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Clonal Anergy Is a Potent Mechanism of Oral Tolerance in the Suppression of Acute Antigen-Induced Arthritis in Rats by Oral Administration of the Inducing Antigen
CELLULAR IMMUNOLOGY ARTICLE NO.
175, 67–75 (1997)
CI961049
Clonal Anergy Is a Potent Mechanism of Oral Tolerance in the Suppression of Acute Antigen-Induced Arthritis in Rats by Oral Administration of the Inducing Antigen1 SHIGEYASU INADA, SHIN YOSHINO, M. AZIZUL HAQUE, YOSHIYASU OGATA,
AND
OSAMU KOHASHI2
Department of Microbiology, Saga Medical School, 5-1-1 Nabeshima, 849 Saga, Japan Received July 19, 1996; accepted October 1, 1996
diseases including encephalomyelitis (8, 9), uveitis (10, 11), diabetes (12), collagen-induced arthritis (13, 14), and adjuvant arthritis (15). Most of the previous studies have focused on the effects of oral administration of antigen on T-cell-mediated diseases, but little is known about the involvement of humoral immune mechanisms in the induction of OT. Antigen-induced arthritis (AIA), originally described by Dumonde and Glynn (16), can be induced by immunization of rabbits, rats, or mice with an antigen such as fibrin (16), ovalbumin (OVA) (17), bovine serum albumin (BSA) (18), or methylated BSA (19) followed by the intraarticular injection of the relevant antigen. The clinical course of AIA can be divided into two different phases, acute and chronic. The acute phase is characterized by strong swelling, redness, heating of the injected joint, and the predominant infiltration of polymorphonuclear cells into the synovium (17, 20). These reactions are noted as early as 1 hr after antigen injection, are more pronounced by 6 hr, and have subsided by 48 hr. The acute reaction in AIA is suggested to be the consequence of the in situ production of immune complexes and complement activation on the surface of the articular collagenous tissues (17, 21, 22) as observed in Arthus reaction (type III hypersensitivity reaction) (17, 20). We have recently demonstrated that the treatment with a monoclonal antibody (mAb) against T cell receptor ab from the time of intraarticular antigenic challenge suppressed the induction as well as the progression of chronic, but not acute, AIA in sensitized rats (23). The suppression of chronic AIA by the mAb was achieved without affecting the levels of antigen-specific antibody titers (23), suggested that acute AIA is mediated by humoral immune responses to the antigen. In the present study, using both the single high-dose or the five daily low-dose feeding protocols, we have shown that the oral administration of OVA before immunization with OVA suppresses the development of acute OVA-induced arthritis (OIA), anti-OVA IgG antibody (Ab) production, and in vitro lymphocyte proliferative responses to OVA, and that the suppression of
The effects of oral administration of ovalbumin (OVA) on acute OVA-induced arthritis (OIA) in rats, which is mediated by Arthus reaction to the antigen in the joint space, were investigated. The oral administration of OVA before immunization with OVA significantly suppressed the development of acute OIA in a dose-dependent manner, in accordance with decreases in both the in vivo anti-OVA IgG antibody production and in vitro lymphocyte proliferative responses to OVA. These results were shown in both the single high-dose (200 mg 1 1) or the multiple low-dose (200 mg 1 5) feeding protocols. In vitro study showed that rat IL-2 could reverse the reduced OVA-specific lymphocyte proliferative responses. The spleen cells obtained from OVA-feeding, unprimed rats neither adoptively transferred the suppression to naive recipient rats nor suppressed the in vitro lymphocyte proliferation. These results demonstrate that the acute OIA can be suppressed by the induction of oral tolerance (OT) to OVA, and strongly suggest that the OT was due to clonal anergy of antigenreactive T lymphocytes, not the active suppression by OVA-specific regulatory cells. q 1997 Academic Press
INTRODUCTION It has been well known that oral administration of antigen induces a state of systemic immunologic hyporesponsiveness termed oral tolerance (OT)3 (1, 2). Three mechanisms of OT have been proposed to be active suppression (3, 4), clonal anergy (5, 6), and clonal deletion (7). It is now well documented that the oral administration of appropriate antigen can suppress the development of various experimental autoimmune 1
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. 2 To whom correspondence should be addressed. Fax: /0952-332518. E-mail: [email protected]. 3 Abbreviations used: OVA, ovalbumin; OIA, ovalbumin-induced arthritis; OT, oral tolerance; KLH, keyhole limpet hemocyanin; LPH, limulus polyphemus hemocyanin; AIA, antigen-induced arthritis. 67
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acute OIA is due to the induction of OT to OVA. Furthermore, we have investigated the mechanisms of OT in acute OIA. MATERIALS AND METHODS Animals Inbred Lewis rats were obtained from Charles River Japan, Inc. (Shiga, Japan). They were housed in our animal breeding facilities, were fed a sterile commercial diet and water ad libitum, and maintained a continuous brother and sister mating under specific pathogen-free conditions. The rats were 7–9 weeks of age at the start of experiments. In each experiment, rats were matched for age and sex, and the data obtained from the experiments using only female rats were included in this report. Antigens Ovalbumin (OVA) grade V, keyhole limpet hemocyanin (KLH), and limulus polyphemus hemocyanin (LPH) were purchased from Sigma Chemical Co. (St. Louis, MO). Induction of Acute Antigen-Induced Arthritis Rats were immunized intradermally (i.d.) in the base of the tail with 1 mg of OVA dissolved in 50 ml of phosphate-buffered saline (PBS) and emulsified with an equal volume of complete Freund’s adjuvant (CFA) (Difco Laboratories, Detroit, MI). Fourteen days later, 100 mg of OVA dissolved in 50 ml of PBS was injected intraarticularly (ia) into the right ankle joint. The left ankle joint was injected with 50 ml of PBS as a control. Every 1 hr after intraarticular challenge of the antigen, the joint thickness was measured by a dial gauge caliper calibrated with 0.01-mm graduations (Ozaki MFG Co., Tokyo, Japan). The net increase in joint thickness attributable to the antigenic challenge was calculated by subtracting the increase in thickness of the left ankle from the increase in thickness of the right ankle. There was no net joint swelling after intraarticular injection of OVA in unprimed rats. In the other experiment, rats were immunized i.d. in the base of the tail with 1 mg of KLH dissolved in 50 ml of PBS and emulsified with an equal volume of CFA. Fourteen days later, 100 mg of KLH dissolved in 50 ml of PBS was injected into ankle joint and the joint thickness was measured as described above. Passive Transfer of Acute OVA-Induced Arthritis (OIA) with Anti-OVA Serum
as described above. Three milliliters of pooled serum from naive rats was used as a control in the passive transfer experiments. Oral Administration of OVA Oral administration of antigen was performed either by the single feeding protocol or by the five daily feedings protocol. In the single feeding protocol, rats were fed different doses (2 mg, 20 mg, 200 mg, 2 mg, 20 mg, or 200 mg) of OVA dissolved in 2 ml of PBS through a syringe fitted with an 18-gauge ball-point needle (Popper and Sons, Inc., New Hyde Park, NY) on Day 07 before immunization with OVA or KLH on Day 0. As controls, rats were fed either 200 mg of LPH dissolved in 2 ml of PBS, or 2 ml of PBS alone. In the five daily feedings protocol, different doses (2 mg, 20 mg, 200 mg, 2 mg, or 20 mg) of OVA or 20 mg of LPH dissolved in 2 ml of PBS, or 2 ml of PBS alone were fed on Days 05, 04, 03, 02, and 01 before immunization with OVA or KLH on Day 0. In some experiments, rats were given 200 mg of OVA or KLH by single feeding as a high-dose feeding protocol, or 200 mg of OVA or KLH by five daily feedings as a low-dose feeding protocol. Measurement of Antigen-Specific Antibody Fourteen days after immunization with OVA or KLH, the serum samples were analyzed for antigenspecific antibodies by ELISA (24). In brief, 96-well flatbottomed microtiter plates (Nunc, Roskilde, Denmark) were incubated with 100 ml/well of OVA or KLH (100 mg/ml in PBS) at 377C for 1 hr and were washed three times with PBS containing 0.05% Tween 20. The wells were then blocked by incubation at 377C for 1 hr with 200 ml of PBS containing 1% casein (Pierce, Rockford, IL). After washing, 100 ml of each serum sample (diluted 4001 in PBS) was added to the wells for further incubation at 377C for 1 hr. The wells were then washed and reacted with alkaline phosphatase-conjugated rabbit anti-rat IgG (Sigma) at 377C for 1 hr, followed by the reaction with p-nitrophenyl-phosphate solution (Bio-Rad Laboratories, Hercules, CA). The amount of bound Ab was measured by colorimetric analysis using a Titertek Multiscan spectrophotometer (EFLAB, Helsinki, Finland) at OD405 . The results were expressed as absorbance units at OD405 { standard errors of the mean (SEM). Measurement of in Vitro Lymphocyte Proliferative Responses
Blood was collected from rats immunized with 1 mg OVA plus CFA 14 days after immunization. After centrifugation, pooled serum was prepared and 3 ml of serum was injected intravenously (iv) into naive recipient rats; the recipients then were injected ia with OVA
Fourteen days after immunization with OVA or KLH, rats were killed, the inguinal lymph nodes were removed, and single-cell suspensions were prepared. The cells (3 1 105) in 100 ml of RPMI 1640 (Flow Laboratories, Inc., McLean, VA) culture medium containing 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml strep-
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tomycin, 5 1 1005 M 2-mercaptoethanol, and 2% heatinactivated autologous rat serum were added to each well of flat-bottomed 96-well plates, followed by addition of 100 ml of 100 mg/ml OVA or KLH. The cells were cultured for 72 hr, each well was pulsed with 0.5 mCi of [3H]thymidine (ICN Pharmaceuticals, Inc., Irvine, CA), and the cells were cultured for another 16 hr. Cultures were harvested onto fiberglass filters using a multiharvester and the amount of [3H]thymidine incorporation was determined using standard liquid scintillation techniques. The results were expressed as mean counts per minute (cpm) { SEM of quadruplicate cultures of cells pooled from five rats. In cell-mixing experiments, inguinal lymph node cells (ILNCs) harvested from unfed, OVA-primed rats on Day 14 after OVA priming were used as responder cells. Spleen cell suspensions from OVA-fed, unprimed rats were also prepared on Day 0, after oral administration of 200 mg of OVA on Day 07 or 200 mg of OVA on Days 05, 04, 03, 02, and 01. Red blood cells in the spleen cells (SPCs) were lysed with Tris–NH4Cl. The SPCs were incubated for 48 hr in complete RPMI medium in the presence of OVA (50 mg/ml), then used as modulator cells (10). In the other experiment, ILNCs from OVA-tolerant (ÅOVA-fed, OVA-primed) rats, which were fed 200 mg of OVA on Day 07 or 200 mg of OVA on Days 05, 04, 03, 02, 01 and primed with OVA on Day 0, were harvested on Day 14, and were used as modulator cells (6, 25). The responder cells were cocultured with the modulator cells at a ratio of 1:1, and proliferative responses to OVA were measured as described above. In the coculture experiments a total of 5 1 105 cells were placed in each well. Adoptive Transfer of Spleen Cells from OVA-Fed, Unprimed Rats The adoptive transfer experiments were carried out according to a previously described method (3). Donor rats were fed 200 mg of OVA on Day 07, or 200 mg of OVA on Days 05, 04, 03, 02, and 01. As controls, rats were given either 200 mg of LPH by single feeding or 200 mg of LPH by five daily feedings at the same time. The rats were killed on Day 0, and the SPCs were harvested. Red blood cells in the SPCs were lysed with Tris-NH4Cl, and single-cell suspensions were prepared. The SPCs were incubated for 48 hr in complete RPMI medium in the presence of concanavalin A (Con A, Sigma) (2 mg/ml). After in vitro culture, the Con Aactivated SPCs were washed and 5 1 108 cells were transferred to naive recipient rats by intraperitoneal injection. Two days after transfer, recipient rats were immunized i.d. with 1 mg of OVA plus CFA. Fourteen days after immunization, the rats were bled to evaluate anti-OVA Ab titers, and then acute OIA was induced and the joint thickness was measured. At the same time proliferative responses to OVA of the ILNCs were measured as described above.
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FIG. 1. Role of the humoral immune response in acute OIA. Acute OIA was induced as described under Materials and Methods. Three milliliters of serum from OVA-primed rats was injected iv into naive recipient rats followed by ia challenge with OVA. Three milliliters of serum from naive untreated rats was injected as a control. (s) Rats immunized i.d. with OVA (time–course of acute OIA); (m) rats received immune serum from OVA-primed rats; (l) rats received serum from naive untreated rats. Vertical bars show SEM of six rats. Data are representative of three experiments.
Reversal of Anergy Rats were fed 200 mg of OVA on Day 07 or 200 mg of OVA on Days 05, 04, 03, 02, and 01, and immunized with 1 mg of OVA plus CFA on Day 0. As controls, rats were fed LPH and immunized with 1 mg of OVA plus CFA at the same time. Fourteen days after immunization, ILNCs were harvested from these rats and incubated for 5 days in complete RPMI medium with or without 50 units/ml of rat interleukin 2 (IL-2) (Becton– Dickinson Labware, Bedford, MA) (6). The incubated ILNCs were washed three times in Hanks’ balanced salt solution and then added to each well in flat-bottomed 96-well plates (2.5 1 105/well) containing irradiated (3000 rad) syngeneic SPCs (2.5 1 105/well) and OVA (50 mg/ml). Plates were incubated for an additional 4 days and proliferative responses to OVA were measured as described above. Statistical Analysis The results were compared by Student’s two-tailed t test. P values õ 0.05 were considered to indicate significance. RESULTS Role of the Humoral Immune Response in Acute OIA As shown in Fig. 1, the rats developed severe arthritis as early as 1 hr after ia challenge with OVA. The joint swelling was rapidly increased and reached its peak by 4 hr. The naive rats which were injected intravenously with immune serum from OVA-primed rats
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examined (data of joint swelling in 2 and 20 mg OVAfed rats were not shown). The rats fed 200 mg, 2 mg, or 20 mg or OVA on Days 05, 04, 03, 02, and 01 also significantly decreased the severities of joint swelling in a dose-dependent manner (Fig. 2B, 25 to 62% suppression at 4 hr after ia injection of OVA, compared with LPH-fed rats). Oral administration of 2 or 20 mg of OVA on the same days did not affect the severities of joint swelling (data of joint swelling in 2 mg OVAfed rats were not shown). Suppression of Anti-OVA Antibody Production by Oral Administration of OVA At Day 14 after immunization, OVA-specific IgG productions in rats fed with 2, 20, or 200 mg of OVA on Day 07 were significantly decreased in a dose-dependent manner (22 to 67% suppression, compared with LPHfed rats), whereas feeding 2, 20, or 200 mg of OVA on the same day did not reduce anti-OVA IgG titers (Fig. 3A). Similarly, OVA-specific IgG productions were significantly suppressed in the rats fed 200 mg, 2 mg, or 20 mg of OVA on Days 05, 04, 03, 02, and 01 (Fig. 3B, 29 to 62% suppression, compared with LPH-fed rats). Oral administration of 2 or 20 mg of OVA on the same days did not reduce anti-OVA IgG titers.
FIG. 2. Suppression of acute OIA by oral administration of OVA. (A) Rats were fed 200 mg (m), 20 mg (s), 2 mg (j), or 200 mg (n) of OVA, or 200 mg of LPH (h), or PBS alone (l) on Day 07 before immunization with OVA on Day 0 followed by ia injection of the antigen on Day 14 as described under Materials and Methods. (B) Rats were fed 20 mg (j), 2 mg (s), 200 mg (m), or 20 mg (n) of OVA, or 20 mg of LPH (h), or PBS alone (l) on Days 05, 04, 03, 02, and 01 before immunization with OVA on Day 0 followed by ia injection of the antigen on Day 14. Vertical bars show SEM of six rats. *P õ 0.05; **P õ 0.01 compared with LPH-fed group. Data are representative of three experiments.
also developed marked arthritis immediately after ia challenge with OVA, and the time course of the disease was indistinguishable from that of the acute OIA. The rats that received serum from naive untreated rats did not show any disease after ia challenge with OVA. Suppression of Acute OIA by Oral Administration of OVA
Suppression of the in Vitro Proliferative Responses to OVA by Oral Administration of OVA ILNCs from rats given 2, 20, or 200 mg of OVA orally on Day 07 showed markedly reduced in vitro proliferative responses to OVA in a dose-dependent manner (Fig. 4A, 80 to 94% suppression, compared with LPHfed rats). ILNCs from rats given 200 mg, 2 mg, or 20 mg of OVA orally on Days 05, 04, 03, 02, and 01 also showed markedly reduced in vitro proliferative responses to OVA in a dose-dependent manner (Fig. 4B, 77 to 94% suppression, compared with LPH-fed rats). In addition, the proliferative responses to OVA of the ILNCs from rats given 200 mg of OVA on Day 07 and those from rats given 20 mg of OVA on Days 05, 04, 03, 02, and 01 were also slightly suppressed (Fig. 4, 27 and 34% suppression respectively, compared with LPH-fed rats). Oral administration of 2 or 20 mg of OVA on Day 07 and that of 2 mg of OVA on Days 05, 04, 03, 02, and 01 did not suppress the proliferative responses to OVA.
Rats fed either LPH or PBS on Day 07 developed severe arthritis within 1 hr of ia challenge and the disease reached its peak by 5 hr. On the other hand, oral administration of 2, 20, or 200 mg of OVA on Day 07 significantly suppressed the severities of joint swelling in a dose-dependent manner at all times examined (Fig. 2A, 24 to 60% suppression at 4 hr after ia injection of OVA, compared with LPH-fed rats). However, oral administration of 2, 20, or 200 mg of OVA on Day 07 did not affect the severities of joint swelling at all times
To determine the antigen specificity of OT, the rats were fed the high dose (200 mg) of OVA or KLH on Day 07, or the low dose (200 mg) of OVA or KLH on Days 05, 04, 03, 02, and 01, then immunized with KLH on Day 0. As control, PBS was given orally at the same time. The onset and severity of KLH-induced arthritis were similar to those of OVA-induced arthritis (Fig. 1). As shown in Table 1, oral administration of
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trol. These results clearly indicate that the induction of oral tolerance to OVA in OIA is antigen specific. Failure of the Suppression of Acute OIA and Immune Responses to OVA by Adoptive Transfer of SPCs from OVA-Fed, Unprimed Rats To investigate the underlying mechanism of the OT in this study, we performed in vivo adoptive cell trans-
FIG. 3. Suppression of anti-OVA antibody production by oral administration of OVA. OVA-fed or control rats were bled 14 days after immunization with OVA, and individual serum was assayed for antiOVA IgG antibody by ELISA. The results are expressed as a mean absorbance units at OD405 { SEM of six rats. (A) Rats were fed indicated doses of OVA, 200 mg of LPH, or PBS alone on Day 07 before immunization on Day 0. (B) Rats were fed indicated doses of OVA, 20 mg of LPH, or PBS alone on Days 05, 04, 03, 02, and 01 before immunization on Day 0. An additional group (CFA-primed) was unfed and primed with CFA alone. *P õ 0.05; **P õ 0.01 compared with LPH-fed group. Data are representative of three experiments.
KLH significantly suppressed the severities of acute KLH-induced arthritis, anti-KLH IgG production, and the lymphocyte proliferative responses to KLH. However, in both the high-dose (200 mg 1 1) and the lowdose (200 mg 1 5) feeding protocols, feeding OVA did not suppress the severities of acute KLH-induced arthritis, anti-KLH IgG production, and lymphocyte proliferative responses to KLH as observed in PBS-fed con-
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FIG. 4. Suppression of the in vitro proliferative responses to OVA by oral administration of OVA. OVA-fed or control rats were immunized with OVA and 14 days later proliferative responses to OVA of ILNCs from the animals were determined as described under Materials and Methods. The results are expressed as mean cpm { SEM of quadruplicate cultures of cells pooled from five rats. Background counts of OVA-immunized cells without OVA added were between 1000 and 2000. (A) Rats were fed indicated doses of OVA, 200 mg of LPH, or PBS alone on Day 07 before immunization on Day 0. (B) Rats were fed indicated doses of OVA, 20 mg of LPH, or PBS alone on Days 05, 04, 03, 02, and 01 before immunization on Day 0. **P õ 0.01 compared with LPH-fed group. Data are representative of three experiments.
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TABLE 1 Effect of Oral Administration of OVA or KLH on Acute KLH-Induced Arthritis and Immune Responses to KLH Feeding regimen
Anti-KLH IgG (OD405)
PBS, alone 1 1 OVA, 200 mg 1 1 KLH, 200 mg 1 1 PBS, alone 1 5 OVA, 200 mg 1 5 KLH, 200 mg 1 5
1.374 1.330 0.960 1.259 1.347 1.047
{ { { { { {
Joint swelling (10.01 mm)
0.028 0.072 0.068** 0.033 0.042 0.081*
291.7 288.0 188.3 281.7 286.0 238.3
{ { { { { {
7.9 6.6 13.5** 9.8 9.3 15.4*
Proliferation to KLH (cpm) 80,404.6 73,642.4 14,672.7 77,540.2 80,564.2 25,688.3
{ { { { { {
3036.2 3458.0 1214.5** 3752.5 5784.3 1792.7**
Note. Rats were fed 200 mg of OVA or KLH on Day 07, or 200 mg of OVA or KLH on Days 05, 04, 03, 02, and 01, then immunized with KLH on Day 0. As control, PBS was given at the same time. On Day 14, the rats were bled to evaluate anti-KLH antibodies, then acute arthritis was induced by intraarticular challenge with KLH and the joint thickness was measured. Lymphocyte proliferative responses to KLH were also measured as described under Materials and Methods. Values of joint swelling are the mean { SEM of six rats at 4 hr after ia injection of KLH. Data are representative of three experiments. * P õ 0.05, **P õ 0.01 compared with PBS-fed group.
fer experiments. SPCs from the rats orally given the high-dose (200 mg 1 1) or the low-dose (200 mg 1 5) of OVA were subjected to in vitro 2-day culture with Con A and then transferred into naive animals. As controls, naive animals received SPCs from LPH-fed rats. As shown in Table 2, the severities of acute OIA, anti-OVA IgG production, and lymphocyte proliferative responses to OVA were indistinguishable from those in the control animals that received SPCs from LPH-fed rats, in both the high-dose and the low-dose feeding protocols. In addition, transfer of disease suppression was observed with neither the unstimulated nor the OVA-stimulated (by in vitro 2-day culture with 50 mg/ ml of OVA) SPCs from OVA-fed, unprimed rats (data not shown). Hence, the SPCs from OVA-fed, unprimed rats failed to transfer protection against acute OIA. Effect of SPCs from OVA-Fed, Unprimed Rats on in Vitro Proliferative Responses to OVA of ILNCs from Unfed, OVA-Primed Rats SPCs from the rats orally given the high dose (200 mg 1 1) or the low dose (200 mg 1 5) of OVA were stimulated with OVA in vitro, then cocultured with an
equal number of ILNCs from unfed, OVA-primed rats. SPCs from LPH-fed rats were used as controls. There were no significant differences of proliferative responses to OVA in all groups (Table 3). When the SPCs from OVA-fed rats were mixed at a 3:1 ratio with the ILNCs from OVA-primed rats, similar results were observed (data not shown). Effect of ILNCs from OVA-Tolerant Rats on in Vitro Proliferative Responses to OVA of ILNCs from Unfed, OVA-Primed Rats In order to elucidate whether antigen-specific regulatory cells are generated in OVA-tolerant (OVA-fed, OVA-primed) rats, ILNCs from OVA-tolerant rats were cocultured at a 1:1 ratio with ILNCs from unfed, OVAprimed rats. The lymphocyte-proliferative responses to OVA were very high in group A (the unfed, OVAprimed ILNCs) but very low in group B (the single high dose of OVA-fed, OVA-primed ILNCs), C (the five daily low doses of OVA-fed, OVA-primed ILNCs), and D (the unfed, CFA-primed ILNCs served as negative controls) (Fig. 5). In coculture experiments, the proliferative responses to OVA of the ILNCs in group A / B and A /
TABLE 2 Failure of the Suppression of Acute OVA-Induced Arthritis and Immune Responses to OVA by Adoptive Transfer of SPCs from OVA-Fed, Unprimed Rats Donor rats fed with
Anti-OVA IgG (OD405)
LPH, 200 mg 1 1 OVA, 200 mg 1 1 LPH, 200 mg 1 5 OVA, 200 mg 1 5
1.045 1.100 1.110 1.135
{ { { {
Joint swelling (10.01 mm)
0.065 0.057 0.070 0.038
256.0 252.0 258.0 264.0
{ { { {
7.5 7.3 10.2 9.3
Proliferation to OVA (cpm) 35,445.2 33,871.3 36,204.6 35,724.5
{ { { {
1535.0 1640.5 2549.0 1400.6
Note. SPCs obtained from 200 mg 1 1 or 200 mg 1 5 of OVA fed rats were subjected to in vitro culture with Con A before transfer to naive animals. As controls, SPCs obtained from LPH-fed rats were transferred to naive animals. Two days after transfer, recipient rats were immunized with OVA. Fourteen days after immunization, the rats were bled to evaluate anti-OVA antibodies, then acute OIA was induced and the joint thickness was measured. Lymphocyte proliferative responses to OVA were also measured as described under Materials and Methods. Values of joint swelling are the mean { SEM of six rats at 4 hr after ia injection of OVA. Data are representative of three experiments.
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TABLE 3 SPCs from OVA-Fed, Unprimed Rats Do Not Suppress the Proliferative Responses to OVA of ILNCs from OVA-Primed Rats ILNCs (responder cells) sensitization
SPCs (modulator cells) feeding regimen
OVA-CFA OVA-CFA OVA-CFA OVA-CFA
LPH, 200 mg 1 1 OVA, 200 mg 1 1 LPH, 200 mg 1 5 OVA, 200 mg 1 5
Proliferation OVA (cpm)
to
43,740.2 { 1322.6 41,652.9 { 1875.7 42,218.3 { 2135.9 43,969.0 { 2170.4
Note. SPCs obtained from 200 mg 1 1 or 200 mg 1 5 of OVA fed rats were stimulated with OVA in vitro, then mixed with an equal number of ILNCs from OVA-primed rats cultured in the presence of OVA, and the proliferative responses to OVA were measured as described under Materials and Methods. SPCs obtained from LPHfed rats were used as controls. Values are the mean cpm { SEM of quadruplicate cultures of cells pooled from five rats. Data are representative of three experiments.
C were not reduced compared with the responses in group A / D even when higher ratios of the ILNCs from OVA-tolerant rats were used at a 3:1 ratio (data not shown). The findings that the responses of group A / B, A / C, and A / D were reduced by 40% compared to group A (Fig. 5) may not be due to active suppression by the ILNCs from OVA-tolerant rats, but rather to the proportional dilution of the responding cell population (group A) by the low-responding cell population (group B, C, or D).
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the five consecutive daily oral administrations with 200 mg, 2 mg or 20 mg of OVA before immunization, significantly suppressed antibody-mediated acute OIA in a dose-dependent manner (Figs. 1, 2). In both cases, serum anti-OVA IgG levels (Fig. 3) and in vitro proliferative responses of the ILNCs to OVA (Fig. 4) were also dose-dependently reduced, comparable to the suppression of acute OIA (Fig. 2). In addition, since acute KLHinduced arthritis was suppressed by prior feeding of KLH but not by OVA (Table 1) and vice versa, oral tolerance of acute AIA was confirmed to be antigen specific. These findings demonstrate that the suppressive effects of feeding OVA on acute OIA were associated with induction of OT to OVA, and are consistent with previous reports of oral treatment by feeding with the appropriate antigen in a variety of experimental cell-mediated diseases (2–5, 8–15, 26), as well as our recent report showing that chronic AIA can be suppressed by prior feeding with the inducing antigen (27). Moreover, studies indicate that oral treatment by prior feeding with the appropriate antigen not only inhibits cell-mediated inflammatory responses but also reduces antigen-specific humoral responses (8, 28, 29), and protect animals from the development of experimental im-
Effect of Culturing with IL-2 on the Decreased Proliferative Responses of the Tolerant Lymph Node Cells We next assessed whether clonal anergy of antigenreactive T lymphocytes is a possible mechanism of the OT in the present study. Tolerized and control ILNCs were cultured for 5 days in the presence of rat IL-2 or medium. Proliferative responses to OVA were measured just before and after a 5-day culture with or without rat IL-2. As shown in Fig. 6, OVA-specific proliferative responses, measured before IL-2 stimulation, were markedly reduced in both the high dose (200 mg 1 1) and the low dose (200 mg 1 5) of OVA-fed tolerant rats (Fig. 6A). These reduced OVA-specific proliferative responses were well restored after IL-2 stimulation in both feeding protocols, but the preculture of cells with IL-2 had little effect on the proliferative responses to OVA of ILNCs derived from CFA-primed rats (Fig. 6C). In addition, the reduced OVA-specific proliferative responses were not restored after culture with medium (Fig. 6B). DISCUSSION We successfully demonstrated that a single bolus oral administration with 2, 20, or 200 mg of OVA, or
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FIG. 5. ILNCs from OVA-tolerant rats do not actively suppress the proliferative responses to OVA of ILNCs from OVA-primed rats. Rats were unfed and immunized with OVA (group A). OVA-tolerant rats were fed 200 mg of OVA on Day 07 (group B), or 200 mg of OVA on Days 05, 04, 03, 02, and 01 (group C), and immunized with OVA on Day 0. ILNCs from unfed, CFA-primed rats served as negative controls (group D). Fourteen days after immunization, proliferative responses to OVA were measured as described under Materials and Methods. Mixed cultures were prepared by mixing unfed, OVAprimed ILNCs with OVA-tolerant ILNCs (group A / B or A / C) or with CFA-primed ILNCs (group A / D) at a 1:1 ratio. The results are expressed as mean cpm { SEM of quadruplicate cultures of cells pooled from five rats. Background counts of OVA-immunized cells without OVA added were between 1000 and 3000. **P õ 0.01 compared with group A. NS, not significant. Data are representative of three experiments.
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FIG. 6. Reversal of anergy by IL-2 stimulation. The lymphocyte proliferative responses to OVA were measured just before (A) and after a 5-day culture in the absence (B) or presence of rat IL-2 (C). Proliferative responses shown are of OVA-primed nontolerant cells fed 200 mg of LPH 1 1 (a) or 200 mg of LPH 1 5 (c), or of OVA-primed tolerant cells fed 200 mg of OVA 1 1 (b), 200 mg of OVA 1 5 (d). An additional group (e) was unfed and immunized with CFA alone. The results are expressed as mean cpm { SEM of quadruplicate cultures of cells pooled from five rats. Background counts of OVA-immunized cells without OVA added were between 1000 and 2000. **P õ 0.01; group a vs group b, and group c vs group d. NS, not significant. Data are representative of three experiments.
mune complex glomerulonephritis, which is also mediated by type III hypersensitivity reactions (30, 31). Although in some cases it appears that B cells themselves may be tolerized (32), it is generally assumed that T cells play the critical role in the induction of OT (33). Different mechanisms of OT have been suggested, and these include the generation of active suppression (3, 4), clonal anergy of antigen-reactive T cells (5, 6), and clonal deletion of antigen-reactive T cells (7). Active suppression is mediated by antigen-specific regulatory cells that produce suppressive cytokines such as transforming growth factor b (TGF-b) (4, 34, 35). Recent reports indicated that the relative roles of the mechanisms of OT are determined primarily by the dose of antigen administered (25, 36). Low doses of antigen that are fed intermittently (up to 1 mg/feeding) favor the active suppression (3, 4, 25, 34–36), whereas high doses of antigen that are fed either as a single bolus or intermittently favor the clonal anergy (5, 6, 25, 36) or the clonal deletion (7). The results of the present study in both the highand the low-dose feeding protocols support the mechanism of clonal anergy rather than active suppression, because [1] in adoptive transfer experiments, the SPCs from OVA-fed, unprimed rats could not suppress the development of acute OIA, anti-OVA Ab production, and lymphocyte proliferative responses to OVA (Table 2); [2] in coculture systems, the SPCs from OVA-fed, unprimed rats failed to exert their suppressive effects on the ILNCs from OVA-primed rats (Table 3); and [3] the in vitro proliferative responses to OVA of the ILNCs from OVA tolerant (OVA-fed, OVA-primed) rats were really suppressed, but in coculture systems, the cells
failed to exert their suppressive effects on the ILNCs from OVA-primed rats (Fig. 5). Direct evidence for the existence of anergic antigenreactive T lymphocytes in peripheral lymph nodes of tolerant animals has been attained by reversal of hyporesponsiveness of the cells with IL-2 stimulation in vitro (6, 37, 38). In our experiments, the involvement of the clonal anergy in the induction of OT was demonstrated further in Fig. 6. The reduced OVA-specific proliferative responses of the ILNCs from OVA tolerant rats could be reversed by rat IL-2 stimulation in vitro, not only in the high-dose feeding protocols but also in the low-dose feeding protocols (Fig. 6). These observations indicate that OVA-reactive T lymphocytes exist in inguinal lymph nodes of OVA tolerant rats, but they are most likely in an anergic state. Since recent studies indicate that orally induced T cell clonal anergy can reduce B cell Ab production through a lack of T cell help from anergic T cells (33) and affect B cell development as demonstrated by restoration of Ab production in the presence of T cell-derived cytokines such as IL4 and IL-5 (29), we therefore suggest that clonal anergy was the principal mechanism of OVA-induced tolerance in this study. However, to confirm this phenomena, it is important to study the existence of TGF-b in the culture supernatant of lymphocyte from OVA-tolerant rats. Although at present we do not know the reason that clonal anergy was induced even in low-dose feeding protocols; we suppose that the discrepancy between our findings and those of others might be caused by experimental differences, including differences of the disease models (3, 4, 25, 34–36), the kind of animals (25, 34), and the antigens (3, 4, 25, 34–36).
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In human clinical trials, it is shown that some patients with multiple sclerosis, uveitis, and rheumatoid arthritis may benefit from oral administration of bovine myelin, bovine S-antigen, and chicken type II collagen (39–41). Our present study suggests that oral administration of the appropriate antigen may be desirable for patients with a disease mediated by humoral immune responses to the antigen, and this hypothesis is supported by several previous reports which showed that tolerance of antibody responses can occur when antigen is fed after immunization (42–45). However, careful consideration is necessary for the clinical application of this strategy, because other reports have indicated that oral administration of antigen enhances preexisting immune responses in some instances (46–48). ACKNOWLEDGMENTS We thank Dr. Kazunori Ohki and Dr. Akiko Kukita for reviewing the manuscript and their helpful comments.
REFERENCES 1. Mowat, A. M., ‘‘Handbook of Mucosal Immunology,’’ p. 185, Academic Press, New York, 1994. 2. Weiner, H. L., Friedman, A., Miller, A., Khoury, S. J., Al-Sabbagh, A., Santos, L., Sayegh, M., Nussenblatt, R. B., Trentham, D. E., and Hafler, D. A., Annu. Rev. Immunol. 12, 809, 1994. 3. Lider, O., Santos, L. M. B., Lee, C. S. Y., Higgins, P. J., and Weiner, H. L., J. Immunol. 142, 748, 1989. 4. Miller, A., Lider, O., Roberts, A. B., Sporn, M. B., and Weiner, H. L., Proc. Natl. Acad. Sci. USA 89, 421, 1992. 5. Whitacre, C. C., Gienapp, I. E., Orosz, C. G., and Bitar, D. M., J. Immunol. 147, 2155, 1991. 6. Melamed, D., and Friedman, A., Eur. J. Immunol. 23, 935, 1993. 7. Chen, Y., Inobe, J., Marks, R., Gonnella, P., Kuchroo, V. K., and Weiner, H. L., Nature 376, 177, 1995. 8. Higgins, P. J., and Weiner, H. L., J. Immunol. 140, 440, 1988. 9. Bitar, D. M., and Whitacre, C. C., Cell. Immunol. 112, 364, 1988. 10. Nussenblatt, R. B., Caspi, R. R., Mahdi, R., Chan, C., Roberge, F., Lider, O., and Weiner, H. L., J. Immunol. 144, 1689, 1990. 11. Singh, V. K., Kalra, H. K., Yamaki, K., and Shinohara, T., Cell. Immunol. 139, 81, 1992. 12. Zhang, Z. J., Davidson, L., Eisenbarth, G., and Weiner, H. L., Proc. Natl. Acad. Sci. USA 88, 10252, 1991. 13. Nagler-Anderson, C., Bober, L. A., Robinson, M. E., Siskind, G. W., and Thorbecke, G. J., Proc. Natl. Acad. Sci. USA 83, 7443, 1986. 14. Thompson, H. S. G., and Staines, N. A., Clin. Exp. Immunol. 64, 581, 1986. 15. Zhang, Z. J., Lee, C. S. Y., Lider, O., and Weiner, H. L., J. Immunol. 145, 2489, 1990. 16. Dumonde, D. C., and Glynn, L. E., Br. J. Exp. Pathol. 43, 373, 1962. 17. Brauer, R., Thoss, K., Henzgen, S., and Waldmann, G., Exp. Pathol. 34, 197, 1988. 18. Jasin, H. E., Clin. Exp. Immunol. 22, 473, 1975.
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19. Brackertz, D., Mitchell, G., and Mackay, I. R., Arthritis Rheum. 20, 841, 1977. 20. Steinberg, M. E., McCrae, C. R., Cohen, L. D., and Schumacher, H. R., Clin. Orthop. Relat. Res. 97, 248, 1973. 21. Ohno, O., Tateishi, H., and Cooke, T. D., Arthritis Rheum. 21, 81, 1978. 22. Ugai, K., Ziff, M., and Jasin, H. E., Arthritis Rheum. 22, 353, 1979. 23. Yoshino, S., and Yoshino, J., Cell. Immunol. 144, 382, 1992. 24. Engvall, E., and Perlmann, P., J. Immunol. 109, 129, 1972. 25. Friedman, A., and Weiner, H. L., Proc. Natl. Acad. Sci. USA 91, 6688, 1994. 26. Yoshino, S., Quattrocchi, E., and Weiner, H. L., Arthritis Rheum. 38, 1092, 1995. 27. Yoshino, S., Cell. Immunol. 163, 55, 1995. 28. Fuller, K. A., Pearl, D., and Whitacre, C. C., J. Neuroimmunol. 28, 15, 1990. 29. Kelly, K. A., and Whitacre, C. C., J. Neuroimmunol. 66, 77, 1996. 30. Devey, M. E., and Bleasdale, K., Clin. Exp. Immunol. 56, 637, 1984. 31. Browning, M. J., and Parrott, D. M. V., Adv. Exp. Med. Biol. 216B, 1619, 1987. 32. Vives, J., Parks, D. E., and Weigle, W. O., J. Immunol. 125, 1811, 1980. 33. Hirahara, K., Hisatsune, T., Nishijima, K., Kato, H., Shiho, O., and Kaminogawa, S., J. Immunol. 154, 6238, 1995. 34. Chen, Y., Kuchroo, V. K., Inobe, J., Hafler, D. A., and Weiner, H. L., Science 265, 1237, 1994. 35. Miller, A., Al-Sabbagh, A., Santos, L. M. B., Prabhu-Das, M., and Weiner, H. L., J. Immunol. 151, 7307, 1993. 36. Gregerson, D. S., Obritsch, W. F., and Donoso, L. A., J. Immunol. 151, 5751, 1993. 37. DeSilva, D. R., Urdahl, K. B., and Jenkins, M. K., J. Immunol. 147, 3261, 1991. 38. Beverly, B., Kang, S.-M., Lenardo, M. J., and Schwartz, R. H., Int. Immunol. 4, 661, 1992. 39. Weiner, H. L., Mackin, G. A., Matsui, M., Orav, E. J., Khoury, S. J., Dawson, D. M., and Hafler, D. A., Science 259, 1321, 1993. 40. Nussenblatt, R. B., de Smet, M. D., Weiner, H. L., and Gery, I., ‘‘Behcet’s Disease: Proceedings of the 6th International Conference on Behcet’s Disease, Paris, France,’’ p. 641, Excerpta Medica, Elsevier, Amsterdam, 1993. 41. Trentham, D. E., Dynesius-Trentham, R. A., Orav, E. J., Combitchi, D., Lorenzo, C., Sewell, K. L., Hafler, D. A., and Weiner, H. L., Science 261, 1727, 1993. 42. Lafont, S., Andre, C., Andre, F., Gillon, J., and Fargier, M.-C., J. Exp. Med. 155, 1573, 1982. 43. Bloch, K. J., Perry, R., Bloch, M., and Walker, W. A., Ann. N. Y. Acad. Sci. 409, 787, 1983. 44. Saklayen, M. G., Pesce, A. J., Pollak, V. E., and Michael, J. G., Int. Arch. Allergy Appl. Immunol. 73, 5, 1984. 45. Peng, H.-J., Turner, M. W., and Strobel, S., Immunol. 67, 425, 1989. 46. Hanson, D. G., Vaz, N. M., Rawlings, L. A., and Lynch, J. M., J. Immunol. 122, 2261, 1979. 47. Titus, R. G., and Chiller, J. M., Int. Arch. Allergy Appl. Immunol. 65, 323, 1981. 48. Lens, J. W., van den Berg, W. B., van de Putte, L. B. A., and van den Bersselaar, L., Clin. Exp. Immunol. 58, 364, 1984.
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Clonal Anergy Is a Potent Mechanism of Oral Tolerance in the Suppression of Acute Antigen-Induced Arthritis in Rats by Oral Administration of the Inducing Antigen1 SHIGEYASU INADA, SHIN YOSHINO, M. AZIZUL HAQUE, YOSHIYASU OGATA,
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OSAMU KOHASHI2
Department of Microbiology, Saga Medical School, 5-1-1 Nabeshima, 849 Saga, Japan Received July 19, 1996; accepted October 1, 1996
diseases including encephalomyelitis (8, 9), uveitis (10, 11), diabetes (12), collagen-induced arthritis (13, 14), and adjuvant arthritis (15). Most of the previous studies have focused on the effects of oral administration of antigen on T-cell-mediated diseases, but little is known about the involvement of humoral immune mechanisms in the induction of OT. Antigen-induced arthritis (AIA), originally described by Dumonde and Glynn (16), can be induced by immunization of rabbits, rats, or mice with an antigen such as fibrin (16), ovalbumin (OVA) (17), bovine serum albumin (BSA) (18), or methylated BSA (19) followed by the intraarticular injection of the relevant antigen. The clinical course of AIA can be divided into two different phases, acute and chronic. The acute phase is characterized by strong swelling, redness, heating of the injected joint, and the predominant infiltration of polymorphonuclear cells into the synovium (17, 20). These reactions are noted as early as 1 hr after antigen injection, are more pronounced by 6 hr, and have subsided by 48 hr. The acute reaction in AIA is suggested to be the consequence of the in situ production of immune complexes and complement activation on the surface of the articular collagenous tissues (17, 21, 22) as observed in Arthus reaction (type III hypersensitivity reaction) (17, 20). We have recently demonstrated that the treatment with a monoclonal antibody (mAb) against T cell receptor ab from the time of intraarticular antigenic challenge suppressed the induction as well as the progression of chronic, but not acute, AIA in sensitized rats (23). The suppression of chronic AIA by the mAb was achieved without affecting the levels of antigen-specific antibody titers (23), suggested that acute AIA is mediated by humoral immune responses to the antigen. In the present study, using both the single high-dose or the five daily low-dose feeding protocols, we have shown that the oral administration of OVA before immunization with OVA suppresses the development of acute OVA-induced arthritis (OIA), anti-OVA IgG antibody (Ab) production, and in vitro lymphocyte proliferative responses to OVA, and that the suppression of
The effects of oral administration of ovalbumin (OVA) on acute OVA-induced arthritis (OIA) in rats, which is mediated by Arthus reaction to the antigen in the joint space, were investigated. The oral administration of OVA before immunization with OVA significantly suppressed the development of acute OIA in a dose-dependent manner, in accordance with decreases in both the in vivo anti-OVA IgG antibody production and in vitro lymphocyte proliferative responses to OVA. These results were shown in both the single high-dose (200 mg 1 1) or the multiple low-dose (200 mg 1 5) feeding protocols. In vitro study showed that rat IL-2 could reverse the reduced OVA-specific lymphocyte proliferative responses. The spleen cells obtained from OVA-feeding, unprimed rats neither adoptively transferred the suppression to naive recipient rats nor suppressed the in vitro lymphocyte proliferation. These results demonstrate that the acute OIA can be suppressed by the induction of oral tolerance (OT) to OVA, and strongly suggest that the OT was due to clonal anergy of antigenreactive T lymphocytes, not the active suppression by OVA-specific regulatory cells. q 1997 Academic Press
INTRODUCTION It has been well known that oral administration of antigen induces a state of systemic immunologic hyporesponsiveness termed oral tolerance (OT)3 (1, 2). Three mechanisms of OT have been proposed to be active suppression (3, 4), clonal anergy (5, 6), and clonal deletion (7). It is now well documented that the oral administration of appropriate antigen can suppress the development of various experimental autoimmune 1
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. 2 To whom correspondence should be addressed. Fax: /0952-332518. E-mail: [email protected]. 3 Abbreviations used: OVA, ovalbumin; OIA, ovalbumin-induced arthritis; OT, oral tolerance; KLH, keyhole limpet hemocyanin; LPH, limulus polyphemus hemocyanin; AIA, antigen-induced arthritis. 67
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acute OIA is due to the induction of OT to OVA. Furthermore, we have investigated the mechanisms of OT in acute OIA. MATERIALS AND METHODS Animals Inbred Lewis rats were obtained from Charles River Japan, Inc. (Shiga, Japan). They were housed in our animal breeding facilities, were fed a sterile commercial diet and water ad libitum, and maintained a continuous brother and sister mating under specific pathogen-free conditions. The rats were 7–9 weeks of age at the start of experiments. In each experiment, rats were matched for age and sex, and the data obtained from the experiments using only female rats were included in this report. Antigens Ovalbumin (OVA) grade V, keyhole limpet hemocyanin (KLH), and limulus polyphemus hemocyanin (LPH) were purchased from Sigma Chemical Co. (St. Louis, MO). Induction of Acute Antigen-Induced Arthritis Rats were immunized intradermally (i.d.) in the base of the tail with 1 mg of OVA dissolved in 50 ml of phosphate-buffered saline (PBS) and emulsified with an equal volume of complete Freund’s adjuvant (CFA) (Difco Laboratories, Detroit, MI). Fourteen days later, 100 mg of OVA dissolved in 50 ml of PBS was injected intraarticularly (ia) into the right ankle joint. The left ankle joint was injected with 50 ml of PBS as a control. Every 1 hr after intraarticular challenge of the antigen, the joint thickness was measured by a dial gauge caliper calibrated with 0.01-mm graduations (Ozaki MFG Co., Tokyo, Japan). The net increase in joint thickness attributable to the antigenic challenge was calculated by subtracting the increase in thickness of the left ankle from the increase in thickness of the right ankle. There was no net joint swelling after intraarticular injection of OVA in unprimed rats. In the other experiment, rats were immunized i.d. in the base of the tail with 1 mg of KLH dissolved in 50 ml of PBS and emulsified with an equal volume of CFA. Fourteen days later, 100 mg of KLH dissolved in 50 ml of PBS was injected into ankle joint and the joint thickness was measured as described above. Passive Transfer of Acute OVA-Induced Arthritis (OIA) with Anti-OVA Serum
as described above. Three milliliters of pooled serum from naive rats was used as a control in the passive transfer experiments. Oral Administration of OVA Oral administration of antigen was performed either by the single feeding protocol or by the five daily feedings protocol. In the single feeding protocol, rats were fed different doses (2 mg, 20 mg, 200 mg, 2 mg, 20 mg, or 200 mg) of OVA dissolved in 2 ml of PBS through a syringe fitted with an 18-gauge ball-point needle (Popper and Sons, Inc., New Hyde Park, NY) on Day 07 before immunization with OVA or KLH on Day 0. As controls, rats were fed either 200 mg of LPH dissolved in 2 ml of PBS, or 2 ml of PBS alone. In the five daily feedings protocol, different doses (2 mg, 20 mg, 200 mg, 2 mg, or 20 mg) of OVA or 20 mg of LPH dissolved in 2 ml of PBS, or 2 ml of PBS alone were fed on Days 05, 04, 03, 02, and 01 before immunization with OVA or KLH on Day 0. In some experiments, rats were given 200 mg of OVA or KLH by single feeding as a high-dose feeding protocol, or 200 mg of OVA or KLH by five daily feedings as a low-dose feeding protocol. Measurement of Antigen-Specific Antibody Fourteen days after immunization with OVA or KLH, the serum samples were analyzed for antigenspecific antibodies by ELISA (24). In brief, 96-well flatbottomed microtiter plates (Nunc, Roskilde, Denmark) were incubated with 100 ml/well of OVA or KLH (100 mg/ml in PBS) at 377C for 1 hr and were washed three times with PBS containing 0.05% Tween 20. The wells were then blocked by incubation at 377C for 1 hr with 200 ml of PBS containing 1% casein (Pierce, Rockford, IL). After washing, 100 ml of each serum sample (diluted 4001 in PBS) was added to the wells for further incubation at 377C for 1 hr. The wells were then washed and reacted with alkaline phosphatase-conjugated rabbit anti-rat IgG (Sigma) at 377C for 1 hr, followed by the reaction with p-nitrophenyl-phosphate solution (Bio-Rad Laboratories, Hercules, CA). The amount of bound Ab was measured by colorimetric analysis using a Titertek Multiscan spectrophotometer (EFLAB, Helsinki, Finland) at OD405 . The results were expressed as absorbance units at OD405 { standard errors of the mean (SEM). Measurement of in Vitro Lymphocyte Proliferative Responses
Blood was collected from rats immunized with 1 mg OVA plus CFA 14 days after immunization. After centrifugation, pooled serum was prepared and 3 ml of serum was injected intravenously (iv) into naive recipient rats; the recipients then were injected ia with OVA
Fourteen days after immunization with OVA or KLH, rats were killed, the inguinal lymph nodes were removed, and single-cell suspensions were prepared. The cells (3 1 105) in 100 ml of RPMI 1640 (Flow Laboratories, Inc., McLean, VA) culture medium containing 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml strep-
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tomycin, 5 1 1005 M 2-mercaptoethanol, and 2% heatinactivated autologous rat serum were added to each well of flat-bottomed 96-well plates, followed by addition of 100 ml of 100 mg/ml OVA or KLH. The cells were cultured for 72 hr, each well was pulsed with 0.5 mCi of [3H]thymidine (ICN Pharmaceuticals, Inc., Irvine, CA), and the cells were cultured for another 16 hr. Cultures were harvested onto fiberglass filters using a multiharvester and the amount of [3H]thymidine incorporation was determined using standard liquid scintillation techniques. The results were expressed as mean counts per minute (cpm) { SEM of quadruplicate cultures of cells pooled from five rats. In cell-mixing experiments, inguinal lymph node cells (ILNCs) harvested from unfed, OVA-primed rats on Day 14 after OVA priming were used as responder cells. Spleen cell suspensions from OVA-fed, unprimed rats were also prepared on Day 0, after oral administration of 200 mg of OVA on Day 07 or 200 mg of OVA on Days 05, 04, 03, 02, and 01. Red blood cells in the spleen cells (SPCs) were lysed with Tris–NH4Cl. The SPCs were incubated for 48 hr in complete RPMI medium in the presence of OVA (50 mg/ml), then used as modulator cells (10). In the other experiment, ILNCs from OVA-tolerant (ÅOVA-fed, OVA-primed) rats, which were fed 200 mg of OVA on Day 07 or 200 mg of OVA on Days 05, 04, 03, 02, 01 and primed with OVA on Day 0, were harvested on Day 14, and were used as modulator cells (6, 25). The responder cells were cocultured with the modulator cells at a ratio of 1:1, and proliferative responses to OVA were measured as described above. In the coculture experiments a total of 5 1 105 cells were placed in each well. Adoptive Transfer of Spleen Cells from OVA-Fed, Unprimed Rats The adoptive transfer experiments were carried out according to a previously described method (3). Donor rats were fed 200 mg of OVA on Day 07, or 200 mg of OVA on Days 05, 04, 03, 02, and 01. As controls, rats were given either 200 mg of LPH by single feeding or 200 mg of LPH by five daily feedings at the same time. The rats were killed on Day 0, and the SPCs were harvested. Red blood cells in the SPCs were lysed with Tris-NH4Cl, and single-cell suspensions were prepared. The SPCs were incubated for 48 hr in complete RPMI medium in the presence of concanavalin A (Con A, Sigma) (2 mg/ml). After in vitro culture, the Con Aactivated SPCs were washed and 5 1 108 cells were transferred to naive recipient rats by intraperitoneal injection. Two days after transfer, recipient rats were immunized i.d. with 1 mg of OVA plus CFA. Fourteen days after immunization, the rats were bled to evaluate anti-OVA Ab titers, and then acute OIA was induced and the joint thickness was measured. At the same time proliferative responses to OVA of the ILNCs were measured as described above.
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FIG. 1. Role of the humoral immune response in acute OIA. Acute OIA was induced as described under Materials and Methods. Three milliliters of serum from OVA-primed rats was injected iv into naive recipient rats followed by ia challenge with OVA. Three milliliters of serum from naive untreated rats was injected as a control. (s) Rats immunized i.d. with OVA (time–course of acute OIA); (m) rats received immune serum from OVA-primed rats; (l) rats received serum from naive untreated rats. Vertical bars show SEM of six rats. Data are representative of three experiments.
Reversal of Anergy Rats were fed 200 mg of OVA on Day 07 or 200 mg of OVA on Days 05, 04, 03, 02, and 01, and immunized with 1 mg of OVA plus CFA on Day 0. As controls, rats were fed LPH and immunized with 1 mg of OVA plus CFA at the same time. Fourteen days after immunization, ILNCs were harvested from these rats and incubated for 5 days in complete RPMI medium with or without 50 units/ml of rat interleukin 2 (IL-2) (Becton– Dickinson Labware, Bedford, MA) (6). The incubated ILNCs were washed three times in Hanks’ balanced salt solution and then added to each well in flat-bottomed 96-well plates (2.5 1 105/well) containing irradiated (3000 rad) syngeneic SPCs (2.5 1 105/well) and OVA (50 mg/ml). Plates were incubated for an additional 4 days and proliferative responses to OVA were measured as described above. Statistical Analysis The results were compared by Student’s two-tailed t test. P values õ 0.05 were considered to indicate significance. RESULTS Role of the Humoral Immune Response in Acute OIA As shown in Fig. 1, the rats developed severe arthritis as early as 1 hr after ia challenge with OVA. The joint swelling was rapidly increased and reached its peak by 4 hr. The naive rats which were injected intravenously with immune serum from OVA-primed rats
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examined (data of joint swelling in 2 and 20 mg OVAfed rats were not shown). The rats fed 200 mg, 2 mg, or 20 mg or OVA on Days 05, 04, 03, 02, and 01 also significantly decreased the severities of joint swelling in a dose-dependent manner (Fig. 2B, 25 to 62% suppression at 4 hr after ia injection of OVA, compared with LPH-fed rats). Oral administration of 2 or 20 mg of OVA on the same days did not affect the severities of joint swelling (data of joint swelling in 2 mg OVAfed rats were not shown). Suppression of Anti-OVA Antibody Production by Oral Administration of OVA At Day 14 after immunization, OVA-specific IgG productions in rats fed with 2, 20, or 200 mg of OVA on Day 07 were significantly decreased in a dose-dependent manner (22 to 67% suppression, compared with LPHfed rats), whereas feeding 2, 20, or 200 mg of OVA on the same day did not reduce anti-OVA IgG titers (Fig. 3A). Similarly, OVA-specific IgG productions were significantly suppressed in the rats fed 200 mg, 2 mg, or 20 mg of OVA on Days 05, 04, 03, 02, and 01 (Fig. 3B, 29 to 62% suppression, compared with LPH-fed rats). Oral administration of 2 or 20 mg of OVA on the same days did not reduce anti-OVA IgG titers.
FIG. 2. Suppression of acute OIA by oral administration of OVA. (A) Rats were fed 200 mg (m), 20 mg (s), 2 mg (j), or 200 mg (n) of OVA, or 200 mg of LPH (h), or PBS alone (l) on Day 07 before immunization with OVA on Day 0 followed by ia injection of the antigen on Day 14 as described under Materials and Methods. (B) Rats were fed 20 mg (j), 2 mg (s), 200 mg (m), or 20 mg (n) of OVA, or 20 mg of LPH (h), or PBS alone (l) on Days 05, 04, 03, 02, and 01 before immunization with OVA on Day 0 followed by ia injection of the antigen on Day 14. Vertical bars show SEM of six rats. *P õ 0.05; **P õ 0.01 compared with LPH-fed group. Data are representative of three experiments.
also developed marked arthritis immediately after ia challenge with OVA, and the time course of the disease was indistinguishable from that of the acute OIA. The rats that received serum from naive untreated rats did not show any disease after ia challenge with OVA. Suppression of Acute OIA by Oral Administration of OVA
Suppression of the in Vitro Proliferative Responses to OVA by Oral Administration of OVA ILNCs from rats given 2, 20, or 200 mg of OVA orally on Day 07 showed markedly reduced in vitro proliferative responses to OVA in a dose-dependent manner (Fig. 4A, 80 to 94% suppression, compared with LPHfed rats). ILNCs from rats given 200 mg, 2 mg, or 20 mg of OVA orally on Days 05, 04, 03, 02, and 01 also showed markedly reduced in vitro proliferative responses to OVA in a dose-dependent manner (Fig. 4B, 77 to 94% suppression, compared with LPH-fed rats). In addition, the proliferative responses to OVA of the ILNCs from rats given 200 mg of OVA on Day 07 and those from rats given 20 mg of OVA on Days 05, 04, 03, 02, and 01 were also slightly suppressed (Fig. 4, 27 and 34% suppression respectively, compared with LPH-fed rats). Oral administration of 2 or 20 mg of OVA on Day 07 and that of 2 mg of OVA on Days 05, 04, 03, 02, and 01 did not suppress the proliferative responses to OVA.
Rats fed either LPH or PBS on Day 07 developed severe arthritis within 1 hr of ia challenge and the disease reached its peak by 5 hr. On the other hand, oral administration of 2, 20, or 200 mg of OVA on Day 07 significantly suppressed the severities of joint swelling in a dose-dependent manner at all times examined (Fig. 2A, 24 to 60% suppression at 4 hr after ia injection of OVA, compared with LPH-fed rats). However, oral administration of 2, 20, or 200 mg of OVA on Day 07 did not affect the severities of joint swelling at all times
To determine the antigen specificity of OT, the rats were fed the high dose (200 mg) of OVA or KLH on Day 07, or the low dose (200 mg) of OVA or KLH on Days 05, 04, 03, 02, and 01, then immunized with KLH on Day 0. As control, PBS was given orally at the same time. The onset and severity of KLH-induced arthritis were similar to those of OVA-induced arthritis (Fig. 1). As shown in Table 1, oral administration of
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trol. These results clearly indicate that the induction of oral tolerance to OVA in OIA is antigen specific. Failure of the Suppression of Acute OIA and Immune Responses to OVA by Adoptive Transfer of SPCs from OVA-Fed, Unprimed Rats To investigate the underlying mechanism of the OT in this study, we performed in vivo adoptive cell trans-
FIG. 3. Suppression of anti-OVA antibody production by oral administration of OVA. OVA-fed or control rats were bled 14 days after immunization with OVA, and individual serum was assayed for antiOVA IgG antibody by ELISA. The results are expressed as a mean absorbance units at OD405 { SEM of six rats. (A) Rats were fed indicated doses of OVA, 200 mg of LPH, or PBS alone on Day 07 before immunization on Day 0. (B) Rats were fed indicated doses of OVA, 20 mg of LPH, or PBS alone on Days 05, 04, 03, 02, and 01 before immunization on Day 0. An additional group (CFA-primed) was unfed and primed with CFA alone. *P õ 0.05; **P õ 0.01 compared with LPH-fed group. Data are representative of three experiments.
KLH significantly suppressed the severities of acute KLH-induced arthritis, anti-KLH IgG production, and the lymphocyte proliferative responses to KLH. However, in both the high-dose (200 mg 1 1) and the lowdose (200 mg 1 5) feeding protocols, feeding OVA did not suppress the severities of acute KLH-induced arthritis, anti-KLH IgG production, and lymphocyte proliferative responses to KLH as observed in PBS-fed con-
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FIG. 4. Suppression of the in vitro proliferative responses to OVA by oral administration of OVA. OVA-fed or control rats were immunized with OVA and 14 days later proliferative responses to OVA of ILNCs from the animals were determined as described under Materials and Methods. The results are expressed as mean cpm { SEM of quadruplicate cultures of cells pooled from five rats. Background counts of OVA-immunized cells without OVA added were between 1000 and 2000. (A) Rats were fed indicated doses of OVA, 200 mg of LPH, or PBS alone on Day 07 before immunization on Day 0. (B) Rats were fed indicated doses of OVA, 20 mg of LPH, or PBS alone on Days 05, 04, 03, 02, and 01 before immunization on Day 0. **P õ 0.01 compared with LPH-fed group. Data are representative of three experiments.
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TABLE 1 Effect of Oral Administration of OVA or KLH on Acute KLH-Induced Arthritis and Immune Responses to KLH Feeding regimen
Anti-KLH IgG (OD405)
PBS, alone 1 1 OVA, 200 mg 1 1 KLH, 200 mg 1 1 PBS, alone 1 5 OVA, 200 mg 1 5 KLH, 200 mg 1 5
1.374 1.330 0.960 1.259 1.347 1.047
{ { { { { {
Joint swelling (10.01 mm)
0.028 0.072 0.068** 0.033 0.042 0.081*
291.7 288.0 188.3 281.7 286.0 238.3
{ { { { { {
7.9 6.6 13.5** 9.8 9.3 15.4*
Proliferation to KLH (cpm) 80,404.6 73,642.4 14,672.7 77,540.2 80,564.2 25,688.3
{ { { { { {
3036.2 3458.0 1214.5** 3752.5 5784.3 1792.7**
Note. Rats were fed 200 mg of OVA or KLH on Day 07, or 200 mg of OVA or KLH on Days 05, 04, 03, 02, and 01, then immunized with KLH on Day 0. As control, PBS was given at the same time. On Day 14, the rats were bled to evaluate anti-KLH antibodies, then acute arthritis was induced by intraarticular challenge with KLH and the joint thickness was measured. Lymphocyte proliferative responses to KLH were also measured as described under Materials and Methods. Values of joint swelling are the mean { SEM of six rats at 4 hr after ia injection of KLH. Data are representative of three experiments. * P õ 0.05, **P õ 0.01 compared with PBS-fed group.
fer experiments. SPCs from the rats orally given the high-dose (200 mg 1 1) or the low-dose (200 mg 1 5) of OVA were subjected to in vitro 2-day culture with Con A and then transferred into naive animals. As controls, naive animals received SPCs from LPH-fed rats. As shown in Table 2, the severities of acute OIA, anti-OVA IgG production, and lymphocyte proliferative responses to OVA were indistinguishable from those in the control animals that received SPCs from LPH-fed rats, in both the high-dose and the low-dose feeding protocols. In addition, transfer of disease suppression was observed with neither the unstimulated nor the OVA-stimulated (by in vitro 2-day culture with 50 mg/ ml of OVA) SPCs from OVA-fed, unprimed rats (data not shown). Hence, the SPCs from OVA-fed, unprimed rats failed to transfer protection against acute OIA. Effect of SPCs from OVA-Fed, Unprimed Rats on in Vitro Proliferative Responses to OVA of ILNCs from Unfed, OVA-Primed Rats SPCs from the rats orally given the high dose (200 mg 1 1) or the low dose (200 mg 1 5) of OVA were stimulated with OVA in vitro, then cocultured with an
equal number of ILNCs from unfed, OVA-primed rats. SPCs from LPH-fed rats were used as controls. There were no significant differences of proliferative responses to OVA in all groups (Table 3). When the SPCs from OVA-fed rats were mixed at a 3:1 ratio with the ILNCs from OVA-primed rats, similar results were observed (data not shown). Effect of ILNCs from OVA-Tolerant Rats on in Vitro Proliferative Responses to OVA of ILNCs from Unfed, OVA-Primed Rats In order to elucidate whether antigen-specific regulatory cells are generated in OVA-tolerant (OVA-fed, OVA-primed) rats, ILNCs from OVA-tolerant rats were cocultured at a 1:1 ratio with ILNCs from unfed, OVAprimed rats. The lymphocyte-proliferative responses to OVA were very high in group A (the unfed, OVAprimed ILNCs) but very low in group B (the single high dose of OVA-fed, OVA-primed ILNCs), C (the five daily low doses of OVA-fed, OVA-primed ILNCs), and D (the unfed, CFA-primed ILNCs served as negative controls) (Fig. 5). In coculture experiments, the proliferative responses to OVA of the ILNCs in group A / B and A /
TABLE 2 Failure of the Suppression of Acute OVA-Induced Arthritis and Immune Responses to OVA by Adoptive Transfer of SPCs from OVA-Fed, Unprimed Rats Donor rats fed with
Anti-OVA IgG (OD405)
LPH, 200 mg 1 1 OVA, 200 mg 1 1 LPH, 200 mg 1 5 OVA, 200 mg 1 5
1.045 1.100 1.110 1.135
{ { { {
Joint swelling (10.01 mm)
0.065 0.057 0.070 0.038
256.0 252.0 258.0 264.0
{ { { {
7.5 7.3 10.2 9.3
Proliferation to OVA (cpm) 35,445.2 33,871.3 36,204.6 35,724.5
{ { { {
1535.0 1640.5 2549.0 1400.6
Note. SPCs obtained from 200 mg 1 1 or 200 mg 1 5 of OVA fed rats were subjected to in vitro culture with Con A before transfer to naive animals. As controls, SPCs obtained from LPH-fed rats were transferred to naive animals. Two days after transfer, recipient rats were immunized with OVA. Fourteen days after immunization, the rats were bled to evaluate anti-OVA antibodies, then acute OIA was induced and the joint thickness was measured. Lymphocyte proliferative responses to OVA were also measured as described under Materials and Methods. Values of joint swelling are the mean { SEM of six rats at 4 hr after ia injection of OVA. Data are representative of three experiments.
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TABLE 3 SPCs from OVA-Fed, Unprimed Rats Do Not Suppress the Proliferative Responses to OVA of ILNCs from OVA-Primed Rats ILNCs (responder cells) sensitization
SPCs (modulator cells) feeding regimen
OVA-CFA OVA-CFA OVA-CFA OVA-CFA
LPH, 200 mg 1 1 OVA, 200 mg 1 1 LPH, 200 mg 1 5 OVA, 200 mg 1 5
Proliferation OVA (cpm)
to
43,740.2 { 1322.6 41,652.9 { 1875.7 42,218.3 { 2135.9 43,969.0 { 2170.4
Note. SPCs obtained from 200 mg 1 1 or 200 mg 1 5 of OVA fed rats were stimulated with OVA in vitro, then mixed with an equal number of ILNCs from OVA-primed rats cultured in the presence of OVA, and the proliferative responses to OVA were measured as described under Materials and Methods. SPCs obtained from LPHfed rats were used as controls. Values are the mean cpm { SEM of quadruplicate cultures of cells pooled from five rats. Data are representative of three experiments.
C were not reduced compared with the responses in group A / D even when higher ratios of the ILNCs from OVA-tolerant rats were used at a 3:1 ratio (data not shown). The findings that the responses of group A / B, A / C, and A / D were reduced by 40% compared to group A (Fig. 5) may not be due to active suppression by the ILNCs from OVA-tolerant rats, but rather to the proportional dilution of the responding cell population (group A) by the low-responding cell population (group B, C, or D).
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the five consecutive daily oral administrations with 200 mg, 2 mg or 20 mg of OVA before immunization, significantly suppressed antibody-mediated acute OIA in a dose-dependent manner (Figs. 1, 2). In both cases, serum anti-OVA IgG levels (Fig. 3) and in vitro proliferative responses of the ILNCs to OVA (Fig. 4) were also dose-dependently reduced, comparable to the suppression of acute OIA (Fig. 2). In addition, since acute KLHinduced arthritis was suppressed by prior feeding of KLH but not by OVA (Table 1) and vice versa, oral tolerance of acute AIA was confirmed to be antigen specific. These findings demonstrate that the suppressive effects of feeding OVA on acute OIA were associated with induction of OT to OVA, and are consistent with previous reports of oral treatment by feeding with the appropriate antigen in a variety of experimental cell-mediated diseases (2–5, 8–15, 26), as well as our recent report showing that chronic AIA can be suppressed by prior feeding with the inducing antigen (27). Moreover, studies indicate that oral treatment by prior feeding with the appropriate antigen not only inhibits cell-mediated inflammatory responses but also reduces antigen-specific humoral responses (8, 28, 29), and protect animals from the development of experimental im-
Effect of Culturing with IL-2 on the Decreased Proliferative Responses of the Tolerant Lymph Node Cells We next assessed whether clonal anergy of antigenreactive T lymphocytes is a possible mechanism of the OT in the present study. Tolerized and control ILNCs were cultured for 5 days in the presence of rat IL-2 or medium. Proliferative responses to OVA were measured just before and after a 5-day culture with or without rat IL-2. As shown in Fig. 6, OVA-specific proliferative responses, measured before IL-2 stimulation, were markedly reduced in both the high dose (200 mg 1 1) and the low dose (200 mg 1 5) of OVA-fed tolerant rats (Fig. 6A). These reduced OVA-specific proliferative responses were well restored after IL-2 stimulation in both feeding protocols, but the preculture of cells with IL-2 had little effect on the proliferative responses to OVA of ILNCs derived from CFA-primed rats (Fig. 6C). In addition, the reduced OVA-specific proliferative responses were not restored after culture with medium (Fig. 6B). DISCUSSION We successfully demonstrated that a single bolus oral administration with 2, 20, or 200 mg of OVA, or
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FIG. 5. ILNCs from OVA-tolerant rats do not actively suppress the proliferative responses to OVA of ILNCs from OVA-primed rats. Rats were unfed and immunized with OVA (group A). OVA-tolerant rats were fed 200 mg of OVA on Day 07 (group B), or 200 mg of OVA on Days 05, 04, 03, 02, and 01 (group C), and immunized with OVA on Day 0. ILNCs from unfed, CFA-primed rats served as negative controls (group D). Fourteen days after immunization, proliferative responses to OVA were measured as described under Materials and Methods. Mixed cultures were prepared by mixing unfed, OVAprimed ILNCs with OVA-tolerant ILNCs (group A / B or A / C) or with CFA-primed ILNCs (group A / D) at a 1:1 ratio. The results are expressed as mean cpm { SEM of quadruplicate cultures of cells pooled from five rats. Background counts of OVA-immunized cells without OVA added were between 1000 and 3000. **P õ 0.01 compared with group A. NS, not significant. Data are representative of three experiments.
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FIG. 6. Reversal of anergy by IL-2 stimulation. The lymphocyte proliferative responses to OVA were measured just before (A) and after a 5-day culture in the absence (B) or presence of rat IL-2 (C). Proliferative responses shown are of OVA-primed nontolerant cells fed 200 mg of LPH 1 1 (a) or 200 mg of LPH 1 5 (c), or of OVA-primed tolerant cells fed 200 mg of OVA 1 1 (b), 200 mg of OVA 1 5 (d). An additional group (e) was unfed and immunized with CFA alone. The results are expressed as mean cpm { SEM of quadruplicate cultures of cells pooled from five rats. Background counts of OVA-immunized cells without OVA added were between 1000 and 2000. **P õ 0.01; group a vs group b, and group c vs group d. NS, not significant. Data are representative of three experiments.
mune complex glomerulonephritis, which is also mediated by type III hypersensitivity reactions (30, 31). Although in some cases it appears that B cells themselves may be tolerized (32), it is generally assumed that T cells play the critical role in the induction of OT (33). Different mechanisms of OT have been suggested, and these include the generation of active suppression (3, 4), clonal anergy of antigen-reactive T cells (5, 6), and clonal deletion of antigen-reactive T cells (7). Active suppression is mediated by antigen-specific regulatory cells that produce suppressive cytokines such as transforming growth factor b (TGF-b) (4, 34, 35). Recent reports indicated that the relative roles of the mechanisms of OT are determined primarily by the dose of antigen administered (25, 36). Low doses of antigen that are fed intermittently (up to 1 mg/feeding) favor the active suppression (3, 4, 25, 34–36), whereas high doses of antigen that are fed either as a single bolus or intermittently favor the clonal anergy (5, 6, 25, 36) or the clonal deletion (7). The results of the present study in both the highand the low-dose feeding protocols support the mechanism of clonal anergy rather than active suppression, because [1] in adoptive transfer experiments, the SPCs from OVA-fed, unprimed rats could not suppress the development of acute OIA, anti-OVA Ab production, and lymphocyte proliferative responses to OVA (Table 2); [2] in coculture systems, the SPCs from OVA-fed, unprimed rats failed to exert their suppressive effects on the ILNCs from OVA-primed rats (Table 3); and [3] the in vitro proliferative responses to OVA of the ILNCs from OVA tolerant (OVA-fed, OVA-primed) rats were really suppressed, but in coculture systems, the cells
failed to exert their suppressive effects on the ILNCs from OVA-primed rats (Fig. 5). Direct evidence for the existence of anergic antigenreactive T lymphocytes in peripheral lymph nodes of tolerant animals has been attained by reversal of hyporesponsiveness of the cells with IL-2 stimulation in vitro (6, 37, 38). In our experiments, the involvement of the clonal anergy in the induction of OT was demonstrated further in Fig. 6. The reduced OVA-specific proliferative responses of the ILNCs from OVA tolerant rats could be reversed by rat IL-2 stimulation in vitro, not only in the high-dose feeding protocols but also in the low-dose feeding protocols (Fig. 6). These observations indicate that OVA-reactive T lymphocytes exist in inguinal lymph nodes of OVA tolerant rats, but they are most likely in an anergic state. Since recent studies indicate that orally induced T cell clonal anergy can reduce B cell Ab production through a lack of T cell help from anergic T cells (33) and affect B cell development as demonstrated by restoration of Ab production in the presence of T cell-derived cytokines such as IL4 and IL-5 (29), we therefore suggest that clonal anergy was the principal mechanism of OVA-induced tolerance in this study. However, to confirm this phenomena, it is important to study the existence of TGF-b in the culture supernatant of lymphocyte from OVA-tolerant rats. Although at present we do not know the reason that clonal anergy was induced even in low-dose feeding protocols; we suppose that the discrepancy between our findings and those of others might be caused by experimental differences, including differences of the disease models (3, 4, 25, 34–36), the kind of animals (25, 34), and the antigens (3, 4, 25, 34–36).
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In human clinical trials, it is shown that some patients with multiple sclerosis, uveitis, and rheumatoid arthritis may benefit from oral administration of bovine myelin, bovine S-antigen, and chicken type II collagen (39–41). Our present study suggests that oral administration of the appropriate antigen may be desirable for patients with a disease mediated by humoral immune responses to the antigen, and this hypothesis is supported by several previous reports which showed that tolerance of antibody responses can occur when antigen is fed after immunization (42–45). However, careful consideration is necessary for the clinical application of this strategy, because other reports have indicated that oral administration of antigen enhances preexisting immune responses in some instances (46–48). ACKNOWLEDGMENTS We thank Dr. Kazunori Ohki and Dr. Akiko Kukita for reviewing the manuscript and their helpful comments.
REFERENCES 1. Mowat, A. M., ‘‘Handbook of Mucosal Immunology,’’ p. 185, Academic Press, New York, 1994. 2. Weiner, H. L., Friedman, A., Miller, A., Khoury, S. J., Al-Sabbagh, A., Santos, L., Sayegh, M., Nussenblatt, R. B., Trentham, D. E., and Hafler, D. A., Annu. Rev. Immunol. 12, 809, 1994. 3. Lider, O., Santos, L. M. B., Lee, C. S. Y., Higgins, P. J., and Weiner, H. L., J. Immunol. 142, 748, 1989. 4. Miller, A., Lider, O., Roberts, A. B., Sporn, M. B., and Weiner, H. L., Proc. Natl. Acad. Sci. USA 89, 421, 1992. 5. Whitacre, C. C., Gienapp, I. E., Orosz, C. G., and Bitar, D. M., J. Immunol. 147, 2155, 1991. 6. Melamed, D., and Friedman, A., Eur. J. Immunol. 23, 935, 1993. 7. Chen, Y., Inobe, J., Marks, R., Gonnella, P., Kuchroo, V. K., and Weiner, H. L., Nature 376, 177, 1995. 8. Higgins, P. J., and Weiner, H. L., J. Immunol. 140, 440, 1988. 9. Bitar, D. M., and Whitacre, C. C., Cell. Immunol. 112, 364, 1988. 10. Nussenblatt, R. B., Caspi, R. R., Mahdi, R., Chan, C., Roberge, F., Lider, O., and Weiner, H. L., J. Immunol. 144, 1689, 1990. 11. Singh, V. K., Kalra, H. K., Yamaki, K., and Shinohara, T., Cell. Immunol. 139, 81, 1992. 12. Zhang, Z. J., Davidson, L., Eisenbarth, G., and Weiner, H. L., Proc. Natl. Acad. Sci. USA 88, 10252, 1991. 13. Nagler-Anderson, C., Bober, L. A., Robinson, M. E., Siskind, G. W., and Thorbecke, G. J., Proc. Natl. Acad. Sci. USA 83, 7443, 1986. 14. Thompson, H. S. G., and Staines, N. A., Clin. Exp. Immunol. 64, 581, 1986. 15. Zhang, Z. J., Lee, C. S. Y., Lider, O., and Weiner, H. L., J. Immunol. 145, 2489, 1990. 16. Dumonde, D. C., and Glynn, L. E., Br. J. Exp. Pathol. 43, 373, 1962. 17. Brauer, R., Thoss, K., Henzgen, S., and Waldmann, G., Exp. Pathol. 34, 197, 1988. 18. Jasin, H. E., Clin. Exp. Immunol. 22, 473, 1975.
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75
19. Brackertz, D., Mitchell, G., and Mackay, I. R., Arthritis Rheum. 20, 841, 1977. 20. Steinberg, M. E., McCrae, C. R., Cohen, L. D., and Schumacher, H. R., Clin. Orthop. Relat. Res. 97, 248, 1973. 21. Ohno, O., Tateishi, H., and Cooke, T. D., Arthritis Rheum. 21, 81, 1978. 22. Ugai, K., Ziff, M., and Jasin, H. E., Arthritis Rheum. 22, 353, 1979. 23. Yoshino, S., and Yoshino, J., Cell. Immunol. 144, 382, 1992. 24. Engvall, E., and Perlmann, P., J. Immunol. 109, 129, 1972. 25. Friedman, A., and Weiner, H. L., Proc. Natl. Acad. Sci. USA 91, 6688, 1994. 26. Yoshino, S., Quattrocchi, E., and Weiner, H. L., Arthritis Rheum. 38, 1092, 1995. 27. Yoshino, S., Cell. Immunol. 163, 55, 1995. 28. Fuller, K. A., Pearl, D., and Whitacre, C. C., J. Neuroimmunol. 28, 15, 1990. 29. Kelly, K. A., and Whitacre, C. C., J. Neuroimmunol. 66, 77, 1996. 30. Devey, M. E., and Bleasdale, K., Clin. Exp. Immunol. 56, 637, 1984. 31. Browning, M. J., and Parrott, D. M. V., Adv. Exp. Med. Biol. 216B, 1619, 1987. 32. Vives, J., Parks, D. E., and Weigle, W. O., J. Immunol. 125, 1811, 1980. 33. Hirahara, K., Hisatsune, T., Nishijima, K., Kato, H., Shiho, O., and Kaminogawa, S., J. Immunol. 154, 6238, 1995. 34. Chen, Y., Kuchroo, V. K., Inobe, J., Hafler, D. A., and Weiner, H. L., Science 265, 1237, 1994. 35. Miller, A., Al-Sabbagh, A., Santos, L. M. B., Prabhu-Das, M., and Weiner, H. L., J. Immunol. 151, 7307, 1993. 36. Gregerson, D. S., Obritsch, W. F., and Donoso, L. A., J. Immunol. 151, 5751, 1993. 37. DeSilva, D. R., Urdahl, K. B., and Jenkins, M. K., J. Immunol. 147, 3261, 1991. 38. Beverly, B., Kang, S.-M., Lenardo, M. J., and Schwartz, R. H., Int. Immunol. 4, 661, 1992. 39. Weiner, H. L., Mackin, G. A., Matsui, M., Orav, E. J., Khoury, S. J., Dawson, D. M., and Hafler, D. A., Science 259, 1321, 1993. 40. Nussenblatt, R. B., de Smet, M. D., Weiner, H. L., and Gery, I., ‘‘Behcet’s Disease: Proceedings of the 6th International Conference on Behcet’s Disease, Paris, France,’’ p. 641, Excerpta Medica, Elsevier, Amsterdam, 1993. 41. Trentham, D. E., Dynesius-Trentham, R. A., Orav, E. J., Combitchi, D., Lorenzo, C., Sewell, K. L., Hafler, D. A., and Weiner, H. L., Science 261, 1727, 1993. 42. Lafont, S., Andre, C., Andre, F., Gillon, J., and Fargier, M.-C., J. Exp. Med. 155, 1573, 1982. 43. Bloch, K. J., Perry, R., Bloch, M., and Walker, W. A., Ann. N. Y. Acad. Sci. 409, 787, 1983. 44. Saklayen, M. G., Pesce, A. J., Pollak, V. E., and Michael, J. G., Int. Arch. Allergy Appl. Immunol. 73, 5, 1984. 45. Peng, H.-J., Turner, M. W., and Strobel, S., Immunol. 67, 425, 1989. 46. Hanson, D. G., Vaz, N. M., Rawlings, L. A., and Lynch, J. M., J. Immunol. 122, 2261, 1979. 47. Titus, R. G., and Chiller, J. M., Int. Arch. Allergy Appl. Immunol. 65, 323, 1981. 48. Lens, J. W., van den Berg, W. B., van de Putte, L. B. A., and van den Bersselaar, L., Clin. Exp. Immunol. 58, 364, 1984.
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