Induction of Oral Tolerance in TGF-β1 Null Mice

RESEARCH ARTICLE | JULY 01 1998 Induction of Oral Tolerance in TGF-β1 Null Mice  1 K. Siobhan Barone; ... et. al https://doi.org/10.4049/jimmunol.161.1.154 Related Content β2-Microglobulin-De cient Background Ameliorates Lethal Phenotype of the TGF-β1 Null Mouse J Immunol (October,1999) Dysregulation of IFN-γ Signaling Pathways in the Absence of TGF-β1 J Immunol (November,2002) Aberrant Toll Receptor Expression and Endotoxin Hypersensitivity in Mice Lacking a Functional TGF-β1 Signaling Pathway J Immunol (March,2004) Downloaded from http://journals.aai.org/jimmunol/article-pdf/161/1/154/1082304/im139800154o.pdf by guest on 04 January 2023 J Immunol (1998) 161 (1): 154–160. Induction of Oral Tolerance in TGF-b1 Null Mice1 K. Siobhan Barone,2* Dana D. Tolarova,† Ilona Ormsby,† Thomas Doetschman,† and J. Gabriel Michael† I ngestion of soluble Ag has long been known to lead to subsequent immunologic hyporesponsiveness to that specific Ag. This phenomenon, referred to as oral tolerance, may have the evolutionary advantage of preventing hypersensitivity reactions to ingested food proteins (1, 2). In addition, it may be responsible for preventing immune responses to the numerous food proteins encountered daily, thus reducing the risk of inducing autimmune reactions via cross-reactivity with self Ags (1). Interest in discovering the underlying mechanism of oral tolerance has recently intensified due to the therapeutic potential it holds in the treatment of certain autoimmune diseases. Therapies based on the concept of oral tolerance have proved effective in treating animals with experimental autoimmune diseases (3– 6). Similar treatments are now being applied to humans in treating such autoimmune diseases as rheumatoid arthritis, multiple sclerosis, and uveitis (7–9). It has been shown that oral tolerance is due primarily to the inactivation of the CD41 cell, which plays a pivotal role in the induction of both humoral and cellular immune responses (10). However, the mechanism by which Ag-specific CD41 cells are inactivated remains a point of considerable controversy. Experiments by others have indicated that the secretion of cytokines is responsible for mediating suppression, a phenomenon termed active or bystander suppression (3, 11). The cytokine primarily responsible for this suppression was suggested to be TGF-b1 (12, 13). In contrast, we and others have produced evidence indicating that direct inactivation of the Ag-specific CD41 cell occurs via a phenomenon known as anergy (the functional inactivation of a cell) rather than by active suppression (14 –20). *Department of Biology, Thomas More College, Crestview Hills, KY 41017; †Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH 45267 Received for publication December 4, 1997. Accepted for publication March 3, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by a Research Enhancement grant from the Kentucky National Science Foundation/Experimental Program to Stimulate Competitive Research (EPSCoR). 2 Address correspondence and reprint requests to Dr. K. Siobhan Barone, Department of Biology, Thomas More College, 333 Thomas More Pkwy., Crestview Hills, KY 41017. Copyright © 1998 by The American Association of Immunologists To resolve the controversy over which mechanism was responsible for the generation of oral tolerance, it was proposed that the dose of Ag fed was the critical factor (21). Feeding high doses of Ag resulted in tolerance characterized by anergy or deletion, with little or no active suppression evident; in contrast, low doses of fed Ag resulted in tolerance induction characterized by the presence of regulatory cells secreting cytokines (21, 22). In addition to TGFb1, increased production of IL-4 and IL-10 has been observed in both lymph node cells and CD41 clones derived from mice fed low doses of Ag (23–26). Given the pivotal role that TGF-b1 appears to play in the generation of low dose oral tolerance, it is essential to clarify its function in this process. Three isoforms of TGF-b exist in mammals: TGF-b1, TGF-b2, and TGF-b3. TGF-b1 has been identified as an imunoregulatory molecule with both immunogenic and immonsuppressive properties depending on the cellular environment (27, 28). Systemic administration of TGF-b1 has been shown to suppress immune responses, while local administration enhanced inflammatory responses (28). Recently, knockout mice were generated using embryonic stem cells with a disrupted TGF-b1 gene (29, 30). TGF-b1 null mutants exhibit inflammation leading to tissue necrosis of numerous organs, particularly heart and stomach. These lesions are characterized by elevated levels of peripheral lymphocytes and immature neutrophils (29) as well as enhanced adhesiveness of the TGF-b1 null leukocytes (31). Gross development of TGF-b1 null mice appears normal until approximately the third week after birth, at which time they exhibit an acute wasting syndrome that quickly leads to death. As it is difficult to induce oral tolerance in mice before 2 to 3 wk of age, it was previously impossible to use these mice as models to assess the role of TGF-b1 in the generation of oral tolerance. However, due to recent advances, the lives of these TGF-b1 null mice can now be extended to approximately 40 days or more by repeated injection of an Ab (anti-CD11a) to LFA-1 (32). LFA-1 is an integrin found on lymphocytes that allows them to bind to other cells, such as ICAM-1 on endothelial cells, thus facilitating transendothelial migration. It has been suggested that the anti-CD11a mAb inhibits the extravasation and migration of lymphocytes into surrounding tissues (i.e., heart and stomach) and thus delays the onset of organ inflammation and subsequent death (32). 0022-1767/98/$02.00 Downloaded from http://journals.aai.org/jimmunol/article-pdf/161/1/154/1082304/im139800154o.pdf by guest on 04 January 2023 Previous studies have suggested that oral tolerance induction by low doses of Ag is mediated by inhibitory cytokines, particularly TGF-b1. To examine the roles of TGF-b1 and other inhibitory cytokines in the induction of oral tolerance, TGF-b1 null mice and controls were gavaged with 10 to 20 mg (high dose) or 1 mg (low dose) of OVA for 3 days. After immunization with OVA, the in vitro proliferative response of OVA-specific popliteal lymph node cells was assessed. Lymphocytes from all TGF-b1 null mice fed high doses of OVA exhibited highly significant suppression compared with controls. A weaker, but still significant, suppression was observed in lymphocytes from the majority of TGF-b1 null mice fed low doses of OVA. In addition, supernatants from these lymphocytes exhibited lower levels of IL-4, IL-10, and IFN-g than those from water-fed control animals. These results indicate that while TGF-b1 may play a role in suppression, inhibitory cytokines are not the exclusive mechanism by which low dose oral tolerance is induced. The Journal of Immunology, 1998, 161: 154 –160. The Journal of Immunology Given the above finding, it is now possible to use antiCD11a-treated TGF-b1 null mice as models to evaluate the role of TGF-b1 in the generation of oral tolerance. Therefore, antiCD11a-treated TGF-b1 null mice were fed both high and low doses of OVA, and the ability of these mice to establish oral tolerance was assessed. The results of our studies indicate that high dose feeding did not interfere with oral tolerance induction in TGF-b1 null mice. However, we also found that a significant number of the TGF-b1 null mice fed low doses of OVA were able to be tolerized; this was an unexpected result, since TGF-b1 is thought to play a primary role in the induction of low dose oral tolerance. In addition, cytokine analysis revealed that levels of other putative inhibitory cytokines (IL-4 and IL-10) were decreased, rather than enhanced, in tolerized TGF-b1 null mice. Mice Male and female (129 3 CF1) mice heterozygous for the TGF-b1 null allele were housed in the animal facility at the University of Cincinnati Medical Center under barrier housing conditions. Neonates from heterozygote intercrosses were identified as TGF-b1 null mice within 48 h of birth using the PCR techniques described below. Neonates identified as either control mice (wild-type, 1/1, or heterozygous, 1/2) or TGF-b1 null mice (2/2) were immediately given injections of antiCD11a (60 mg/injection) every other day from day 2 after birth until weaning (approximately day 18 after birth). All experiments used 3- to 4-wk-old mice. RT-PCR genotyping RT-PCR genotyping was performed as described previously (29). Briefly, mRNA was isolated from tail clips of newborn mice; the isolated mRNA was reverse transcribed and examined by PCR analysis using an upstream primer (59-GAGAAGAACTGCTGTGTGCG-39) and a downstream primer (59-GTGTCCAGGCTCCAAATATAGG-39) corresponding to the exon 6 sequences flanking the neo insert in the TGF-b1 null mutant. PCR was performed on a thermal cycler in a 20-ml volume at 95°C for 20 s, at 55°C for 50 s, and at 72°C for 1 min for 30 cycles. Amplified products were size fractionated by electrophoresis through agarose and visualized by UV illumination on ethidium bromide-stained gels. Positive controls consisted of total RNA from spleen cells cultured for 48 h with Con A. Reagents Chicken egg albumin (OVA; grade V) and Con A were obtained from Sigma (St. Louis, MO). Anti-CD11a was a gift from Genentech (San Francisco, CA). Effect of anti-CD11a treatment on immune responsiveness Neonatal mice (1/1) were divided into two groups and injected with 0.02 ml of either PBS or anti-CD11a (60 mg) from day 2 until day 18 after birth. On day 31 after birth, all mice were injected with 10 mg of OVA in IFA in the footpad and tail base. Ten days later, mice were bled via the retroorbital plexus and then sacrificed; their popliteal lymph nodes (PLN)3 were removed, and cells were incubated in vitro with OVA (100 mg/ml, final concentration). Serum Ab responses were assessed using ELISA, and in vitro lymphocyte responsiveness was determined using liquid scintillation spectrometry. Induction of immune responsiveness in anti-CD11a-treated TGF-b1 null mice Anti-CD11a-treated TGF-b1 null (2/2) mice and control (1/1 or 1/2) mice were injected with 10 mg of OVA in IFA in the footpad and tail base on day 31 after birth; 10 days later, mice were sacrificed, PLN were removed, and cells were incubated in vitro with either Con A (2.5 mg/ml, final concentration) or OVA (1000 or 100 mg/ml, final concentration). In vitro lymphocyte responsiveness was determined using liquid scintillation spectrometry. 3 Abbreviation used in this paper: PLN, popliteal lymph nodes. Induction of oral tolerance in anti-CD11a-treated TGF-b1 null mice Anti-CD11a-treated TGF-b1 null (2/2) mice and control (1/1 or 1/2) mice were each divided into two groups; mice in one group were orally tolerized by gavaging either 10 to 20 mg (high dose) or 1 mg (low dose) of OVA/mouse dissolved in 0.5 ml of water three times over a 3-day period, while mice in the second group received water only. Eight days after the last feeding, all mice were injected in the tail base and footpad with 10 mg of OVA in IFA. Ten days after immunization, mice were then sacrificed, and lymphocytes were obtained from the PLN to assess the OVA-specific proliferative response. Ab titer determination Ab titer was determined as described previously (19). Briefly, serum samples were serially diluted on plates coated with OVA and incubated for 1 h at 37°C. Goat anti-mouse IgG coupled to alkaline phosphatase (1/1000; Boehringer Mannheim, Indianapolis, IN) was then added, and the plate was incubated for an additional 1 h at 37°C. 4-Nitrophenylphosphate (Boehringer Mannheim), at a concentration of 1 mg/ml, was added as substrate, and the color change was measured using a Bio-Rad microplate reader (Bio-Rad, Hercules, CA) at a wavelength of 405 nm. All samples were pooled, assayed in duplicate, and expressed as Ab titer 6 SD. Standard curves were run each time an assay was performed using hyperimmune anti-OVA antisera, and sample titers were calculated based on dilutions that resulted in absorbance equal to 1/400 diluted standard sera. Serum samples were considered positive if their titers exhibited a fivefold or greater increase over titers of unimmunized mice (previous studies have consistently shown that unimmunized mice exhibit titers ,50; data not shown). Assessment of in vitro lymphocyte proliferative responses Proliferative responses were assessed as described previously (19). Briefly, 10 days after footpad and tail base immunization, draining PLN were removed, and single cell suspensions were prepared in supplemented RPMI 1640 medium and 5% heat-inactivated FCS. Cells were then aliquoted at 5 3 105 cells/well in 96-well flat-bottom plates along with OVA at a concentration of 1000 or 100 mg/ml (maximal stimulation from previous experiments was seen with these doses) and incubated at 37°C in humidified 5% CO2 for 48 h. Cells were pulsed for 18 h with 1 mCi of [3H]thymidine and harvested onto glass-fiber filtermats using a Skatron multiple automated sample harvester (Skatron, Sterling, VA). Radioactivity was determined using liquid scintillation spectrometry. Addition of 100 mg/ml of an unrelated Ag, cytochrome c, was added to control wells to assess nonspecific proliferation. Assessment of cytokine secretion in anti-CD11a-treated TGF-b1 null mice fed low doses of Ag Anti-CD11a-treated TGF-b1 null mice (2/2) and control (1/1 or 1/2) mice were divided into two groups. Mice in one group were orally tolerized by gavaging 1.0 mg of OVA/mouse dissolved in 0.5 ml of water three times over a 3-day period, while mice in the second group received water only. Eight days after the last feeding, all mice were injected in the tail base and footpad with 10 mg of OVA in IFA. Ten days after immunization, mice were sacrificed, lymphocytes were obtained from the spleen and PLN, and single cell suspensions were prepared. RBC were lysed from spleen cell suspensions using Tris-buffered ammonium chloride. Spleen cells and PLN cells were then aliquoted at 8 3 105 and 5 3 105 cells/well, respectively, in 24-well flat-bottom plates along with either medium or 100 mg/ml OVA. All cells were incubated at 37°C in humidified 5% CO2 for 48 h. Supernatants were then harvested and stored at 270°C. IL-4, IL-10, and IFN-g production was quantified using sandwich ELISA techniques. The concentrations of IL-4 and IFN-g in cell supernatants were quantified using murine IL-4 and IFN-g ELISA kits, respectively (Genzyme, Cambridge, MA). The concentration of IL-10 in cell supernatants was quantified using an Ag capture ELISA with Abs (capture and secondary biotinylated Abs) purchased from PharMingen (San Diego, CA); murine rIL-2 was also purchased from PharMingen. All ELISAs were conducted according to the manufacturer’s recommendation. Statistical analysis Due to the small number of TGF-b1 null mice available from each litter, experiments were repeated a minimum of three times. Results obtained from the in vitro lymphocyte responsiveness experiments are indicated as the counts per minute 6 SD of triplicate cultures. The statistical significance of differences among experimental values was determined using the Downloaded from http://journals.aai.org/jimmunol/article-pdf/161/1/154/1082304/im139800154o.pdf by guest on 04 January 2023 Materials and Methods 155 156 Student’s t test for multiple comparisons. A value of p # 0.05 was considered significant. Stimulation indexes were generated for each experimental group or mouse by dividing the total counts per minute by the background counts per minute. For cytokine analysis experiments, cell supernatants from mice in each group were pooled and run in triplicate; the SD was determined. The statistical significance of differences among experimental values was determined using Student’s t test for multiple comparisons. A value of p # 0.05 was considered significant. Results Effect of anti-CD11a treatment on immune responsiveness and the induction of oral tolerance Initially, it was necessary to determine whether anti-CD11a treatment affected the ability of treated mice to mount an immune response. To assess this, wild-type mice were treated with antiCD11a up until day 18 after birth (the treatment time used in all subsequent experiments), while controls were injected with PBS. As seen from the results in Figure 1, treatment with anti-CD11a, while having a slight inhibitory effect, did not result in a drastic reduction in cellular proliferation. In contrast, humoral responses of the mice in the anti-CD11a-treated group exhibited profound inhibition and were not used for assessment purposes in future investigations. Although the ability to induce oral tolerance in CD11a-treated mice was slightly less effective than that in untreated mice (data not shown), the results shown in Figures 2 and 3 (for wild-type FIGURE 2. Induction of high dose tolerance in TGF-b1 null mice. Each mouse was injected s.c. with approximately 60 mg of anti-LFA1 from day 2 after birth until weaning. On day 18 after birth, both wild-type and mutant mice were fed 10 to 20 mg of OVA for 3 consecutive days. On day 31 after birth, the mice were injected with 10 mg of OVA in IFA in the footpad and tail base. Ten days later, the mice were sacrificed, PLN were removed, and the cellular proliferation assay was performed in vitro with OVA at the concentrations indicated. A, B, and C represent three independent experiments (wild-type mice, n 5 3, 2/2 mice, n 5 1). Background counts for each group were subtracted to give the final counts per minute. Background counts are as follows: A, 1/1 or 1/2 mice fed water, 4,524; 1/1 or 1/2 mice fed OVA, 1,552; 2/2 mice fed water, 16,171; 2/2 mice fed OVA, 15,590, 16,521, and 15,881, respectively. B, 1/1 or 1/2 mice fed water, 7,260; 1/1 or 1/2 mice fed OVA, 6,340; 2/2 mice fed water, 12,392 and 10,179, respectively; 2/2 mice fed OVA, 10,294. C, 1/1 or 1/2 mice fed water, 2,200; 1/1 or 1/2 mice fed OVA, 1,552; 2/2 mice fed water, 14,464 and 14,486, respectively; 2/2 mice fed OVA, 12,101. The asterisk indicates Student’s t test values that significantly differ ( p # 0.05) from the corresponding value for the water-fed control. mice) clearly show that significant oral tolerance can be induced in these mice using both high and low dose feeding regimens. Immune responsiveness of anti-CD11a-treated TGF-b1 null mice The prolonged life of TGF-b1 null mice treated with anti-CD11a permitted these animals to be the subject of oral tolerance experiments. However, since previous reports have indicated that lymphocytes from TGF-b1 null mutant mice (without anti-CD11a treatment) may be maximally stimulated inherently (33), we first determined whether lymph nodes from anti-CD11a-treated TGF-b1 null mutant mice exhibited an increased proliferative response to a given stimulus. To test this, cells were incubated with either the mitogen Con A or various doses of OVA Ag. The results showed that the response of PLN from anti-CD11atreated TGF-b1 null mutant mice to Con A stimulation was similar to that of anti-CD11a-treated wild-type mice (214,219 6 8,046 and 170,114 6 19,098, respectively; counts are over background). These data suggested that lymphocytes from anti-CD11a-treated TGF-b1 null mutant mice were not maximally stimulated and were capable of proliferating in response to antigenic stimulation. This was confirmed by results from experiments showing that TGF-b1 null mutant mice were able to respond specifically to various doses Downloaded from http://journals.aai.org/jimmunol/article-pdf/161/1/154/1082304/im139800154o.pdf by guest on 04 January 2023 FIGURE 1. The effect of anti-LFA-1 on the immune response. Neonatal mice were divided into two groups and injected with 0.02 ml of either PBS or anti-LFA-1 (CD11a) from day 2 until day 18 after birth. On day 31, all mice were injected with 10 mg of OVA in IFA in the footpad and tail base. Ten days later, mice were bled via the retro-orbital plexus and then sacrificed; their PLN were removed, and cells were incubated in vitro with 100 mg/ml OVA for 48 h. In vitro lymphocyte responsiveness was determined using liquid scintillation spectrometry (A). The anti-OVA serum Ab responses were assessed using ELISA (B). Statistical significance of differences among experimental values was determined using Student’s t test for multiple comparisons. p # 0.05 was considered significant. ORAL TOLERANCE IN TGF-b1 NULL MICE The Journal of Immunology 157 Table I. Immune responsiveness of anti-LFA1-treated TGF-b1 null micea Cellular Proliferation (cpm) OVA Concentration (mg/ml) 2/2 TGF-b1 null mouse (n 5 1) Indexb 50,991 6 6,056 28,222 6 1,042 NA 83,762 6 15,064 61,305 6 14,764 NA 1.64* 2.17* NA NA 26,128 6 759 15,388 6 4,126 NA 43,334 6 10,698 39,510 6 3,076 NA 1.66* 2.57* 49,696 6 10,738 31,699 6 4,480 22,754 6 4,961 65,451 6 4,622 56,028 6 9,135 42,986 6 3,300 1.32 1.77* 1.89* a Each experiment consisted of one wild-type mouse and one TGF-b1 null mutant mouse from the same litter. Each mouse was injected s.c. with approximately 60 mg of anti-LFA1 from day 2 after birth until weaning. On day 31 after birth, the mice were injected with 10 mg of OVA in IFA in the footpad and tail base. Ten days later, the mice were sacrificed, popliteal lymph nodes were removed, and the cellular proliferation assay was performed in vitro with the indicated amounts of OVA. Background counts were subtracted out to give the final cpm count. Background cpm counts for 1/1 wild-type mice in Expts. 1, 2, and 3 were 8359, 7231, and 8757, respectively. Background cpm counts for 2/2 TGF-ß1 null mice in Expts. 1, 2, and 3 were 15,948, 14,554 and 13,889, respectively. b Indices were calculated by dividing the cpm value for the mutant mouse by the cpm value of the control mouse. Asterisk indicates Student’s t test values that significantly differ (p # 0.05) from the corresponding value for wild-type mice. FIGURE 3. Induction of low dose tolerance in TGF-b1 null mice. The experiment was performed as described in Figure 2, except that wild-type and mutant mice were fed 1 mg of OVA for 3 consecutive days. A, B, C, and D represent four independent experiments (wild-type mice, n 5 3; 2/2 mice, n 5 1). Background counts for each group were subtracted to give the final counts per minute. Background counts are as follows: A, 1/1 or 1/2 mice fed water, 2,200; 1/1 or 1/2 mice fed OVA, 3,463; 2/2 mice fed water, 14,464 and 14,486, respectively; 2/2 mice fed OVA, 18,424. B, 1/1 or 1/2 mice fed water, 8,809; 1/1 or 1/2 mice fed OVA, 7,554; 2/2 mice fed water, 16,096 and 15,916, respectively; 2/2 mice fed OVA, 13,420, 13,348, and 15,598, respectively. C, 2/2 mice fed water, 15,616 and 15,366, respectively; 2/2 mice fed OVA, 13,523 and 12,283, respectively. D, 1/1 or 1/2 mice fed water, 785; 1/1 or 1/2 mice fed OVA, 1,508; 2/2 mice fed water, 2,510; 2/2 mice fed OVA, 3,082, 2,295, and 3,005, respectively. The asterisk indicates Student’s t test values that significantly differ ( p # 0.05) from the corresponding value for the water-fed control. of a given Ag (Table I); the in vitro proliferative response to an unrelated Ag, cytochrome c, was ,10% over background (data not shown). It is interesting to note that the proliferative responses of the PLN from TGF-b1 null mutant mice were almost always significantly higher than those of PLN from the wild-type controls (Table I). However, when stimulation indexes were generated for comparative purposes, little if any difference between wild-type and mutant mice could be observed. Stimulation indexes for each group were as follows. In Experiment 1, values for lymphocytes from 1/1 and 2/2 mice at an in vitro concentration of 1000 mg/ml were 7.1 and 6.3, respectively, and at an in vitro concentration of 100 mg/ml, they were 4.4 and 4.8, respectively. In Experiment 2, values for lymphocytes from 1/1 and 2/2 mice at an in vitro concentration of 100 mg/ml were 4.6 and 4.0, respectively, and at an in vitro concentration of 10 mg/ml, they were 3.1 and 3.7, respectively. In Experiment 3, values for lymphocytes from 1/1 and 2/2 mice at an in vitro concentration of 1000 mg/ml were 6.7 and 5.7, respectively; at an in vitro concentration of 100 mg/ml, they were 4.6 and 5.0, respectively; and at an in vitro concentration of 10 mg/ml, they were 3.6 and 4.0, respectively. Induction of high dose oral tolerance in anti-CD11a-treated TGF-b1 null mice To test whether TGF-b1 played a significant role in the generation of high dose oral tolerance, PLN from mice fed 10 to 20 mg/mouse of OVA for 3 days were stimulated in vitro with either 100 or 1000 mg/ml of OVA; the results are shown in Figure 2. In all experiments performed, high doses of OVA resulted in Ag-specific tolerance in the anti-CD11a-treated wild-type/heterozygote mice, indicating that the high dose feeding regimen used was capable of inducing tolerance. Similarly, all the TGF-b1 null OVA-fed mice showed highly significant levels of suppression. In addition, all OVA-fed mice had lower stimulation indexes than the corresponding water-fed control mice (data not shown). It should be noted that the suppression observed in the TGF-b1 null OVA-fed mice did not appear to be as profound as that in the wild-type mice. Induction of low dose oral tolerance in anti-CD11a-treated TGF-b1 null mice To test whether TGF-b1 played a significant role in the generation of low dose oral tolerance, the procedure described above was used, except that 1 mg/mouse of OVA was fed for 3 consecutive days. Results showed that anti-CD11a-treated wild-type/heterozygote mice were tolerized compared with water-fed controls, indicating that the low dose feeding regimen used was capable of inducing tolerance (Fig. 3). Of the nine TGF-b1 null OVA-fed mice, seven exhibited significant suppression compared with the TGF-b1 null H20-fed control(s) when stimulated with 100 mg/ml of OVA in vitro. The lymph node cells from one TGF-b1 null OVA-fed mice mouse showed no significant change in proliferation compared with controls (Fig. 3A), while another showed a significant increase in proliferation (Fig. 3D). When the PLN of TGF-b1 null OVA-fed mice were stimulated with 1000 mg/ml of OVA in vitro, only four of the nine mutant mice exhibited a suppressed proliferative response; three showed no change in the proliferative response compared with the water-fed controls, and one Downloaded from http://journals.aai.org/jimmunol/article-pdf/161/1/154/1082304/im139800154o.pdf by guest on 04 January 2023 Expt. 1 1000 100 10 Expt. 2 1000 100 10 Expt. 3 1000 100 10 1/1 Wild-type mouse (n 5 4) 158 ORAL TOLERANCE IN TGF-b1 NULL MICE Table II. Cytokine production in spleen cells from low dose orally tolerized TGFb1 null mice Cytokine Production (pg/ml)a IL-4 IL-10 IFN-g WT WT WT WT H2O OVA H2O OVA OVA OVA Medium Medium 212.9 6 6.2 13.9 6 4.5b 109.8 6 10.3 6.2 6 8.3 753.9 6 140.3 314.7 6 47.0 474.4 6 116.2 403.7 6 94.5 189,070.0 6 170.3 4,186.3 6 309.4 37,991.7 6 5,608.3 2,734.0 6 526.5 2/2 2/2 2/2 2/2 H2O OVA H2O OVA OVA OVA Medium Medium 22.6 6 1.4 16.2 6 1.1 11.0 6 1.9 12.4 6 3.3 424.3 6 101.3 196.1 6 58.9 283.9 6 97.5 347.8 6 181.9 9,341.7 6 1,111.7 7,320.7 6 505.0 3,790.0 6 902.6 4,409.0 6 307.7 WT WT WT WT H2O OVA H2O OVA OVA OVA Medium Medium 3.2 6 2.5 4.9 6 0.1 0.0 0.9 6 1.6 336.4 6 96.2 27.3 6 47.3 155.0 6 83.6 63.2 6 41.1 28,142.3 6 2,329.6 2,412.7 6 324.8 7,782.3 6 947.8 2,230.7 6 252.7 2/2 2/2 2/2 2/2 H2O OVA H2O OVA OVA OVA Medium Medium 44.9 6 2.7 4.4 6 0.6 3.2 6 0.6 0.3 6 0.6 574.4 6 97.8 42.1 6 20.0 301.0 6 12.3 109.4 6 63.4 25,246.7 6 3,126.4 4,837.7 6 309.0 7,122.7 6 766.6 2,049.3 6 289.0 Genotype Expt. 1 Expt. 2 a Each mouse was injected s.c. with approximately 60 mg of anti-LFA1 from day 2 after birth until weaning. On day 21 after birth, both wild-type and mutant mice were fed either water or 1 mg of OVA for 3 consecutive days. (Expt. 1: WT mice, n 5 3; 2/2 mice, H2O fed, n 5 4; Ova fed, n 5 5; Expt. 2: WT mice, n 5 3; 2/2 mice, H2O fed, n 5 5; Ova fed, n 5 3.) On day 31 after birth, the mice were injected with 10 mg of OVA in IFA in the foot pad and tail base. Ten days later, the mice were sacrificed, and spleen cells were removed and incubated in vitro with a final concentration of 100 mg/ml of OVA for 48 h. Cytokine analysis was performed as described in Materials and Methods. b Values in bold indicate Student’s t test values that significantly differ (p # 0.05) from the corresponding value for stimulated or unstimulated water-fed control. showed a significant increase in the proliferative response (Fig. 3). All OVA-fed mice that had counts per minute values significantly lower then the corresponding water-fed control mice also had lower stimulation indexes (data not shown). Taken together, these results indicate that TGF-b1 null mice fed low doses of OVA are able to be tolerized, but less effectively than the controls. Assessment of IL-4, IL-10, and IFN-g levels in antiCD11a-treated TGF-b1 null mice fed low doses of OVA Since seven of nine anti-CD11a-treated TGF-b1 null mutant mice fed a low dose of OVA exhibited a significantly decreased proliferative response when stimulated with 100 mg/ml of OVA in vitro, this indicated that factors other than TGF-b1 must be involved in the induction of low dose oral tolerance. Studies by others have shown that secretion of cytokines other than TGF-b1 may play a role in inducing oral tolerance. Therefore, IL-4 and IL-10 levels were quantitated in stimulated spleen cells and lymph node cells from wild-type/heterozygote and TGF-b1 null mutant mice fed low doses of OVA. The results presented in Table II show that IL-4, IL-10, and IFN-g levels were reduced in spleen cells from mice fed OVA compared with those in spleen cells from mice fed water (with the exception of IL-4 in the wild-type control group, Expt. 2). Similarly, levels of IL-10 and IFN-g were reduced in PLN from mice fed OVA compared with those in PLN from mice fed water (Table III); levels of IL-4 could not be assessed in PLN due to the lack of detectable levels. Discussion Recent studies by others have indicated that active suppression, particularly TGF-b1, plays a major role in the generation of low dose oral tolerance, while T cell anergy and/or deletion may be the primary mechanism involved in the induction of high dose oral tolerance. However, it is possible that these mechanisms are not mutually exclusive and that components of both anergy and active suppression are involved in high and low dose oral tolerance, with one mechanism assuming the dominant role (34). To clarify the primary mechanism involved in low dose oral tolerance, TGF-b1 null mice were used. Previously, it has not been possible to use TGF-b1 null mice in oral tolerance experiments due to the fact that these mutants rarely live beyond 3 wk of age; however, treatment of TGF-b1 null mice with anti-CD411a in vivo significantly prolonged their life-span (32). Thus, it is now possible to feed Ag to TGF-b1 null mice and determine the role of TGF-b1 in the generation of oral suppression. The results of preliminary studies showed that mice treated with anti-CD11a were capable of mounting an immune response to parenterally administered Ag at the level of cellular proliferation. Table III. Cytokine production in PLN from low dose orally tolerized TGF-b1 null mice Cytokine Production (pg/ml)a Genotype Fed In Vitro Stimulus WT WT WT WT H2O OVA H2O OVA OVA OVA Medium Medium 535.5 6 50.8 90.6 6 2.4b 246.3 6 38.7 114.5 6 55.7 2/2 2/2 2/2 2/2 H2O OVA H2O OVA OVA OVA Medium Medium 1,086.5 6 84.6 189.8 6 21.8 306.2 6 99.2 88.9 6 58.1 IL-10 IFN-g 4,920.0 6 795.1 713.0 6 87.3 828.3 6 89.0 1,034.7 6 299.4 14,390.7 6 2,170.2 1,447.0 6 89.2 218.3 6 378.2 334.0 6 578.5 a Each mouse was injected s.c. with approximately 60 mg of anti-LFA-1 from day 2 after birth until weaning. On day 21 after birth, both wild-type and mutant mice were fed either water or 1 mg of OVA for 3 consecutive days. (WT mice, n 5 3; 2/2 mice, H2O fed, n 5 4; Ova fed, n 5 5.) On day 31 after birth, the mice were injected with 10 mg of OVA in IFA in the foot pad and tail base. Ten days later, the mice were sacrificed, PLN were removed, and the cells were incubated in vitro with a final concentration of 100 mg/ml of OVA for 48 h. Cytokine analysis was performed as described in Materials and Methods. b Values in bold indicate Student’s t test values that significantly differ (p # 0.05) from the corresponding value for stimulated or unstimulated water-fed control. One of three experiments with similar results is shown. Downloaded from http://journals.aai.org/jimmunol/article-pdf/161/1/154/1082304/im139800154o.pdf by guest on 04 January 2023 Fed In Vitro Stimulus The Journal of Immunology vitro stimulation, levels of all three of these cytokines decreased, rather than increased, in OVA-fed mice compared with those in water-fed controls. In addition, although constitutive production of IL-4, IL-10, and IFN-g was observed, no significant increase in the levels of these cytokines was found in any of the unstimulated lymphocytes from OVA-fed mice (wild type or mutant). These findings imply that a generalized suppression mechanism was induced in low dose OVA-fed mice that affects both Th1 and Th2 cells. Such a mechanism is more consistent with the phenomenon of anergy and/or deletion rather than active suppression. Repeated attempts were made to determine whether anergy served as a mechanism for the observed suppression by incubating PLNs from OVA-fed mutant mice in vitro with rIL-2. However, due to extensive cell death during the incubation period, conclusive results could not be obtained. The inability of enough cells to survive during the experimental period could be due in part to the fact that lymphocytes were obtained from 129 3 CF1 mice; the CF1 mouse is a partially outbred strain. Using the TGF-b1 null mice in our studies has the primary advantage of eliminating a major variable that has been implicated in generating low dose oral tolerance. Both TGF-b1 null mice as well as wild-type mice were used to ensure that the results observed in mutant mice were not due to compensatory mechanisms unique to the TGF-b1 null mice. Our data support the findings of others, which indicate that clonal anergy is the primary mechanism involved in inducing oral tolerance regardless of whether high or low doses of Ag are fed (34, 35). It is uncertain why our results differ from those of others, which suggest that active suppression is the primary mechanism for inducing low dose oral tolerance (36). The discrepancy could be due to the fact that different strains of mice were used or that mice in our system were treated with anti-CD11a. In preliminary experiments it was shown that antiCD11a treatment had a profound, detrimental effect on the production of humoral Ab. It is conceivable that this treatment may have also altered the immune response at the T cell level, thus favoring anergy over active suppression in our model. It is also possible that the amount of Ag fed (1 mg, three times) was too high to be considered low dose in 129 3 CF1 mice. However, in previous studies by Weiner and colleagues, 1 mg of Ag fed five times was considered a low dose (21, 37). In addition, preliminary studies designed to assess the concentration of a low dose in our experimental model indicated inconsistent suppression of cellular proliferation in wild-type mice if fed doses were lowered to 0.5 mg, three times (data not shown). Whether additional feedings of OVA (i.e., 1 or 0.5 mg of OVA fed five times) are needed to observe increased cytokine levels remains to be determined. In summary, our results indicate that both high and low dose oral tolerance can be generated in mice in the absence of TGF-b1. Our findings suggest that mechanisms other than the secretion of inhibitory cytokines may be associated with low dose oral tolerance and that these mechanisms may be the primary ones involved in our model system. References 1. Kagnoff, M. F. 1987. Antigen handling by intestinal mucosa: humoral and cellmediated immunity, tolerance and genetic control of local immune responses. In Immunopathology of the Small Intestine. M. N. Marsh, eds, John Wiley and Sons, p. 73. 2. Mowat, A. 1987. The regulation of immune responses to dietary protein antigens. Immunol. Today 8:93. 3. Lider, O., L. M. B. Santos, C. S. Y. Lee, P. J. Higgins, and L. L. Weiner. 1989. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. II. Suppression of disease and in vitro immune responses is mediated by antigen-specific CD81 T lymphocytes. J. Immunol. 142:748. Downloaded from http://journals.aai.org/jimmunol/article-pdf/161/1/154/1082304/im139800154o.pdf by guest on 04 January 2023 However, humoral responses were drastically reduced in antiCD11a-treated mice, suggesting that the concentration of antiCD11a used in our experiments interfered with critical LFA-1dependent associations needed for Ab production. Therefore, assessment of oral tolerance induction was restricted to cellular proliferation assays. Lymphocytes from TGF-b1 null mutant mice treated with antiCD11a in vivo were capable of responding to Ag-specific stimulation in a dose-dependent fashion. It is interesting to note that in almost all cases, cells from anti-CD11a-treated mutant mice proliferated more vigorously in response to antigenic stimulation than did anti-CD11a-treated wild-type mice. However, when stimulation indexes were calculated, a consistent pattern of enhanced proliferation in anti-CD11a-treated mutant mice was not observed. With respect to the induction of oral tolerance, we have shown that, compared with controls, lymph node cells from anti-CD11atreated TGF-b1 null mutant mice fed high doses of Ag exhibited suppressed proliferation in response to in vitro stimulation with 1000 and 100 mg/ml of OVA. These results indicate that lymphocytes from anti-CD11a-treated TGF-b1 null mutant animals are being suppressed and confirm the findings of others that cytokines do not play a primary role in the induction of high dose oral tolerance (14 –19, 21). However, it should be noted that although lymph node cells from all anti-CD11a-treated TGF-b1 null mice fed high doses of OVA were significantly suppressed, this suppression appeared to be less effective than that in similarly treated wild-type controls. Such results may be explained by the aforementioned increase in responsiveness of lymphocytes from TGF-b1 null mice. A generalized increase in the proliferative response might tend to diminish and thus obscure any observable suppression occurring in these cells. An alternative explanation is that TGF-b1 may also play a role (albeit a minor one) in high dose oral tolerance. In contrast to results obtained with high dose feeding regimens, anti-CD11a-treated TGF-b1 null mutant mice fed low doses of OVA varied in their ability to generate oral tolerance. Lymph node cells from mutant mice fed low doses of OVA were not tolerized as efficiently as those from their wild-type controls; lymph node cells from seven of nine and four of nine mice were significantly suppressed when incubated in vitro with 100 and 1000 mg/ml OVA, respectively. However, the ability of the majority of mice to exhibit a suppressed proliferative response when incubated in vitro with 100 mg/ml of OVA would argue that TGF-b1 may play a partial, but not exclusive, role in the induction of low dose oral tolerance. The possibility that suppressed proliferative responses in TGF-b1 null mutant mice may be obscured due to heightened cellular responsiveness cannot be excluded. The significant suppression observed in the majority of TGF-b1 null mice fed low doses of OVA suggests that factors other than the production and secretion of TGF-b1 are involved in the induction of low dose oral tolerance. It has been reported that feeding low doses of OVA stimulates the production of IL-4 and IL-10 in addition to that of TGF-b1 with or without in vitro stimulation (21, 23). It has been proposed that these cytokines, in a manner similar to that of TGF-b1, may play a role in inhibition of the immune response via active suppression (21, 23). Such a mechanism assumes the preferential generation of Th2 cells (or putative Th3 cells) while Th1 cells are down-regulated. It was possible that in our system, Th2 cells secreting elevated levels of IL-4 and IL-10 were at least partially responsible for the suppressed proliferative response in TGF-b1 null mice fed low doses of OVA. To test for this possibility, levels of IFN-g (a Th1 cytokine), IL-4, and IL-10 were assessed in anti-CD11a-treated TGF-b1 null mutant mice fed either water or low doses of OVA. Our results show that after in 159 160 22. Chen, Y., J. Inobe, R. Marks, P. Gonnella, V. K. Kuchroo, and H. L. Weiner. 1995. Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature 376:177. 23. Chen, Y., V. K. Kuchroo, J. Inobe, D. A. Hafler, and H. L. Weiner. 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265:1237. 24. Chen, Y., J. Inobe, and H. L. Weiner. 1995. Induction of oral tolerance to myelin basic protein in CD8-depleted mice: both CD41 and CD81 cells mediate active suppression. J. Immunol. 155:910. 25. Caspi, R. R., L. R. Stiff, R. Morawetz, N. E. Miller-Rivero, C. C. Chan, B. Wigger, R. B. Nussenblatt, H. C. Morse, and L. V. Rizzo. 1996. Cytokinedependent modulation of oral tolerance in murine model of autoimmune uveitis. Ann. NY Acad. Sci. 778:315. 26. Chen, Y., J. Inobe, V. K. Kuchroo, J. L. Baron, C. A. Janeway, and H. L. Weiner. 1996. Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells. Proc. Natl. Acad. Sci. USA 93:388. 27. Wahl, S. M., N. McCartney-Francis, and S. E. Mergenhagen. 1989. Inflammatory and immunoregulatory role for transforming growth factor b. Immunol. Today 10:258. 28. McCartney-Francis, N. L., and S. M. Wahl. 1994. TGF-b: a matter of life and death. J. Leukocyte Biol. 55:401. 29. Shull, M., I. Ormsby, A. B. Kier, S. Pawlowski, R. J. Diebold, M. Yin, R. Allen, C. Sidman, G. Proetzel, D. Calvin, N. Annunziata, and T. Doetschman. 1992. Targeted disruption of the mouse transforming growth factor-b1 gene results in multifocal inflammatory disease. Nature 359:693. 30. Kulkarni, A. B., C. G. Huh, D. Becker, A. Geiser, M. Lyght, K. C. Flanders, A. B. Roberts, M. B. Sporn, J. M. Ward, and S. Karlsson. 1993. Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc. Natl. Acad. Sci. USA 90:770. 31. Hines, K. L., A. B. Kulkarni, J. B. McCartney-Francis, L. T. Furcht, S. Karlsson, and S. M. Wahl. 1994. Synthetic fibronectin peptides interrupt inflammatory cell infiltration in TGF-b1 knockout mice. Proc. Natl. Acad. Sci. USA 91:5187. 32. Diebold, R. J., M. J. Eis, M. Yin, I. Ormsy, G. P. Boivin, B. J. Darrows, J. E. Saffitz, and T. Doetschman. Early-onset multifocal inflammation in the transforming growth factor b1-null mouse is lymphocyte mediated. Proc. Natl. Acad. Sci. USA 92:12215. 33. Christ, M., N. L. McCartney-Francis, A. B. Kulkarni, J. M. Ward, D. E. Mizel, C. L. Mackall, R. E. Gress, K. L. Hines, H. Tian, S. Karlsson, and S. M. Wahl. 1994. Immune dysregulation in TGF-b1-deficient mice. J. Immunol. 153:1936. 34. Garside, P., and A. M. Mowat. 1997. Mechanisms of oral tolerance. Crit. Rev. Immunol. 17:119. 35. Inada, S., S. Yoshino, M. A. Haque, Y. Ogata, and O. Kohashi. 1997. Clonal anergy is a potent mechanism of oral tolerance in suppression of acute antigeninduced arthritis in rats by oral administration of the inducing antigen. Cell. Immunol. 175:67. 36. Weiner, H. L. 1997. Oral tolerance: Immune mechanisms and treatment of autoimmune diseases. Immunol. Today 18:335. 37. Fishman-Lobell, J., A. Friedman, and H. L. Weiner. 1994. Different kinetic patterns of cytokine gene expression in vivo in orally tolerant mice. Eur. J. Immunol. 24:2720. Downloaded from http://journals.aai.org/jimmunol/article-pdf/161/1/154/1082304/im139800154o.pdf by guest on 04 January 2023 4. Nagler-Anderson, C., A. Bober, M. Robinson, G. Siskind, and G. Thorbecke. 1983. Suppression of type II collagen-induced arthritis by intragastric administration of soluble type II collagen. Proc. Natl. Acad. Sci. USA 83:7443. 5. Nussenblatt, R., R. Aspi, R. Mahdi, C. Chan, F. Rogere, O. Lider, and H. Weiner. 1989. Inhibition of S-antigen induced experimental autoimmune uveoretinitis by oral induction of tolerance by S-antigen. J. Immunol. 144:1889. 6. Zhang, Z. J., L. E. Davidson, G. Eisenbarth, and H. L. Weiner. 1991. Suppression of diabetes in NOD mice by oral administration of porcine insulin. Proc. Natl. Acad. Sci. USA 88:10252. 7. Yoon, C. K. 1993. MS study yields mixed results. Science 259:1263. 8. Weiner, H. L., G. A Mackin, M. Matsui, E. J. Orav, S. J. Khoury, D. M. Dawson, and D. A. Hafler. 1993. Double-blind pilot trial of oral tolerization with myelin antigens in multiple sclerosis. Science 259:1321. 9. Trentham, D. E., R. A. Dynesius-Trentham, E. J. Orav, D. Combithi, C. Lorenzo, K. L. Sewell, D. Hafler, and H. L. Weiner. 1993. Effects of oral administration of type II collagen on rheumatoid arthritis. Science 261:1727. 10. Titus, R. G., and J. M. Chiller. 1981. Orally induced tolerance: definition at the cellular level. Int. Arch. Allergy Appl. Immunol. 65:323. 11. Miller, A., O. Lider, and H. L. Weiner. 1991. Antigen-driven bystander suppression after oral administration of antigens. J. Exp. Med. 174:791. 12. Miller, A., O. Lider, A. B. Robers, M. Sporn, and H. L. Weiner. 1992. Suppressor T cells generated by oral tolerization to myelin basic proteins suppress both in vitro and in vivo responses by the release of transforming growth factor b after antigen-specific triggering. Proc. Natl. Acad. Sci. USA 89:421. 13. Khoury, S. J., W. W. Hancock, and H. L. Weiner. 1992. Oral tolerance to myelin basic protein and natural recovery from experimental autoimmune encephalomyelitis are associated with downregulation of inflammatory cytokines and differential upregulation of transforming growth factor b, interleukin 4 and prostaglandin E expression in the brain. J. Exp. Med 176:1355. 14. Hanson, D. G., and S. D. Miller. 1982. Inhibition of specific immune responses by feeding protein antigens. V. Induction of the tolerant state in the absence of specific suppressor T cells. J. Immunol. 128:2379. 15. Whitacre, C. C., I. E. Gienappm, C. G. Orosz and D. M. Bitar. 1991. Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy. J. Immunol. 147:2155. 16. Whitacre, C., I. Gienapp, K. Cox, S. Jewell, M. Javed, S. Goldman, and E. Hebber-Katz. 1993. Oral tolerance in experimental autoimmune encephalomyelitis (EAE): T cell anergy. J. Immunol. 150:245A. 17. Melamed, D., and A. Friedman. 1993. Direct evidence for anergy in T lymphocytes tolerized by oral administration of ovalbumin. Eur. J. Immunol. 23:935. 18. Melamed, D., and A. Friedman. 1994. In vivo tolerization of Th1 lymphocytes following a single feeding with ovalbumin: anergy in the absence of suppression. Eur. J. Immunol. 24:1974. 19. Barone, K. S., S. L. Jain, and J. G. Michael. 1995. Effect of in vivo depletion of CD41 and CD81 cells on the induction and maintenance of oral tolerance. Cell. Immunol. 163:19. 20. Garside, P., M. Steel, E. A. Worthey, A. Satoskar, J. Alexander, H. Bluethmann, F. Y. Liew, and A. McI. Mowat. 1995. T helper 2 cells are subject to high dose oral tolerance and are not essential for its induction. J. Immunol. 154:5649. 21. Freidman, A., and H. L. Weiner. 1994. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc. Natl. Acad. Sci. USA 91:6688. ORAL TOLERANCE IN TGF-b1 NULL MICE