis revealed complete deletion of CD4/ TCR bdim T cells in the mesenteric lymph nodes and lamina propria during this period. The absence of CD4/ TCR bdim T cells was always associated with the striking reduction in the numbers of IgA, IgG, and IgM antibody-producing cells in the lamina propria and spleen of the anti-TCRb MAb-treated mice. On the other hand, starting at 4 months of age, 67% of the hamster IgG–treated TCR a chain–deficient mice developed IBD characterized by anorectal prolapse and hyperplasia of the colon. Histological examination of the colon dem-
Table 3. Monoclonal Anti-TCRb Treatment Inhibited the Development of CD4/ TCRbdim T Cells and IBD MAb treatment
Frequency (% gated) of CD4/ TCRbdim T cellsa
Development of IBDb
Anti-TCRb IgG
0.7 { 0.4 13.2 { 4.8
0/9 6/9
a
Data represent mean values { SD from intestinal lamina propria of 3 treated mice. b Numbers of mice developed IBD.
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onstrated that there were hyperplasia of crypts and infiltration of inflammatory lymphocytes within the lamina propria of the hamster IgG-treated TCR a chain–deficient mice (data not shown). In addition, increase of numbers of antibody-forming cells in the lamina propria and spleen were associated with the development of IBD in the hamster IgG–treated mice.
Discussion Experimental IBD has been spontaneously developed in mice incapable of synthesizing specific components involved in the immune systems because of, e.g., alterations in TCR or targeted disruption of cytokine gene.1,2 These experimental IBD models provide us with information about the cellular and molecular mechanisms in development of IBD. For example, there is direct evidence for the involvement of a specific subset of CD4/ T cells in the development of IBD. Powrie et al.4,18 observed that adoptive transfer of CD45 RBhigh CD4/ T cells from BALB/c mice to C.B-17 mice with severe combined immunodeficiency disease results in the intestinal inflammation. Furthermore, cotransfer of the reciprocal CD45 RBlow CD4/ T cells prevented the development of colitis.6 The mature lesions of IBD in TCR a–deficient mice were characterized by a mixed lymphocyte infiltrate in the lamina propria of the colon and a marked enlargement of mucosa-associated peripheral lymphoid tissues such as Peyer’s patch and spleen. We also have found that striking increases in plasma cells producing IgA, IgG, and IgM were always accompanied with the increase of the aberrant CD4/ TCR bdim T cells in gut-associated lymphoid tissues and spleen of the diseased mice (Figures 1 and 2, and Table 1). These enhanced levels of CD4/ T cells that were stained weakly with MAb to TCR b in the periphery of TCR a–deficient mice were first reported by Mombaerts et al.5,19,33 followed by Viney et al.34 Most recently, it was demonstrated that elevation of TCR b/ T cells was always noted in TCR a0/0 mice with IBD but not without the disease.17 To this end, possible association of TCR bdim T cells for the development and regulation of IBD has been suggested.17 The pathological role of the aberrant T cells was clearly supported by the finding that the CD4/ TCR bdim T cells enhanced the synthesis of IgA, IgG, and IgM antibodies from the B cells of TCR a–deficient mice in vitro (Figure 8). Consistent with the Ig class switching of the B cells, the CD4/ TCR bdim T cells were found to exclusively produce IL-4 (Table 2). In addition, depletion of the CD4/ TCR bdim T cells by the treatment of TCR a0/0 mice with MAb TCR b completely suppressed the polyclonal B-cell activation occurring in the mucosal and / 5e1d$$0011
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systemic lymphoid organs and the development of IBD in the TCR a0/0 mice (Table 3). Thus, the appearance of the CD4/ TCR bdim T cells can lead to severe pathological changes in the large intestine and peripheral lymphoid tissues in TCR a0/0 mice. The equivalent subset of the CD4/ TCR bdim T cells in normal mice or humans has not yet been detected; however, rearrangement and expression of the TCR b chain genes is epistatic to those of TCR a chain genes.35 The CD4/ TCR bdim T cells might be positively selected during development both in thymus and extrathymic lymphoid tissues. The TCR bdim T cells increased with age (Figure 2) and exclusively used limited TCR Vb receptors (Figures 3 and 4). These findings suggest that the increase in the CD4/ T cells among peripheral T cells may be the result of a secondary expansion to yet unidentified major histocompatibility complex class II–restricted antigens. The CD4/ TCR bdim T cells are not detected in TCR a 1 major histocompatibility complex class II double– deficient mice, suggesting that they are dependent on major histocompatibility complex class II molecules.31 Because TCR a0/0 mice with IBD showed humoral immune responses to food and self antigens (Figures 6 and 7), it is likely that the CD4/ TCR bdim T cells have specificities to food or self antigens. Development and activation of the CD4/ TCR bdim T cells might be positively regulated by bacterial superantigens. We show in the present study that the CD4/ TCR bdim T cells could massively proliferate by the stimulation with SEB (Figure 5). Our preliminary results show that coagulase-negative, but mannitol-fermenting, staphylococci were always recovered from the stool of mice with IBD. In addition, the CD4/ TCR bdim T cells exposed to bacterial sonicates from the staphylococci strongly proliferated (unpublished observation). Superantigen-like products from the staphylococci might modulate or positively regulate the development of the aberrant CD4/ T cells in TCR a0/0 mice. Hyperreactivity of gd T cells may also lead to the aberrant B-cell responses in the mucosal effector sites such as the lamina propria of the intestine. The T-cell compartment of the TCR a–deficient mice with IBD is composed of largely gd T cells with minor population of the CD4/ TCR bdim T cells.5,7,34 We recently reported that gd T cells serve an important regulatory role for mucosal IgA responses in that TCR d–deficient mice possess normal numbers of functional ab T cells but display markedly decreased IgA responses to oral immunization.36 Mombaerts et al. also reported that severity of disease was significantly reduced when mice lacking ab T cells were crossed with mice lacking gd T cells, WBS-Gastro
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suggesting that gd T cells may also play a role in the pathogenesis of the colitis because of a lack of the ordinary ab T cell–mediated down-regulation of gd T cells.5 It has been suggested that autoantibodies mediate the disease as an organ-specific autoimmune disease in IBD.37 Other investigators reported that antibodies specific for epithelial cell–associated components were found in patients with IBD.38 Other antibodies were reported to be reactive with a 40-kilodalton epithelial cell cytoskeletal microfilament protein belonging to the tropomyosin family.17,39 Along similar lines, it was shown that IBD was associated with antineutrophil cytoplasmic antibodies.40 The antibodies might cause tissue damage in mice with IBD via complement-mediated cytolysis.17,41 Our study clearly indicates that TCR a–deficient mice with IBD produced serum and mucosal antibodies against food and self-antigens (Figures 6 and 7). It is possible that the CD4/ TCR bdim T cells could be key effector T cells that induce these autoantigen-specific antibody responses. A unique feature of the mucosal immune system is the maintenance of appropriate IgA responses to orally administered antigens in the presence of systemic unresponsiveness to these antigens, commonly termed oral tolerance.21,26 Thus, the finding that the IgE antibodies from the diseased mice are strongly reactive with myelin basic protein and milk casein provides good evidence that systemic unresponsiveness to self-antigen and food antigens are abrogated in the diseased mice.
References 1. Strober W, Ehrhardt RO. Chronic intestinal inflammation: an expected outcome in cytokine or T cell receptor mutant mice. Cell 1993;75:203–205. 2. Powrie F. T cells in inflammatory bowel disease: protective and pathogenic roles. Immunity 1995;3:171–174. 3. Philpott KL, Viney JL, Kay G, Rastan S, Gardiner EM, Chae S, Hayday AC, Owen MJ. Lymphoid development in mice congenitally lacking T cell receptor ab-expressing cells. Science 1992;256: 1448–1452. 4. Powrie F, Leach MW, Mauze S, Caddle LB, Coffman RL. Phenotypically distinct subsets of CD4/ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice. Int Immunol 1993;5:1461–1471. 5. Mombaerts P, Mizoguchi E, Grusby MJ, Glimcher LH, Bhan AK, Tonegawa S. Spontaneous development of inflammatory bowel disease in T cell receptor mutant mice. Cell 1993;75:275–282. 6. Morrissey PJ, Charrier K, Braddy S, Liggitt D, Watson JD. CD4/ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice. Disease development is prevented by cotransfer of purified CD4/ T cells. J Exp Med 1993;178:237–244. 7. Wen L, Roberts SJ, Viney JL, Wong FS, Mallick C, Findly RC, Peng Q, Craft JE, Owen MJ, Hayday AC. Immunoglobulin synthesis and generalized autoimmunity in mice congenitally deficient in ab(/) T cells. Nature 1994;369:654–658. 8. Holla¨nder GA, Simpson SJ, Mizoguchi E, Nichogiannopoulu A, She J, Gutierrez-Ramos JC, Bhan AK, Burakoff SJ, Wang B, Terhorst C. Severe colitis in mice with aberrant thymic selection. Immunity 1995;3:27–38.
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9. Ku¨hn R, Lo¨hler J, Rennick D, Rajewsky K, Mu¨ller W. Interleukin10-deficient mice develop chronic enterocolitis. Cell 1993;75: 263–274. 10. Sadlack B, Merz H, Schorle H, Schimpl A, Feller AC, Horak I. Ulcerative colitis-like disease in mice with a disrupted interleukin2 gene. Cell 1993;75:253–261. 11. Sadlack B, Lo¨hler J, Schorle H, Klebb G, Haber H, Sickel E, Noelle RJ, Horak I. Generalized autoimmune disease in interleukin-2deficient mice is triggered by an uncontrolled activation and proliferation of CD4/ T cells. Eur J Immunol 1995;25:3053–3059. 12. Ma A, Datta M, Margosian E, Chen J, Horak I. T cells, but not B cells, are required for bowel inflammation in interleukin 2-deficient mice. J Exp Med 1995;182:1567–1572. 13. Kra¨mer S, Schimpl A, Hu¨nig T. Immunopathology of interleukin (IL) 2-deficient mice: thymus dependence and suppression by thymus-dependent cells with an intact IL-2 gene. J Exp Med 1995; 182:1769–1776. 14. Willerford DM, Chen J, Ferry JA, Davidson L, Ma A, Alt FW. Interleukin-2 receptor a chain regulates the size and content of the peripheral lymphoid compartment. Immunity 1995;3:521–530. 15. Suzuki H, Ku¨ndig TM, Furlonger C, Wakeham A, Timms E, Matsuyama T, Schmits R, Simard JJL, Ohashi PS, Grisser H, Taniguchi T, Paige CJ, Mak TW. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor b. Science 1995;268: 1472–1475. 16. Cao X, Shores EW, Hu-Li J, Anver MR, Kelsall BL, Russell SM, Drago J, Noguchi M, Grinberg A, Bloom ET, Paul WE, Katz SI, Love PE, Leonard WJ. Defective lymphoid development in mice lacking expression of the common cytokine receptor g chain. Immunity 1995;2:223–238. 17. Mizoguchi A, Mizoguchi E, Chiba C, Spiekerman GM, Tonegawa S, Nagler-Anderson C, Bhan AK. Cytokine imbalance and autoantibody production in T cell receptor-a mutant mice with inflammatory bowel disease. J Exp Med 1996;183:847–856. 18. Powrie F, Leach MW, Mauze S, Caddle LB, Coffman RL. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4/ T cells. Immunity 1994; 1:553–562. 19. Mombaerts P, Clarke AR, Rudnicki MA, Iacomini J, Itohara S, Lafaille JJ, Wang L, Ichikawa Y, Jaenisch R, Hooper ML, Tonegawa S. Mutations in T-cell antigen receptor genes a and b block thymocyte development at different stages. Nature 1992;360:225– 231. 20. Takahashi I, Marinaro M, Kiyono H, Jackson RJ, Nakagawa I, Fujihashi K, Hamada S, Clements JD, Bost KL, McGhee JR. Mechanisms for mucosal immunogenicity and adjuvancy of Escherichia coli labile enterotoxin. J Infect Dis 1996;173:627–635. 21. Fujihashi K, Taguchi T, Aicher WK, McGhee JR, Bluestone JA, Eldridge JH, Kiyono H. Immunoregulatory functions for murine intraepithelial lymphocytes: g/d T cells receptor-positive (TCR/) T cells abrogate oral tolerance, while a/b TCR/ T cells provide B cell help. J Exp Med 1992;175:695–707. 22. Jackson RJ, Fujihashi K, Xu-Amano J, Kiyono H, Elson CO, McGhee JR. Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant. Infect Immun 1993;61:4272– 4279. 23. Hiroi T, Fujihashi K, McGhee JR, Kiyono H. Polarized Th2 cytokine expression by both mucosal gd and ab T cells. Eur J Immunol 1995;25:2743–2751. 24. VanCott JL, Staats HF, Pascual DW, Roberts M, Chatfield SN, Yamamoto M, Coste M, Carter PB, Kiyono H, McGhee JR. Regulation of mucosal and systemic antibody responses by T helper cell subsets, macrophages, derived cytokines following oral immunization with live recombinant Salmonella. J Immunol 1996; 156:1504–1514. 25. Kaye J, Porgelli S, Tite J, Jones B, Janeway CA. Both a monoclonal
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antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen-presenting cells in the activation of T cells. J Exp Med 1983;158:836–856. Takahashi I, Nakagawa I, Kiyono H, McGhee JR, Clements JD, Hamada S. Mucosal T cells induce systemic anergy for oral tolerance. Biochem Biophys Res Commun 1995;206:414–420. Nakagawa I, Takahashi I, Kiyono H, McGhee JR, Hamada S. Oral immunization with the B subunit of the heat-labile enterotoxin of Escherichia coli induces early Th1 and late Th2 cytokine expression in Peyer’s patches. J Infect Dis 1996;173:1428–1436. Koide Y, Kaidoh T, Yanagawa T, Yoshida TO. A comparative study on T cell receptor Vb gene usages: spleen cells from the nonobese diabetic (NOD) mouse and its non-diabetic sister strain, the ILI mouse, and infiltrating T cells into pancreata of NOD mice. Microbiol Immunol 1993;37:653–659. Yokoi H, Natsuyama S, Iwai M, Noda Y, Mori T, Mori K, Fujita K, Nakayama H, Fujita J. Non-radioisotopic quantitative RT-PCR to detect changes in mRNA levels during early mouse embryo development. Biochem Biophys Res Commun 1993;195:769–775. Chelly J, Kaplan JC, Marie P, Gautron S, Kahn S. Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature 1988;333:858–860. Takahashi I, Kiyono H, Jackson RJ, Fujihashi K, Staats HF, Hamada S, Clements JD, Bost KL, McGhee JR. Epitope maps of the Escherichia coli heat-labile toxin B subunit for development of a synthetic oral vaccine. Infect Immun 1996;64:1290–1298. Mega J, Bruce MG, Beagley KW, McGhee JR, Taguchi T, Pitts AM, McGhee ML, Bucy RP, Eldridge JH, Mestecky J, Kiyono H. Regulation of mucosal responses by CD4/ T lymphocytes: effects of anti-L3T4 treatment on the gastrointestinal immune system. Int Immunol 1991;3:793–805. Mombaerts P, Mizoguchi E, Ljunggren HG, Iacomini J, Ishikawa H, Wang L, Grusby MJ, Glimcher LH, Winn HJ, Bahn AK, Tonegawa S. Peripheral lymphoid development and function in TCR mutant mice. Int Immunol 1994;6:1061–1070. Viney JL, Dianda L, Roberts SJ, Wen L, Mallick CA, Hayday AC, Owen MJ. Lymphocyte proliferation in mice congenitally deficient
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in T-cell receptor ab/ cells. Proc Natl Acad Sci USA 1994;91: 11948–11952. Kishi H, Borgulya P, Scott B, Karjalainen K, Traunecker A, Kaufman J, von Boehmer H. Surface expression of b T cell receptor chain (TCR) in absence of other TCR or CD3 proteins on immature T cells. EMBO J 1991;10:93–100. Fujihashi K, McGhee JR, Kweon M, Cooper MD, Tonegawa S, Takahashi I, Hiroi T, Mestecky J, Kiyono H. g/d T cell-deficient mice have impaired mucosal immunoglobulin A responses. J Exp Med 1996;183:1929–1935. Brandtzaeg P, Halstensen TS, Kett K, Krajci P, Kvale D, Rognum TO, Scott H, Sollid LM. Immunobiology and immunopathology of human gut mucosa: humoral immunity and intraepithelial lymphocytes. Gastroenterology 1989;97:1562–1584. Roche JK, Fiocchi C, Youngman K. Sensitization to epithelial antigens in chronic mucosal inflammatory disease: characterization of human intestinal mucosa-derived mononuclear cells reactive with purified epithelial cell-associated components in vitro. J Clin Invest 1985;75:522–530. Das KM, Dasgupta A, Mandal A, Geng X. Autoimmunity to cytoskeletal protein tropomyosin: a clue to the pathogenic mechanism for ulcerative colitis. J Immunol 1993;150:2487–2493. Snook JA, Chapman RW, Fleming K, Jewell DP. Anti-neutrophil nuclear antibody in ulcerative colitis, Crohn’s disease and primary sclerosing cholangitis. Clin Exp Immunol 1989;76:30–33. Halstensen TS, Mollnes TE, Brandtzaeg P. Persistent complement activation in submucosal blood vessels of active inflammatory bowel disease: immunohistochemical evidence. Gastroenterology 1989;97:10–19.
Received August 9, 1996. Accepted February 27, 1997. Address requests for reprints to: Shigeyuki Hamada, D.D.S., Ph.D., Department of Oral Microbiology, Osaka University, Faculty of Dentistry, 1-8 Yamadaoka, Suita-Osaka 565, Japan. Fax: (81) 6-8784755. Supported by Grant-in-Aid 07771604 and 08771572 from the Ministry of Education, Science, and Culture of Japan (to I. T.).
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CD4/ T-cell Population Mediates Development of Inflammatory Bowel Disease in T-cell Receptor a Chain– Deficient Mice ICHIRO TAKAHASHI,*,‡ HIROSHI KIYONO,‡ and SHIGEYUKI HAMADA* *Department of Oral Microbiology, Faculty of Dentistry, and ‡Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita-Osaka, Japan
Background & Aims: Increase of T cells expressing CD4 and T-cell receptor (TCR) a0b/ (bdim) was observed in the mucosal and peripheral lymphoid tissues of TCR a0/0 mice with inflammatory bowel disease (IBD). The aim of this study was to characterize the CD4/ TCR a0b/ T cells. Methods: Cytokine production, TCR Vb usage, and helper function for Peyer’s patch B cells by the CD4/ TCR a0b/ T cells were assessed. Results: The CD4/ TCR a0b/ T cells purified from mesenteric lymph nodes and lamina propria of the intestine of IBD mice exclusively produced interleukin 4, used selected subsets (Vb6, Vb8, Vb14, and Vb15) of TCR, and massively proliferated after stimulation with staphylococcal enterotoxin B. Addition of the CD4/ TCR a0b/ T cells to Peyer’s patch B-cell cultures markedly enhanced immunoglobulin (Ig) A, IgG, and IgM antibody responses. Furthermore, depletion of the TCR a0b/ T cells with monoclonal antibody against TCR b chain completely suppressed the onset of IBD and polyclonal B-cell activation in the TCR a0/0 mice. Conclusions: These findings suggest the CD4/ TCR a0b/ T cells–mediated development of IBD in TCR a0/0 mice.
I
n the last 3 years, a number of murine models of inflammatory bowel disease (IBD) have been developed.1,2 These IBD mice showed the common feature of disrupting a T cell–dependent regulatory system that includes alterations in T-cell subpopulations and T-cell selection3 – 8 as well as those with targeted disruption of cytokine genes9 – 13 and cytokine receptor genes.14,15 Results obtained from these experimental IBD models indicate that either destruction of homeostasis in regulatory T cells or emergence of the forbidden T cells plays a crucial role in the development of intestinal immune disorders. It has also been shown that ubiquitous nonpathogenic microorganisms in the gut flora worsen IBD, whereas IBD is much milder in symptoms and slower in progress, or even completely absent, in germfree mice.10 Accumulated results from a number of these different experimental models of IBD indicated that a subpopulation of CD4/ T cells and their cytokines play a critical / 5e1d$$0011
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role for the induction of IBD. For example, either Thelper type 1 (Th1) or Th2 subset supports the development of IBD. Th2 cells and their cytokines, particularly interleukin (IL)-4, have been suggested to enhance the development of IBD.7,16,17 IL-2 gene–disrupted mice that harbored abnormal B-cell responses, including production of autoantibodies and anti–gut constituent antibodies, developed IBD.10 On the other hand, it was shown that CD4//CD45RBhigh T-cell transfer model of colitis in mice with severe combined immunodeficiency disease resembles a dysregulated Th1 response.4,18 In this study, we investigated the cellular and molecular characteristics of CD4/ T-cell receptor (TCR) a0b/ (bdim) T cells in mucosal and systemic compartments of TCR a0/0 mice with IBD. We show that CD4/ TCR bdim T cells become increasingly detectable with age in the mucosal (Peyer’s patches, intestinal lamina propria, and mesenteric lymph nodes) and peripheral lymphoid organs (spleen) of TCR a0/0 mice. The increase of the aberrant CD4/ TCR bdim T cells accompanied the elevated immunoglobulin (Ig) secretion, anti–self constituent and food antibodies, including those of IgE isotype (i.e., disruption of naturally induced peripheral tolerance), and development of ulcerative colitis in the TCR a–deficient mice. These aberrant T cells selectively produced IL-4, used limited Vb genes, and strongly responded on stimulation with staphylococcal enterotoxin B (SEB). They could also support the development of germinal centers of Peyer’s patches and spleen and provide helper function for B-cell differentiation to IgM-, IgG-, and IgA-producing cells. Erroneous selection and expansion of the CD4/ TCR bdim T cells in extrathymic lymphoid tissue may be responsible for the aberrant imAbbreviations used in this paper: CCD, charge-coupled device; ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme-linked immunospot; IFN-g, interferon gamma; IL, interleukin; MAb, monoclonal antibody; PBS-T, PBS containing 0.05% Tween 20; RT-PCR, reverse-transcription polymerase chain reaction; SEB, staphylococcal enterotoxin B; TCR, T-cell receptor; Th, T helper. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
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mune responses occurring in TCR a0/0 mice that may account for the development of IBD.
phosphatase buffer (100 mmol/L Tris-HCl [pH 9.5] containing 100 mmol/L NaCl and 5 mmol/L MgCl2 ).
Cytokine-Specific ELISPOT
Materials and Methods Mice TCR a– and TCR d–deficient (TCR a0/0 and TCR d ) mice with a background of (129 1 C57BL/6) were obtained from the Jackson Laboratory (Bar Harbor, ME). C57BL/ 6 mice were purchased from Charles River Japan (Kanagawa, Japan). TCR a0/0 and TCR d0/0 mice were originally generated by a targeted disruption of TCR genes in embryonic stem cells.19 The mice were housed in two rooms in the Experimental Animal Facility at the Research Institute for Microbial Diseases at Osaka University. All mice were kept on a 12hour light-dark cycle, housed under specific pathogen–free conditions, and received sterilized food (Certified diet MF; Oriental Yeast Co., Osaka, Japan) and autoclaved distilled water ad libitum. The cages were changed every 5 days, and the mice were handled with sterilized gloves. 0/0
Reagents SEB was purchased from Sigma Chemical Co. (St. Louis, MO). Purified a-casein from bovine milk was obtained from Wako Pure Chemicals (Osaka, Japan). Myelin basic protein from bovine brain was purchased from Sigma. Protein extracts from soy bean and wheat, the main constituents of the sterilized food, were provided by Oriental Yeast Co.
Isolation of Lymphoid Cells From MucosaAssociated Tissues and Spleen The spleen and mesenteric lymph nodes were aseptically removed, and single cell suspensions were prepared by a standard mechanical disruption procedure as described previously.20,21 Single-cell suspensions of Peyer’s patches and the lamina propria lymphocytes were prepared by an enzymatic dissociation method using Dispase (Boehringer Mannheim Biochemicals, Indianapolis, IN) as described previously.20,21 In these procedures, the cell viability of Peyer’s patches and spleen were ú98% and that of lamina propria lymphocytes were ú95% viable. Antigen-reactive and total IgA, IgG, and IgM antibodyforming cells were evaluated in mononuclear cells isolated from mucosa-associated and peripheral lymphoid tissues by a modified enzyme-linked immunospot (ELISPOT) assay as described previously.20,22 Nitrocellulose microtiter plates (Millipore Co., Bedford, MA) were coated with 100 mL of anti-mouse IgA, IgE, IgG, or IgM (Southern Biotechnology Associates, Birmingham, AL) for total Ig antibody-forming cells or various antigens (e.g., protein extracts from soybean and wheat) for antigen-reactive antibody-forming cells at a concentration of 5 mg/mL in phosphate-buffered saline (PBS). For the detection of isotype and antigen-reactive antibody-producing cells, alkaline phosphatase–conjugated anti-mouse IgA, IgG, or IgM (1 mg/mL; Southern Biotechnology Associates) was added and then visualized with the substrate, 5-bromo-4-chloro-3-indolyl phosphate (Wako)/nitroblue tetrazolium (Wako) in alkaline
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For the assessment of cytokine production by CD4/ TCR b T cells at the single-cell level, cytokine-specific ELISPOT assay was used. Purified CD4/ TCR bdim T cells were sorted by FACStar Plus (Becton Dickinson, Palo Alto, CA) from the TCR a0/0 mice with IBD. Cells producing IL-2, interferon (IFN)g, IL-4, IL-6, and IL-10 were detected by the cytokine-specific ELISPOT assay as described previously.23,24 In brief, 96-well Multiscreen HA plates equipped with 0.45-mm cellulose ester membrane (Millipore) were coated with the rat monoclonal antibodies (MAbs) anti-mouse IL-2 (clone no. JES6-1A12), IL-4 (BVD4-1D11), IL-6 (MP5-20F3), IL-10 (JES5-2A5), or IFN-g (R4-6A2) (20 mg/mL) at 47C for 16 hours. Then the plates were washed three times with PBS, followed by preincubation with RPMI 1640 (Sigma) containing 10% fetal calf serum for 1 hour at 377C. Thereafter, different concentrations of purified CD4/ TCR bdim T cells (105 –103 cells in 100 mL of RPMI 1640 supplemented with insulin-transferrin-selenium-S [Life Technologies, Gaithersburg, MD]) were added to the plates and incubated at 377C overnight in a humidified incubator at 5% CO2 . As positive controls for cytokine-specific ELISPOT assay, a Th clone (D10.G4.1) specific for conalbumin25 and SEB-stimulated Peyer’s patch CD4/ T cells from TCR a/// mice were used. The plates were then washed with PBS followed by PBS containing 0.05% Tween 20 (PBS-T) (Wako). Subsequently, biotinylated rat antimouse IL-2 (JES6-5H4), IL-4 (BVD6-24G2), IL-6 (MP5-32C11), IL-10 (SXC-1), or IFN-g (XMG1.2) MAbs (5 mg/mL) were added, and the incubation was performed at 47C overnight. After washing with PBS-T, streptavidin-peroxidase (1:2000 dilution; Zymed Laboratories, San Francisco, CA) was added for 1 hour at 377C. The plates were washed again in PBS-T and the substrate solution containing 3-amino-9-ethylcarbazole (Aldrich Chemical Co., Milwaukee, WI) and H2O2 was added. Cytokine-specific spots were quantitated by using an Olympus SZH stereo-zoom microscope (Olympus, Tokyo, Japan). dim
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Enzyme-Linked Immunosorbent Assay Antibody concentrations in sera from TCR a0/0 mice with or without IBD were determined by enzyme-linked immunosorbent assay (ELISA).26 ELISA plates (Nunc, Naperville, IL) were coated with 100 mL of 5 mg/mL of goat anti-mouse IgM, IgG, or IgA or rat anti-mouse IgE (Southern Biotechnology Associates) in PBS. Serial twofold dilutions of sera or fecal extracts from 5 individual mice were added (100 mL/well). Fecal extracts were prepared by mixing fresh feces with PBS containing 0.5% sodium azide (100 mg wet weight of feces per milliliter), and the centrifuged supernatant was used as fecal extract for ELISA.27 After 2-hour incubation at 377C, unbound antibodies were removed and alkaline phosphatase– conjugated goat or rat anti-mouse m, g, a, and e heavy chain (Southern Biotechnology Associates) was added. The plates were incubated at 377C for 2 hours and developed with pnitrophenyl phosphate (1 mg/mL; Wako) in 10 mmol/L di-
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ethanolamine buffer (pH 9.6). Antibody concentrations were calculated from the standard curve using purified mouse IgA, IgG, IgM, or IgE (Southern Biotechnology Associates).
In Vitro T-cell Proliferative Responses Mesenteric lymph nodes and spleen were aseptically removed, and single-cell suspensions of the tissues were prepared by mechanical disruption, washed extensively, and resuspended in RPMI 1640 (Sigma) supplemented with sodium bicarbonate, HEPES (15 mmol/L), L-glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (100 mg/mL), and gentamicin (100 mg/mL). The CD3/ T cells were enriched by negative panning on Petri dishes coated with goat anti-mouse IgG F(ab*)2 (20 mg/mL; Jackson ImmunoResearch Laboratories, West Grove, PA). CD4/ T cells were further purified by positive panning on a Petri dish coated with anti-CD4 MAb.27 The MAb specific for CD4 (clone GK1.5, American Type Culture Collection, Rockville, MD) was purified from ascites through a protein G column (Pharmacia LKB, Uppsala, Sweden). The purified CD4/ T cell population was ú95% CD4/ CD80 as determined by flow cytometry.27 The CD4/ T cells (1 1 105/well) were cultured in flat-bottom 96-well microculture plates (Corning, Corning, NY) in the presence of mitomycin C–treated feeder spleen cells (1 1 104 cells/well) for 3 days.26 SEB-specific CD4/ T-cell proliferative responses were assessed by addition of 0.5 mCi of [3H]thymidine (ICN, Costa Mesa, CA) 6 hours before the cell collection. The level of proliferation was determined by liquid scintillation counting. As a control, CD4/ T cells were cultured with feeder cells in the absence of SEB.
Reverse-Transcription Polymerase Chain Reaction Reverse-transcription polymerase chain reaction (RTPCR) analysis was performed to detect cytokine and TCR Vb messenger RNA (mRNA) expression according to the method previously described.23,27 To isolate RNA from the fluorescenceactivated cell sorter (FACS) purified CD4/ T cells, TRIzol reagent (Life Technologies) was used. The RNA was reverse-transcribed into complementary DNA (cDNA) by use of 10 mL of an RT mixture containing 10 ng of total cellular RNA, 11 PCR buffer composed of 10 mmol/L Tris-HCl (pH 8.3) and 50 mmol/L KCl, 5 mmol/L MgCl2 , 1 mmol/L each deoxynucleoside triphosphate, 10 U of RNase inhibitor (Promega, Madison, WI), 0.1 mg of oligo(dT16), and 2 U of Superscript II reverse transcriptase (Life Technologies). The mixture was incubated at 427C for 30 minutes, heated to 957C for 5 minutes, and chilled quickly on ice. The PCR mixture for TCR Vb–specific mRNA analysis consisted of 11 PCR buffer, 2 mmol/L MgCl2 , 50 mmol/L deoxynucleoside triphosphate, 0.4 mmol/L Vb-specific sense primer28 and Cb antisense primers, and 1.25 U of AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Branchburg, NJ) in a total volume of 50 mL. PCR amplification was performed in sequential 21–33 cycles at 947C for 1 minute and 607C for 1 minute with a 727C extension step for 7 minutes after the last cycle with GeneAmp PCR System 9600 (Perkin-Elmer Cetus). One fifth of the amplified products
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were separated by electrophoresis in 2% agarose gel, and amplified products were visualized with ethidium bromide (1 mg/mL). The intensity of ethdium bromide fluorescence of each TCR Vb–Cb bands was measured by the charge-coupled device (CCD) imaging system (Densitograph AE 6900-F; Atto Co., Tokyo, Japan) as previously described.29 To normalize the condition of gel staining and CCD imaging, a constant amount of control DNA (b-actin cDNA) was electrophoresed in every time. Quantitation of PCR products was performed by a formula described by Chelly et al.30 Briefly, the extent of amplification (Y) is given by the formula: Y Å A(1 / R)n, where A is the initial amount of the original template (cDNA), R is the efficiency, and n is the number of cycles. It is possible to estimate Y by measuring the intensity of ethidium bromide fluorescence of amplified products by CCD imaging system, and R can be deduced from the slope of the semi-log plot. Thus, the proportion of a certain Vb gene expressed to total Vb genes expressed was calculated: %Vbx Å A(Vbx) 1 100/ [A(Vb1) / A(Vb2) / rrrrr / A(Vb19)].
Helper Assay To examine helper functions of CD4/ TCR bdim T cells in intestinal lamina propria, Peyer’s patch B cells were prepared by the treatment of lymphocytes with two cycles of anti-Thy 1.2 MAb (Cedarlane, Ontario, Canada) and low-toxic guinea pig complement (Cedarlane), followed by fractionation on discontinuous gradient of Percoll (Pharmacia LKB) as described previously23 and resuspended in complete RPMI 1640 consisting of sodium bicarbonate, L-glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (100 mg/mL), and 10% fetal calf serum. Purified CD4/ TCR bdim T cells (105 cells per well) were added to individual B-cell cultures (2 1 106 cells per well). In some experiments, 100 mg/mL of MAb anti– IL-4 (BVD4-1D11) was added to these cultures. As controls, CD4/ T cells purified from intestinal lamina propria of TCR a/// mice were added to Peyer’s patch B-cell cultures. These cultures were incubated for 4 days at 377C in humidified atmosphere of 5% CO2 . Nonadherent, viable B cells were fractionated by discontinuous Percoll density gradient centrifugation, and the number and isotype of Ig-producing cells were determined by ELISPOT assay.
Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Western Immunoblotting Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed as described previously.31 Purified casein and myelin basic protein (1 mg each) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred electrophoretically to a nitrocellulose sheet. The sheet was then treated either with a 1:40 dilution of sera from the mice with or without IBD for 2 hours at 257C. The antibody bound to the immobilized replica proteins was incubated with a 1:1000 dilution of alkaline phosphatase– conjugated rat anti-mouse e heavy chain (Southern Biotechnology Associates) for 2 hours at 257C. The sheet was then washed
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with PBS-T and developed with the substrate 5-bromo-4chloro-3-indolyl phosphate (Wako)/nitroblue tetrazolium (Wako) in alkaline phosphatase buffer (100 mmol/L Tris-HCl [pH 9.5] containing 100 mmol/L NaCl and 5 mmol/L MgCl2 ).
Flow Cytometric Analysis and Cell Sorting Immunofluorescent analysis was performed using a FACScan flow cytometry (Becton Dickinson). Cells stained with single-color reagent were used to set the appropriate compensation levels, and at least 10,000 events were analyzed. Cell sorting was performed on a FACStar (Becton Dickinson). The following MAb from Pharmingen (San Diego, CA) were used: anti-CD4 (RM4-5), anti-CD5 (53-7.3), anti-TCRb (H57-597), anti-TCR Vb4 (KT4), anti-TCR Vb5.1, 5.2 (MR9-4), anti-TCR Vb6 (RR4-7), anti-TCR Vb7 (TR310), anti-TCR Vb8.1, 8.2 (MR5-2), anti-TCR Vb9 (MR 10-2), anti-TCR Vb10b (B21.5), anti-TCR Vb11 (RR3-15), antiTCR Vb12 (MR11-1), anti-TCR Vb13 (MR12-3), anti-TCR Vb14 (14-2), and anti-TCRd (GL3). For two-color flow cytometry, 1 1 106 cells in 20 mL PBS containing 2% fetal calf serum and 0.02% sodium azide were first incubated with antiFc receptor MAb (2.4G2) to prevent nonspecific staining and then stained with appropriate fluorescein isothiocyanate–conjugated MAb and phycoerythrin-conjugated MAb or biotinylated MAb followed by streptavidin-phycoerythrin.21 All MAbs were used at the saturating concentrations. Flow cytometric analysis was performed with a FACScan flow cytometer using Lysis II software (Becton Dickinson). Results are shown as loglog dot plots. For cell sorting, mononuclear cells were stained simultaneously for CD4 (RM4-5) and ab TCR (H57-597) and were positively selected with a FACStar (Becton Dickinson).
Treatment With Anti-ab TCR (Clone H57597) MAb Young healthy TCR a0/0 mice (4–5 weeks of age) were treated with hamster MAb anti-TCRb (H57-597) purified by protein G–Sepharose CL-4B (Pharmacia LKB) as described previously.26 Mice were injected once a week with the MAb (1 mg in a volume of 200 mL) intraperitoneally for 12 weeks according to the protocol described previously.32 Control mice were injected with hamster IgG (Pharmingen).
had extended to the ileum, colon, and rectum with marked hyperplasia, dilatation, and thickening of the colon wall compared with the TCR a0/0 mice without IBD. Histological examination showed marked hyperplasia, elongation, and distortion of crypts, with areas of microabscesses, reduced numbers of goblet cells, and infiltration of inflammatory lymphocytes within the lamina propria of the colon wall (data not shown). Enlargement of Peripheral Lymphoid Tissues Associated With Enhanced B-cell Responses TCR a–deficient mice between 4 and 6 months of age with IBD were found to have developed massive enlargement of Peyer’s patch, mesentric lymph nodes, and spleen. Histological examination of Peyer’s patch revealed a marked expansion of lymphoid tissues with abnormally developed germinal centers (data not shown). There were striking elevations of serum and fecal antibodies in the TCR a0/0 mice with IBD (Table 1). The level of IgA in fecal sample of the mice with IBD was comparable to those of the normal C57BL/6 (TCR a///) mice. In contrast, extremely lower levels of IgA were seen in fecal samples obtained from the TCR a0/0 mice without IBD. A similar pattern was also observed with serum IgA (Table 1). The levels of serum IgG and IgM in the TCR a0/0 mice with IBD were also dramatically increased to the levels of the TCR a/// mice. Furthermore, elevated IgE antibodies were seen in the serum of the TCR a0/0 mice with IBD but not the TCR a0/0 mice without IBD and the TCR a/// mice (Table 1). Concerning antibody-producing cells in lamina propria of the small and large intestines, Peyer’s patch, mesenteric lymph nodes, and spleen from the TCR a0/0 mice with
Table 1. Comparison of Ig Levels in Feces and Serum of TCR a0/0 Mice With and Without IBD TCR a0/0 mice
Statistical Method Significant differences between mean values were determined by Student’s t test. P values of õ0.05 were considered statistically significant.
Sample Feces (ng/100 mg)
Results Morphometric Assessment of the Large Intestine of TCR a0/0 Mice
Serum (mg/mL)
Starting at 4–6 months of age, approximately 60% of the TCR a0/0 mice were found to have developed IBD, characterized by anorectal prolapse, diarrhea, and wasting syndrome such as weight loss and hunched posture. The necropsy revealed that there was bowel inflammation, which / 5e1d$$0011
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Isotype
With IBD
IgA IgE IgG IgM
10,000 { 86 0a 640 { 45 310 { 20
IgA IgE IgG IgM
164 { 11 2.5 { 0.2 640 { 30 320 { 12
Without IBD
TCR a///
1240 { 48 9800 { 24 0 0 644 { 65 84 { 7 0 0 5.0 { 1 0 5.1 { 0.4 5.2 { 0.2
204 { 5 0 820 { 60 200 { 16
NOTE. Data represent mean { SD from 5 mice per group of three separate experiments. a Zero indicated that antibody was undetectable.
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Figure 1. Correlation of IBD induction in the TCR a0/0 mice with selected increases in IgA(j), IgG- (h), and IgM-producing ( ) cells. Mononuclear cells isolated from spleen, mesenteric lymph nodes, Peyer’s patch, and lamina propria of the small intestine of TCR a0/0 mice with or without IBD were examined by isotype-specific ELISPOT.
IBD, increase in IgA antibody–producing cells was mainly found in the lamina propria followed by mesenteric lymph nodes and Peyer’s patch, while IgG-producing cells were predominantly found in mesenteric lymph nodes followed by spleen and lamina propria (Figure 1). Although the frequency of IgM-producing cells was lower compared with the other two isotypes, these isotype antibody-producing cells were noted in lamina propria, mesenteric lymph nodes, and spleen. The number of total Ig-producing cells in lamina propria of the small intestine were similar to those in the large intestine. Increase of CD4/ TCR bdim T Cells in TCR a0/0 Mice With IBD A unique subset of T cells that could be co-stained with MAb anti-CD4 (RM4-5) and MAb H57-597 spe-
cific for pan TCRb became increasingly detectable in the mucosa-associated and peripheral lymphoid tissues in TCR a–deficient mice with IBD (Figure 2). The intensity of staining with MAb H57-597 was weak, and these T-cell populations (approximately 6%–9% of lymphocyte population) were termed CD4/ TCR bdim T cells. These cells had developed in both mucosa-associated lymphoid tissues (e.g., intestinal epithelium, lamina propria, and mesenteric lymph nodes) and the spleen of the TCR a0/0 mice with IBD. The Peyer’s patch of the mice also contained a similar level (6% of lymphocyte population) of CD4/ TCR bdim T cells. In contrast, only a few cells of this subset were seen in the TCR a0/0 mice without IBD (0%–0.8%). Furthermore, these T cells were undetectable in the normal TCR a/// mice (data not shown).
Figure 2. Flow cytometric analysis of lymphocytes isolated from spleen, mesenteric lymph nodes (MLN), and lamina propria (LP) in the large intestine and intestinal intraepithelial lymphocytes (IEL) in the TCR a0/0 mice with IBD (left panels ) and the TCR a0/0 mice without IBD (right panels ). Cell suspensions were stained with MAbs specific for fluorescein isothiocyanate–TCR b and phycoerythrin-CD4.
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Table 2. Numbers of Th1- and Th2-Type Cytokine-Producing CD4/ TCR bdim T Cells Isolated From MucosaAssociated Tissues of TCR a0/0 Mice With IBD Numbers of cytokine-producing cells per 106 cells
Cytokine
LPL
MLN
PP
IFN-g IL-2 IL-4 IL-6 IL-10
0 0 18,300 { 1700 0 0
0 0 520 { 140 0 0
0 0 1300 { 50 0 0
D10.G4.1 (Th2 clone)
2450 6780 25,620 5170
0 { { { {
210 540 1740 420
NOTE. The results are representative of five separate experiments. LPL, lamina propria lymphocytes; MLN, mesenteric lymph nodes; PP, Peyer’s patches.
Histochemical examination of colon and spleen also revealed TCR bdim cells. TCR bdim T cells were observed in the parafollicular region (i.e., T-cell area) of spleen (data not shown). The CD4/ TCR bdim T cells in spleen might support the development of germinal centers of these lymphoid tissues. Focal clusters of the TCR bdim T cells were found in the lamina propria of the colon, suggesting that clonal expansion of the TCR bdim T cells was induced by prolonged exposure to luminal food and bacterial antigens (data not shown). Cytokine Production by CD4/ TCR bdim T Cells The CD4/ TCR bdim T cells isolated from mucosa-associated lymphoid tissues of the TCR a0/0 mice with IBD prominently contained IL-4–producing cells (Table 2). However, none of the IL-2, IFN-g, IL-6, and IL-10 cytokine-producing cells were detected in the FACS-purified CD4/ TCR bdim T cells. To confirm valid-
Figure 4. RT-PCR analysis of TCR Vb repertoire usage of CD4/ TCRbdim cells from intestinal lamina propria of TCR a0/0 mice with IBD. The percentages of each TCR Vb mRNA expression were calculated from PCR products as described in Materials and Methods. The percentage of T cells was the mean of five separate experiments { SD.
ity of our ELISPOT assay, a Th clone (D10.G4.1) was used as a positive control; the Th clone produced spots in IL-2–, IL-4–, IL-6–, and IL-10–specific cytokine assay (Table 2). SEB-stimulated Peyer’s patch CD4/ T cells from TCR a/// mice could form spots specific for IFN-g (1530 { 210/104 cells), IL-2 (980 { 57/104 cells), and IL-4 (680 { 72/104 cells). Furthermore, cytokinespecific RT-PCR analysis revealed the expression of IL4–specific message in RNA extracted from the purified CD4/ TCR bdim T cells (data not shown). This finding is the first direct demonstration that the CD4/ TCR bdim T cells predominantly produce IL-4 in the TCR a0/0 mice with IBD. Responses of the CD4/ TCR bdim T Cells to SEB TCR Vb usage of these CD4/ TCR bdim T cells was then examined by flow cytometry and Vb family-
Figure 3. Flow cytometric analysis of TCR Vb repertoire of CD4/ TCR bdim cells from the lamina propria of intestine of TCR a0/0 mice with IBD. Cell suspensions were stained with MAbs specific for fluorescein isothiocyanate–TCR Vb and phycoerythrin-CD4. The percentages of T cells bearing each TCR Vb were calculated as 100 1 (% Vbx)/%(Vb4 / Vb5 / Vb6 / Vb7 / Vb8 / Vb9 / Vb10 / Vb11 / Vb12 / Vb13 / Vb14). The percentage of T cells was the mean of five separate experiments { SD.
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Figure 5. T-cell proliferative responses to SEB in CD4/ T-cell cultures prepared from the TCR a–deficient mice with IBD and from the TCR a/// mice. [3H]thymidine uptake in CD4/ T-cell cultures in the absence of SEB was 1780 { 145 for mesenteric lymph nodes (MLN) and 2300 { 230 for spleen (SP) in diseased TCR a0/0 mice and 1035 { 61 for mesenteric lymph nodes and 1473 { 85 in TCR a/// mice, respectively. The results presented are representative of five separate experiments. Data are expressed as mean values { SD for triplicate cultures.
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Figure 6. Comparison of antibody responses reactive with soybean and wheat in TCR a0/0 mice with IBD and TCR a/// mice. Mononuclear cells isolated from spleen and lamina propria of both TCR a0/0 mice with IBD and TCR a/// mice were subjected to the soybeanand wheat-reactive ELISPOT assay. Results are expressed as mean values { SD.
specific RT-PCR. The flow cytometric analysis revealed that the CD4/ TCR bdim T cells used TCR Vb8 most frequently, followed by TCR Vb14 and TCR Vb6 (Figure 3). To examine the potential expression of TCR Vb for which antibodies were not available to us, analysis of TCR Vb gene usage by the CD4/ TCR bdim T cells was also conducted by RT-PCR amplification using a panel of TCR Vb–specific oligonucleotide primers (Vb1–Vb19). The RT-PCR analysis revealed Vb6, Vb11, Vb14, and Vb15 mRNA were frequently expressed in the infiltrating CD4/ TCR bdim T cells from lamina propria of the
intestine, while Vb8 message was most frequently detected (Figure 4). Because of the latter finding, it was important to examine whether these CD4/ TCR bdim T cells from the IBD mice proliferated in response to SEB. It was found that these cells massively proliferated in response to SEB (0–10 mg per well) in a dose-dependent manner (Figure 5). The intensity of the [3H]thymidine uptake by the CD4/ T cells from the diseased mice was well over the level of CD4/ T cells from TCR a/// mice. However, CD3/ T cells isolated from the nondiseased TCR a0/0 mice did not respond to SEB (data not shown).
Figure 7. Immunoblots showing reactivity of serum IgE toward (A ) milk casein (food antigen) and (B ) purified myelin basic protein (self antigen). One microgram of purified proteins was analyzed by 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose sheets by electroblotting. The proteins on the sheet allowed to react with serum IgE from the TCR a0/0 mice with IBD, the TCR a0/0 mice without IBD, or the TCR d0/0 mice were detected by alkaline phosphatase–conjugated rat anti-mouse IgE. Molecular sizes are indicated on the left.
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Figure 8. Assessment of helper activity for Peyer’s patch B cells by CD4/ TCR bdim T cells isolated from lamina propria of colon of the TCR a0/0 mice. The CD4/ TCR bdim T cells (1 1 105 cells per well) purified by flow cytometry were cultured with Peyer’s patch B cells (2 1 106 cells per well) for 4 days. Nonadherent cells were collected and analyzed by the isotype-specific ELISPOT assay. To some of the wells, 100 mg per well of anti–IL-4 MAb was added. As controls, CD4/ T cells isolated from TCR a/// mice were added to the culture. Numbers of antibody-forming cells was determined in triplicate cultures and is expressed as the mean value { SD. Numbers of antibodyforming cells in cultures containing Peyer’s patch B cells only were IgA (j), 65 { 4.0/105 antibody-forming cells; IgG (h), 40 { 3.5/105 antibody-forming cells; and IgM ( ), 28 { 2.5/105 antibody-forming cells. The results are representative of five separate experiments.
Unique B-cell Responses in TCR a Mice With IBD
Effect of CD4/ TCR bdim T-cell Depletion on the Development of IBD in TCR a0/0 Mice
0/0
The role of CD4/ TCR bdim T cells for B-cell responses in the TCRa0/0 mice with regard to the development of IBD was examined. The TCR a0/0 mice with IBD were found to show marked B-cell responses reactive with food proteins such as soybean and wheat in the lamina propria of the intestine, mesenteric lymph nodes, and spleen (Figure 6). In particular, lamina propria lymphocytes of the intestine from the mice with IBD contained high numbers of plasma cells that produced IgA antibody reactive with soybean and wheat. It was also interesting to note that high numbers of soybean-reactive IgG-producing cells were detected in the intestinal lamina propria of the TCR a0/0 mice with IBD. In contrast, low numbers of antibody-producing cells reactive with food antigen were seen in the lamina propria and mesenteric lymph nodes of the TCR a/// (C57BL/6) mice. In regard to the systemic lymphoid tissues of the TCR a0/0 mice with IBD, our results revealed the presence of antibody-producing cells reactive with food antigen in spleen (Figure 6). In contrast, spleen of TCR a/// mice did not contain these antibody-producing cells. Furthermore, serum IgE from the TCR a0/0 mice with IBD was found to react strongly with milk casein and myelin-basic protein (Figure 7). Neither the TCR a0/0 mice without IBD nor the TCR a/// mice developed IgE responses against milk casein and myelin-basic protein in serum. Based on the above results, we examined whether the CD4/ TCR bdim T cells found in the TCR a0/0 mice with IBD were capable of providing helper function for / 5e1d$$0011
Peyer’s patch B cells. When the FACS-purified CD4/ TCR bdim T cells were added to Peyer’s patch B-cell cultures without any stimulants, IgA-, IgG-, and IgMproducing cells were prominently demonstrated (Figure 8). The levels of helper function of the CD4/ TCR bdim T cells were comparable to those of Peyer’s patch CD4/ T cells from intestinal lamina propria of TCR a/// mice. Because the CD4/ TCR bdim T cells prominently contained IL-4–producing cells (Table 2), it was important to investigate whether IL-4 was involved in the helper function. When anti–IL4 MAb were added to the Peyer’s patch B-cell cultures in the presence of the CD4/ TCR bdim T cells, induction of IgA- and IgM-producing cells was markedly suppressed, while IgG-producing cells were only partially inhibited by this MAb treatment (Figure 8). These results clearly suggested that the CD4/ TCR bdim T cells exclusively producing IL-4 supported Peyer’s patch B cells to synthesize IgA, IgG, and IgM antibody.
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Young TCR a0/0 mice without IBD were treated with weekly injection of anti-TCRb MAb or hamster IgG (1 mg/mouse). Onset of IBD and appearance of CD4/ TCR bdim T cells were then assessed. Monthly histopathological assessment of TCR a0/0 mice revealed that there were no signs of IBD in the anti-TCRb MAb– treated mice up to 6 months of age (Table 3). In addition, flow cytometric analysis revealed complete deletion of CD4/ TCR bdim T cells in the mesenteric lymph nodes and lamina propria during this period. The absence of CD4/ TCR bdim T cells was always associated with the striking reduction in the numbers of IgA, IgG, and IgM antibody-producing cells in the lamina propria and spleen of the anti-TCRb MAb-treated mice. On the other hand, starting at 4 months of age, 67% of the hamster IgG–treated TCR a chain–deficient mice developed IBD characterized by anorectal prolapse and hyperplasia of the colon. Histological examination of the colon dem-
Table 3. Monoclonal Anti-TCRb Treatment Inhibited the Development of CD4/ TCRbdim T Cells and IBD MAb treatment
Frequency (% gated) of CD4/ TCRbdim T cellsa
Development of IBDb
Anti-TCRb IgG
0.7 { 0.4 13.2 { 4.8
0/9 6/9
a
Data represent mean values { SD from intestinal lamina propria of 3 treated mice. b Numbers of mice developed IBD.
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onstrated that there were hyperplasia of crypts and infiltration of inflammatory lymphocytes within the lamina propria of the hamster IgG-treated TCR a chain–deficient mice (data not shown). In addition, increase of numbers of antibody-forming cells in the lamina propria and spleen were associated with the development of IBD in the hamster IgG–treated mice.
Discussion Experimental IBD has been spontaneously developed in mice incapable of synthesizing specific components involved in the immune systems because of, e.g., alterations in TCR or targeted disruption of cytokine gene.1,2 These experimental IBD models provide us with information about the cellular and molecular mechanisms in development of IBD. For example, there is direct evidence for the involvement of a specific subset of CD4/ T cells in the development of IBD. Powrie et al.4,18 observed that adoptive transfer of CD45 RBhigh CD4/ T cells from BALB/c mice to C.B-17 mice with severe combined immunodeficiency disease results in the intestinal inflammation. Furthermore, cotransfer of the reciprocal CD45 RBlow CD4/ T cells prevented the development of colitis.6 The mature lesions of IBD in TCR a–deficient mice were characterized by a mixed lymphocyte infiltrate in the lamina propria of the colon and a marked enlargement of mucosa-associated peripheral lymphoid tissues such as Peyer’s patch and spleen. We also have found that striking increases in plasma cells producing IgA, IgG, and IgM were always accompanied with the increase of the aberrant CD4/ TCR bdim T cells in gut-associated lymphoid tissues and spleen of the diseased mice (Figures 1 and 2, and Table 1). These enhanced levels of CD4/ T cells that were stained weakly with MAb to TCR b in the periphery of TCR a–deficient mice were first reported by Mombaerts et al.5,19,33 followed by Viney et al.34 Most recently, it was demonstrated that elevation of TCR b/ T cells was always noted in TCR a0/0 mice with IBD but not without the disease.17 To this end, possible association of TCR bdim T cells for the development and regulation of IBD has been suggested.17 The pathological role of the aberrant T cells was clearly supported by the finding that the CD4/ TCR bdim T cells enhanced the synthesis of IgA, IgG, and IgM antibodies from the B cells of TCR a–deficient mice in vitro (Figure 8). Consistent with the Ig class switching of the B cells, the CD4/ TCR bdim T cells were found to exclusively produce IL-4 (Table 2). In addition, depletion of the CD4/ TCR bdim T cells by the treatment of TCR a0/0 mice with MAb TCR b completely suppressed the polyclonal B-cell activation occurring in the mucosal and / 5e1d$$0011
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systemic lymphoid organs and the development of IBD in the TCR a0/0 mice (Table 3). Thus, the appearance of the CD4/ TCR bdim T cells can lead to severe pathological changes in the large intestine and peripheral lymphoid tissues in TCR a0/0 mice. The equivalent subset of the CD4/ TCR bdim T cells in normal mice or humans has not yet been detected; however, rearrangement and expression of the TCR b chain genes is epistatic to those of TCR a chain genes.35 The CD4/ TCR bdim T cells might be positively selected during development both in thymus and extrathymic lymphoid tissues. The TCR bdim T cells increased with age (Figure 2) and exclusively used limited TCR Vb receptors (Figures 3 and 4). These findings suggest that the increase in the CD4/ T cells among peripheral T cells may be the result of a secondary expansion to yet unidentified major histocompatibility complex class II–restricted antigens. The CD4/ TCR bdim T cells are not detected in TCR a 1 major histocompatibility complex class II double– deficient mice, suggesting that they are dependent on major histocompatibility complex class II molecules.31 Because TCR a0/0 mice with IBD showed humoral immune responses to food and self antigens (Figures 6 and 7), it is likely that the CD4/ TCR bdim T cells have specificities to food or self antigens. Development and activation of the CD4/ TCR bdim T cells might be positively regulated by bacterial superantigens. We show in the present study that the CD4/ TCR bdim T cells could massively proliferate by the stimulation with SEB (Figure 5). Our preliminary results show that coagulase-negative, but mannitol-fermenting, staphylococci were always recovered from the stool of mice with IBD. In addition, the CD4/ TCR bdim T cells exposed to bacterial sonicates from the staphylococci strongly proliferated (unpublished observation). Superantigen-like products from the staphylococci might modulate or positively regulate the development of the aberrant CD4/ T cells in TCR a0/0 mice. Hyperreactivity of gd T cells may also lead to the aberrant B-cell responses in the mucosal effector sites such as the lamina propria of the intestine. The T-cell compartment of the TCR a–deficient mice with IBD is composed of largely gd T cells with minor population of the CD4/ TCR bdim T cells.5,7,34 We recently reported that gd T cells serve an important regulatory role for mucosal IgA responses in that TCR d–deficient mice possess normal numbers of functional ab T cells but display markedly decreased IgA responses to oral immunization.36 Mombaerts et al. also reported that severity of disease was significantly reduced when mice lacking ab T cells were crossed with mice lacking gd T cells, WBS-Gastro
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suggesting that gd T cells may also play a role in the pathogenesis of the colitis because of a lack of the ordinary ab T cell–mediated down-regulation of gd T cells.5 It has been suggested that autoantibodies mediate the disease as an organ-specific autoimmune disease in IBD.37 Other investigators reported that antibodies specific for epithelial cell–associated components were found in patients with IBD.38 Other antibodies were reported to be reactive with a 40-kilodalton epithelial cell cytoskeletal microfilament protein belonging to the tropomyosin family.17,39 Along similar lines, it was shown that IBD was associated with antineutrophil cytoplasmic antibodies.40 The antibodies might cause tissue damage in mice with IBD via complement-mediated cytolysis.17,41 Our study clearly indicates that TCR a–deficient mice with IBD produced serum and mucosal antibodies against food and self-antigens (Figures 6 and 7). It is possible that the CD4/ TCR bdim T cells could be key effector T cells that induce these autoantigen-specific antibody responses. A unique feature of the mucosal immune system is the maintenance of appropriate IgA responses to orally administered antigens in the presence of systemic unresponsiveness to these antigens, commonly termed oral tolerance.21,26 Thus, the finding that the IgE antibodies from the diseased mice are strongly reactive with myelin basic protein and milk casein provides good evidence that systemic unresponsiveness to self-antigen and food antigens are abrogated in the diseased mice.
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Received August 9, 1996. Accepted February 27, 1997. Address requests for reprints to: Shigeyuki Hamada, D.D.S., Ph.D., Department of Oral Microbiology, Osaka University, Faculty of Dentistry, 1-8 Yamadaoka, Suita-Osaka 565, Japan. Fax: (81) 6-8784755. Supported by Grant-in-Aid 07771604 and 08771572 from the Ministry of Education, Science, and Culture of Japan (to I. T.).
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