HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blank, M. Articles by Shoenfeld, Y. Search for Related Content PubMed PubMed Citation Articles by Blank, M. Articles by Shoenfeld, Y.

Oral Tolerance to Low Dose ß₂-Glycoprotein I: Immunomodulation of Experimental Antiphospholipid Syndrome¹

Miri Blank^*, Jacob George^*, Vivian Barak, Angela Tincani, Takao Koike^§ and Yehuda Shoenfeld^2,*

^* Research Unit of Autoimmune Diseases, Department of Medicine B, Sheba Medical Center, Tel-Hashomer, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Immunology Laboratory for Diagnosis, Oncology Department, Hadassah Medical Center, Jerusalem, Israel; Clinical Immunology Unit, Spedali Civili, Brescia, Italy; and ^§ Department of Medicine II, Hokkaido University School of Medicine, Sapporo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Oral tolerance was induced in BALB/c mice by feeding low dose ß₂-glycoprotein I (ß₂GPI). The ß₂GPI-fed mice did not develop serologic and clinical markers of experimental antiphospholipid syndrome (APS) upon immunization with the autoantigen. The treated group was characterized by low titers of serum anti-ß₂GPI and anticardiolipin Abs in the serum, lack of fetal resorptions, low incidence of thrombocytopenia, and normal aPTT (activated partial thromboplastin time) values. ß₂GPI given orally before priming with ß₂GPI resulted in complete prevention of experimental APS development; ß₂GPI given at an early stage of the disease reduced clinical manifestations. However, administration of ß₂GPI 70 days postimmunization had a less significant effect on disease expression. Tolerized mice exhibited a diminished T lymphocyte proliferation response to ß₂GPI in comparison with ß₂GPI-immunized mice fed with OVA. When nontolerant ß₂GPI-primed T lymphocytes were mixed with T lymphocytes derived from tolerized mice, a significant inhibition of proliferation upon exposure to ß₂GPI was observed. The induction of suppression was ß₂GPI specific and driven, as well as TGF-ß mediated. The ß₂GPI-specific response of T lymphocytes from the ß₂GPI-fed mice was reversed by anti-TGF-ß Abs. The tolerance was adoptively transferred by CD8⁺ T cells from the tolerized mice into naive mice. Those CD8⁺ cells were MHC class I restricted, found to secrete TGF-ß, and had no cytolytic activity. Oral administration of ß₂GPI suppressed priming of CTLs in the recipient mice. In sum, ß₂GPI-induced oral tolerance has an immunomodulatory effect in experimental APS, demonstrating the importance of ß₂GPI in the pathogenesis of the disease.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The antiphospholipid syndrome (APS)³ is characterized by the presence of high titers of IgG anticardiolipin Abs (aCL) and/or lupus anticoagulant associated with thromboembolic phenomena, thrombocytopenia, and recurrent fetal loss, as well as other multisystem involvement emerging either in the context of systemic lupus erythematosus or as the primary APS (1, 2, 3, 4, 5, 6).

Human ß₂-glycoprotein I (ß₂GPI) is a 50-kDa plasma glycoprotein. It has recently been suggested that pathogenic antiphospholipid Abs such as aCL present in the sera of patients with APS bind to the negatively charged phospholipids via the ß₂GPI cofactor (7, 8). The molecule belongs to the complement control proteins and is composed of five respective consensus ("sushi") repeats. ß₂GPI binds the negatively charged phospholipids through a lysine-rich locus in the fifth domain and possesses several in vitro properties, which renders it an antigcoagulant (9, 10).

In contrast to the phospholipids (e.g., cardiolipin), the ß₂GPI molecule was found to be immunogenic in vivo. Immunization of BALB/c, PL/J mice, or New Zealand white rabbits with ß₂GPI resulted in generation of anti-ß₂GPI Abs (11, 12, 13, 14). ß₂GPI-immunized mice developed high titers of ß₂GPI-dependent aCL, associated with an increased percentage of fetal resorptions (the equivalent of fetal loss in human APS) in utero, thrombocytopenia, and prolonged activated partial thromboplastin time (prolonged activated partial thromboplastin time (aPTT)), indicating the presence of lupus anticoagulant, a presentation characterized as experimental APS (13). Acceleration of APS manifestations was observed in MRL/lpr mice (a mouse model of APS with a genetic background) immunized with ß₂GPI (15).

Oral administration of Ag or autoantigen induces a state of Ag-specific systemic immunologic hyperresponsiveness termed oral tolerance (16). Many investigators studied this natural route of tolerization as a means to suppress experimental autoimmune diseases including experimental autoimmune encephalomyelitis, uveitis, collagen-induced arthritis, and diabetes in NOD mice (17, 18, 19). Furthermore, oral tolerance has been recently applied as a treatment for human autoimmune diseases (20, 21).

The mechanisms by which orally administered Ag/autoantigen induces tolerance entail active suppression (16, 19), clonal anergy (22), and clonal deletion (23). The relative roles of these mechanisms are primarily determined by the Ag dose administered, with low doses of Ag favoring active suppression, probably via induction of CD4^-CD8⁺ suppressor T cells (24) or mediated by CD4⁺CD8⁺ T cells (25), whereas high doses of Ag favor clonal anergy or deletion (23, 26). Both dosages of Ag administration affect the cytokine secretion profile; animals fed high doses of Ag secrete more IL-4 and less TGF-ß, whereas those fed low doses secrete more TGF-ß and less IL-4 (26).

Several variables are known to affect oral tolerance response. These include type of Ag (proteins, contact allergens, viruses, bacterial proteins, etc.), Ag dose, age, and genetic background as well as species (27). Studies have shown that oral tolerance is enhanced by varying the delivery vehicle or by incorporating immunomodulators such as LPS (28) and cholera toxin B subunit (29) together with the fed Ag.

Cardiolipin is not an immunogenic molecule, and there is doubt as to whether it is the target autoantigen in APS. The current study was designed to test whether oral feeding of naive mice with low dose ß₂GPI, before induction of experimental APS, can induce tolerance and to characterize the mechanism involved in the process. The results may support the notion that ß₂GPI is the autoantigen in experimental APS and could propose a novel therapeutic modality in APS.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Female BALB/c mice, age 10–12 wk, were purchased from the Tel-Aviv University animal facility. The females were caged overnight with BALB/c males and examined for vaginal plug next morning. The presence of the plug was considered as day 1 of pregnancy in all of the studied groups.

Feeding and immunization regimens

Oral tolerance was induced by multiple low dose feeding regimens. Each mouse was fed with 1, 0.5, 0.25, 0.1, or 0.01 mg of either ß₂GPI, produced as previously described (30), or PBS by gastric intubation with an 18-gauge stainless steel feeding needle (Thomas Scientific, Swedesboro, NJ). Mice were fed every other day for a total of five times. BALB/c female mice were primed by injection of 20 µg/mouse of ß₂GPI emulsified in CFA (Difco, Detroit, MI) or 20 µg/mouse OVA/CFA in the hind footpads 2 days after the last feeding. In some experiments, mice tolerized to ß₂GPI were primed with egg OVA, grade VI (Sigma Chemical, St. Louis, MO). In addition, oral administration of ß₂GPI was tested 21 days and 70 days after immunization with ß₂GPI.

The mice were bled every month, and aCL and anti-ß₂GPI titers in the sera were assayed by ELISA. The immunized mice were matted 3 mo after immunization, and fetal resorptions, thrombocytopenia, and aPTT were tested at day 15 of pregnancy, as we have previously described (13).

T lymphocyte proliferation

Ten days after immunization, popliteal lymph node cells (LNCs) were prepared in DMEM containing 100 µg/ml penicillin and 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 5 x 10^-5 M 2-ME, and 5% FCS or 1% syngeneic serum and specific (ß₂GPI) and nonspecific (OVA) Ag (10 µg/ml). In some experiments, LNCs, CD8⁺, or CD4⁺ T cells from tolerized mice were added to the proliferation assays. The CD8⁺ or CD4⁺ T cells were separated by positive selection employing mouse CD4⁺ (L3T4) Microbeads or mouse CD8a⁺ (Ly-2) Microbeads columns, respectively (magnetically activated cell separation columns were purchased from Miltenyi Biotec, Bergisch-Gladbach Germany). In several experiments, proliferation was measured in the presence of mouse anti-H-2^kd (provided by Dr. Lea Isenbach, Weizmman Institute of Rehovot, Israel), mouse anti-H-2^kb (provided by Dr. Avi Ben-Nun, Weizmman Institute of Science), goat anti-mouse IL-4 or mouse anti-mouse TGF-ß, and control mouse IgG (10 µg/ml) (Genzyme Diagnostics, Cambridge, MA). [³H]thymidine (0.5 µCi/well; NEN Products, Boston, MA) was added to cultures at 72 h, and proliferation was measured at 96 h. Results, expressed in cpm, are the mean of quadruplicate cultures of LNCs pooled from three to four mice.

Cytokine concentration assays

IL-2, IL-4, and TGF-ß were measured by ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. IL-2 and IL-4 secretion was measured in 18-h supernatants of cultured splenocytes or LNCs, and TGF-ß secretion was measured in 72-h supernatants of cultured splenocytes or LNCs incubated in serum-free medium ± ß₂GPI or OVA (50 µg/ml).

Adoptive transfer of CD4⁺/CD8⁺ T cells from tolerized donor mice

In vivo transfer of CD4⁺ or CD8⁺ T cells from ß₂GPI-fed mice was performed according to a modification of a method previously described (25). Donor mice were fed with 0.5 mg of ß₂GPI or PBS every other day for a total of five feedings. Two days after the last feeding, the animals were killed, and a spleen cell suspension was prepared. Cells were activated with Con A (2 µg/ml) for 48 h. Spleen cells were then depleted of CD4⁺ or CD8⁺ T cells by positive selection employing CD4⁺ (L3T4) Microbeads or CD8a⁺ (Ly-2) Microbeads columns. The CD4⁺ or CD8⁺ T cells were adoptively transferred by an i.v. injection of 10⁸ cells into syngeneic mice. One day after adoptive transfer, recipient mice were primed in the hind footpads with ß₂GPI/CFA, PBS/CFA, or OVA/CFA. Ten days after transfer, CTL activity and ß₂GPI-specific T cell proliferation were studied in some of the recipient mice, and clinical manifestations of experimental APS were followed up in the rest of the recipient mice.

Cytotoxic activity of the CD8⁺ T cells from ß₂GPI-tolerized mice

CTL activity of the CD8⁺ T cells from ß₂GPI-tolerized mice was studied by direct cytotoxic assay using ⁵¹Cr-labeled ß₂GPI macrophages (ß₂GPI-MAC) target cells from BALB/c or C57BL/6 origin. In addition, the tested cells were incubated for 6 days in vitro with anti-CD3 for induction of nonspecific CTLs, then tested by 4-h chromium release assay, employing ⁵¹Cr-labeled target cells: EL4 thymoma (provided by Dr. Gideon Berke, Weizmman Institute of Science), ß₂GPI-MAC, and OVA-MAC. ß₂GPI-MAC or OVA-MAC were prepared by i.p. injection of thioglycolate into naive mice and isolation of macrophages 7 days later. The macrophages were loaded with 10 mg/ml ß₂GPI or OVA by osmotic lysis of pinosomes (37). The potential of oral feeding with ß₂GPI to suppress CTL priming in the recipient mice was studied as follow. Spleen cells from ß₂GPI-primed (10 days earlier) mice, were incubated for 6 days in vitro with an irradiated (30,000 rad) ß₂GPI-MAC, irrelevant OVA-MAC, or macrophages alone. Cytolytic activity of the cultured cells was determined in a standard 4-h chromium release assay using ⁵¹Cr-labeled EL4, ß₂GPI-MAC, or OVA-MAC target cells at different effector:target cell ratios. Data were calculated by the formula: % specific lysis = (⁵¹Cr release by effector cells - spontaneous ⁵¹Cr release)/(maximal ⁵¹Cr release - spontaneous ⁵¹Cr release). Maximal ⁵¹Cr release was determined by the addition of 1% Triton X-100 (Sigma). Spontaneous ⁵¹Cr release in the absence of effector cells was generally <15% of the maximal release in all experiments. Representative experiments are shown at an effector:target ratio of 20:1.

Student’s t test was used to evaluate differences between the binding properties of the various studied groups; p < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Low dose feeding regimen with ß₂GPI induces active suppression

For the purpose of definition, the mice fed with ß₂GPI and immunized with ß₂GPI/CFA will be referred to as tolerized mice. Mice fed with low dose ß₂GPI (1, 0.5, 0.25, 0.1, and 0.01 mg) showed a diminished humoral response, evidenced by lower titers of anti-ß₂GPI and aCL Abs in the sera, in comparison with mice fed with PBS and primed with ß₂GPI/CFA (e.g., in ß₂GPI, 0.5 mg-fed mice 0.374 ± 0.057, 0.413 ± 0.142 OD at 405 nm, compared with PBS-fed mice 1.479 ± 0.189, 1.401 ± 0.168, respectively; p < 0.001; Table I). The ß₂GPI-fed and anti-ß₂GPI/CFA-primed mice did not develop significant titers of anti-ß₂GPI and -aCL when compared with the PBS-fed PBS/CFA-primed mice or compared with ß₂GPI-fed OVA/CFA-primed mice (p > 0.05). Kinetic studies (Fig. 1), point to the fact that the elevated titers of anti-ß₂GPI in ß₂GPI-fed and ß₂GPI/CFA-primed mice decreased 2 mo after immunization, while the titers of anti-ß₂GPI in the PBS-fed ß₂GPI/CFA-primed mice remained high at least 4 mo after immunization (p < 0.001). This effect was associated with disability of the ß₂GPI-fed ß₂GPI/CFA-primed mice to develop other manifestations of experimental APS (Table I), such as high percentages of fetal resorptions (e.g., 7 ± 2% in ß₂GPI (0.5 mg)-fed ß₂GPI/CFA-primed mice in comparison with 43 ± 2% in the group of mice fed with PBS and primed with ß₂GPI/CFA (p < 0.01; nonsignificant when compared with PBS-fed PBS/CFA-primed mice or with ß₂GPI-fed OVA/CFA-primed mice, p > 0.05). Thrombocytopenia was detected in the mice fed with PBS and primed with ß₂GPI (487 ± 131 cells/mm³ x 10³ compared with 1121 ± 201 cells/mm³ x 10³ platelet count in the PBS-fed PBS/CFA-primed mice; p < 0.05). An increase in platelet count was observed in mice fed with ß₂GPI (1 mg and 0.5 mg, p < 0.005; and 0.250 mg, p < 0.04), while no significant change in the number of platelets was detected in mice fed with 0.1 or 0.01 mg ß₂GPI (Table I). No prolongation in aPTT was noticed in the ß₂GPI (1 and 0.5 mg)-fed and -primed mice in comparison with mice fed with PBS and primed with ß₂GPI/CFA or with the group of ß₂GPI-fed OVA/CFA-primed mice (Table I). Characterization of the isotypes involved in the ß₂GPI-tolerized mice revealed a decrease in the titer of anti-ß₂GPI IgG2b isotype (3.462 ± 0.327 mg/ml in the APS mice compared with 1.826 ± 0.411 mg/ml in the tolerized mice, p < 0.05; Table II). Significant abrogation in the anti-ß₂GPI IgG3 isotype (p < 0.02) as well as the IgM isotype (p < 0.05) was documented.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Kinetic study of the anti-ß2GPI titers in the sera of mice during induction of oral tolerance to ß2GPI. The following groups of mice were tested: ß2GPI-fed 10 days before priming with ß2GPI (-10D), ; PBS-fed (-10D) and ß2GPI-primed, ; ß2GPI-immunized and fed 21 days later (+21D) with ß2GPI, •; ß2GPI-immunized and fed 70 days later (+70D) with ß2GPI, . Each point represents the mean ± SD of OD at 405 nm of three different experiments.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Low dose ß2GPI feeding induces active suppression. A, Proliferative responses of LNCs from PBS- or ß2GPI-fed mice, primed with ß2GPI/CFA, to ß2GPI ( and , respectively) or to OVA () were measured. Open circles demonstrate proliferative responses to OVA of LNCs derived from ß2GPI-fed and -primed mice immunized with OVA/CFA. B, ß2GPI-primed T lymphocytes cultured in the presence of 10 µg/ml of ß2GPI were mixed with PBS/CFA-primed LNCs () or with ß2GPI-primed LNCs derived from ß2GPI-fed mice (). OVA-primed T lymphocytes cultured in the presence of 20 µg/ml of OVA were mixed with ß2GPI-primed LNCs derived from ß2GPI-fed mice in the absence () or presence () of 20 µg/ml of ß2GPI. Each point represent the mean ± SD of triplicate cultures of popliteal LNCs pooled from three to four mice in two experiments. Proliferation in response to medium alone ranged from 1800–2600 cpm for all groups.

Data presented in Fig. 3 show that anti-TGFß reversed the ß₂GPI-specific response of T lymphocytes from ß₂GPI-tolerized mice (from 8,675 ± 1,643 cpm to 34,108 ± 3,076 cpm in the presence of anti-TGF-ß, by 3.9-fold; p < 0.001). No statistical significance was noticed when anti-TGFß was added to proliferation assay of T cell derived from PBS-fed ß₂GPI-primed mice (hatched bars) in comparison with those cells in the absence of anti-TGFß (shaded bars; p > 0.05). The effect of anti-TGFß was specific, since an equivalent dosage of normal mouse IgG had no effect on the diminished response of tolerized T lymphocytes to ß₂GPI (cpm of nontolerant controls was 43,419 ± 3,387 cpm in the presence of mouse IgG compared with 49,448 ± 2,387 cpm in the absence of mouse IgG, p > 0.05; cpm of proliferation response of T cells from ß₂GPI-tolerized mice was 7,546 ± 1,007 cpm compared with 6,701 ± 899 cpm when mouse IgG was added to the proliferation assays, p > 0.05).

View larger version (45K): [in this window] [in a new window]   FIGURE 3. TGF-ß involvement in active suppression. Proliferative responses to ß2GPI were measured in the presence of either anti-IL-4 or anti- TGFß. Proliferative responses shown are of nontolerant cells primed by ß2GPI/CFA (shaded bars denote the absence of and hatched bars the presence of tested Ab); ß2GPI-primed tolerant cells (solid bars denote the absence of and open bars the presence of tested Ab). Each point represents the mean ± SD of quadruplicate cultures of popliteal LNCs pooled from three to four mice in two experiments. Proliferation in response to medium alone ranged from 1200–2500 cpm for all groups.

To confirm the role of TGF-ß in the suppression mediated by LNCs derived from ß₂GPI-tolerized mice, cell-mixing experiments were performed in the presence or absence of anti-TGF-ß. As shown in Fig. 4, anti-TGF-ß totally abrogated the suppression by LNCs derived from ß₂GPI-fed mice. LNCs from ß₂GPI (0.5 mg)-fed ß₂GPI-primed mice suppressed ß₂GPI proliferative response of LNCs from PBS-fed ß₂GPI-primed mice at a ratio of 1:1 (8,434 ± 1,217 cpm compared with 47,321 ± 3,756 cpm of responder proliferative response in the presence of modulator cells derived from PBS-fed PBS/CFA-primed mice (p < 0.001). The addition of anti-TGF-ß enhanced the proliferative response of LNCs from PBS-fed ß₂GPI-primed mice in the presence of the modulator LNCs from ß₂GPI-tolerized mice compared with the absence of anti-TGF-ß (p < 0.001). Anti-TGF-ß had no significant effect when added to responder cells in the presence of modulator cells from mice PBS-fed PBS/CFA-primed (p > 0.05).

View larger version (50K): [in this window] [in a new window]   FIGURE 4. The effect of anti-TGFß on the proliferative responses of ß2GPI-primed T lymphocytes to ß2GPI in the presence of T modulator cells from ß2GPI-tolerized mice. Modulator cells, at a responder:modulator ratio of 1:1, from ß2GPI-tolerized mice in the absence (solid bar) or presence of anti-TGFß (shaded bar); modulator cells from PBS-fed PBS/CFA-primed mice in the absence (hatched bar) or presence of anti-TGFß (open bar) were added to the responder ß2GPI-primed nontolerant T lymphocytes in the presence of ß2GPI. Each point represent the mean ± SD of quadruplicate cultures of popliteal LNCs pooled from three to four mice in two experiments. Proliferation in response to medium alone ranged from 1500–2400 cpm for all groups.

Spleen cells from ß₂GPI-fed mice (CD4⁺ or CD8⁺) were infused i.v. into syngeneic naive mice. One day later, mice were primed with ß₂GPI/CFA, PBS/CFA, or OVA/CFA. Data summarized in Table IV indicate that the oral tolerance induced by low dose ß₂GPI feeding (0.5 mg), could be adoptively transferred to syngeneic mice. CD8⁺ cells rather than CD4⁺ cells were responsible for the transfer of the suppression. Donor CD8⁺ cells from ß₂GPI-tolerized mice infused into naive mice suppressed the induction of experimental APS in recipient mice, as evidenced by the serologic and clinical parameters described in Table IV. 1) Low titer of anti-ß₂GPI and aCL were detected in the sera in comparison with mice infused with CD8⁺ cells from PBS-fed ß₂GPI-primed mice (p < 0.001) or with mice infused with CD4⁺ cells derived from ß₂GPI-tolerized mice (p < 0.001). 2) A decrease in the percentage of fetal resorption was observed in the recipient mice infused with CD8⁺ cells from ß₂GPI-tolerized mice in comparison with recipient mice infused with CD8⁺ cells from PBS-fed ß₂GPI-primed mice (p < 0.004) or compared with mice infused with CD4⁺ from ß₂GPI-tolerized mice (p < 0.004). 3) An increase in platelet count was detected in recipient mice infused with CD8⁺ cells from ß₂GPI-tolerized mice in comparison with infusion of CD8⁺ cells from PBS-fed ß₂GPI-primed mice (p < 0.01) or compared with recipient mice infused with CD4⁺ from ß₂GPI-tolerized mice (p < 0.05). 4) Normal aPTT values were detected in mice infused with CD8⁺ cells from ß₂GPI-tolerized mice compared with prolonged aPTT values found in recipient mice infused with CD8⁺ cells from PBS-fed ß₂GPI-primed mice (p < 0.04) or compared with recipient mice infused with CD4⁺ from ß₂GPI-tolerized mice (p < 0.04). The adoptive transfer was ß₂GPI specific. Recipient mice that were infused with CD4⁺ or CD8⁺ cells from ß₂GPI-fed and OVA/CFA-primed mice developed selectively high titers of anti-OVA and no serologic or clinical manifestations of experimental APS.

CD8⁺ T cells from the ß₂GPI-tolerized mice had the potential to adoptively transfer ß₂GPI tolerance into naive mice, which were found to be ß₂GPI specific (Fig. 5) and to recognize ß₂GPI presented by MHC class I molecules (Fig. 6). Data described in Fig. 5 demonstrate that the CD8⁺ T cell population derived from the ß₂GPI-tolerized mice ameliorated the responder cell proliferation in comparison with modulator CD8⁺ T cells derived from the PBS-fed PBS/CFA-primed mice or in comparison with CD4⁺ T modulator cells separated from ß₂GPI-tolerized mice (e.g., 8,345 ± 956 cpm compared with 29,321 ± 1,455 cpm and 27,515 ± 2,532 cpm, respectively, at a responder:modulator ratio of 1:1; p < 0.002). The CD8⁺ cells from the tolerized mice were ß₂GPI specific (Fig. 5). Those cells did not reduce the proliferation of LNCs from OVA-primed mice in the presence of OVA (p > 0.05) compared with CD4⁺ modulator cells from the tolerized mice or compared with CD8⁺ modulator cells from PBS/CFA-primed mice. To study the CD8⁺ cells’ MHC class I restriction, anti-H-2^kd Abs were added to the responder LNCs and ß₂GPI-specific CD8⁺ modulator cells (Fig. 6). Prevention of the inhibitory effect of the modulator CD8⁺ cells from ß₂GPI-tolerized mice on the responder proliferative activity was documented in the presence of anti-H-2^kd (p < 0.02), while addition of anti-H-2^kb Abs failed to do so (p > 0.05).

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Specificity of suppression of ß2GPI proliferation response in the presence of CD8+ T cells from ß2GPI-tolerized mice. Modulator cells that were added to responder cells from ß2GPI-primed mice were: CD8+ cells from ß2GPI-tolerized mice (); CD8+ cells from PBS/CFA-immunized mice (); CD4+ cells from ß2GPI-tolerized mice (); responder OVA-primed T cells in the presence of OVA (); responder OVA-primed T cells in the presence of OVA and CD8+ cells from ß2GPI-tolerized mice (•). Each point represent the mean ± SD of triplicate cultures of two separate experiments.

View larger version (15K): [in this window] [in a new window]   FIGURE 6. MHC class I restriction of the ß2GPI proliferation response suppression in the presence of CD8+ T cells from ß2GPI-tolerized mice. Responder ß2GPI T cells were mixed with CD8+ T cells from ß2GPI-tolerized mice at different responder:modulator ratios. Solid squares represent proliferation response with modulator CD8+ cells from PBS/CFA primed mice. Open circles represent proliferation response with modulator CD8+ cells from ß2GPI-tolerized mice. Anti H-2kd added to the proliferation response with modulator CD8+ cells from ß2GPI-tolerized mice (). Anti H-2kb added to the proliferation response with modulator CD8+ cells from ß2GPI-tolerized mice (). Each point represent the mean ± SD of triplicate cultures of two separate experiments.

View larger version (21K): [in this window] [in a new window]   FIGURE 7. Suppression of CD8+CTL priming by oral ß2GPI. CD8+ cells were isolated on day 10 after immunization and stimulated in vitro with irradiated ß2GPI-MAC or OVA-MAC. Six day later, cytolitic activity was tested on 51Cr-labeled target cells: ß2GPI-MAC (hatched bars for CD8+ cells from primed mice; shaded bars for CD8+ cells from nonprimed mice); OVA-MAC (solid bars). Each point represent the mean ± SD of triplicate cultures of two separate experiments.

Oral feeding with ß₂GPI was performed at two stages after disease induction, 21 days after immunization with ß₂GPI or 70 days following ß₂GPI priming, when all the clinical manifestations of experimental APS had been expressed (late stage of the disease; data presented in Fig. 1 and Table VI). The results show that successful oral treatment with ß₂GPI occurred mainly at an early stage of the disease. The kinetic study of anti-ß₂GPI titers in the serum of mice that received ß₂GPI orally (0.5 mg) after immunization with ß₂GPI is described in Fig. 1. Anti-ß₂GPI titers in the group of mice treated orally 21 days after priming with ß₂GPI were significantly decrease when compared with titers in the PBS-fed (-10 days) ß₂GPI/CFA-immunized mice (p < 0.02) or compared with the sera of ß₂GPI-fed (-10 days) and ß₂GPI/CFA-immunized mice (p < 0.04). As described in Table VI, the percentage of fetal resorption in the group of mice that received ß₂GPI orally at an early stage of disease (+21 days) was reduced to 12 ± 2% compared with 42 ± 3% (p < 0.01) in the same group fed with PBS or 5 ± 2% in the group of mice fed ß₂GPI (-minus]10 days) and then immunized with ß₂GPI/CFA (p > 0.05). A moderate but significant elevation in the platelet count and a decrease in aPTT were observed in the group of mice treated orally with ß₂GPI 21 days after immunization compared with the PBS-treated group (p < 0.03).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
During the last decade, several groups demonstrated that oral administration of an exogenous autoantigen induces tolerance in experimental autoimmune diseases by down-regulating both humoral and cell-mediated responses (16, 17, 18, 19, 22, 23, 24, 25, 26). In the present study, we directly assessed the question whether feeding naive BALB/c mice with low dose ß₂GPI can induce oral tolerance in those mice. Data presented here demonstrate that oral administration of low dose ß₂GPI inhibited Ab response (generation of anti- ß₂GPI and aCL), and the induction of experimental APS was prevented upon priming with ß₂GPI (characterized by high percentage of fetal resorptions and prolonged aPTT). The platelet counts significantly improved. Suppression of anti-ß₂GPI and aCL production in our experimental model confirmed previous studies in which oral tolerance was conducted with different Ags (31, 32, 33, 34).

Amelioration in the humoral response in our model was associated with a reduced cellular response in the tolerized mice. It seems, in the current study, that low dose ß₂GPI-fed mice developed cytokine-mediated active suppression, supporting previous reports with similar observations that oral tolerance is induced by feeding with low dose hen egg lysozyme (HEL) or myelin basic protein or with Torpedo acetylcholine receptor in myasthenia gravis (19, 33, 35).

T lymphocyte proliferation to ß₂GPI was diminished in the mice fed ß₂GPI. The induction of suppression was ß₂GPI specific and ß₂GPI driven. The active suppression of the tolerance response to ß₂GPI was mediated via TGF-ß. These data support an early report on the important role of TGF-ß in active suppression by low dose feeding with HEL Ag through active suppression mediated via TGF-ß (36).

Two main, distinct mechanisms have been demonstrated for systemic Ag-specific immune suppression associated with oral tolerance. Providing a strong TCR signal to circulating T cells by feeding high doses of Ag induces either anergy or apoptosis of Ag-specific cells (23, 36). By contrast, feeding multiple low doses of the Ag in experimental models—such as OVA, HEL, or myelin basic protein in experimental autoimmune encephalomyelitis and multiple sclerosis, S-Ag in uveitis, collagen type II in adjuvant arthritis, or Torpedo acetylcholine receptor in myasthenia gravis (summarized in 37 —induces regulatory T cells, which mediate suppression by induction of CD8⁺ suppressor T cells (24, 37). Recent works have suggested that production of inhibitory cytokines (such as IFN- and TGF-ß by CD8⁺ T cells) following Ag-specific activation may play an important role in induction of oral tolerance by multiple low dose feeding (22, 26, 33, 35). Conversely, other findings have suggested that oral tolerance is associated with preferential production of IL-4, IL-10 by the Th2 subset, and TGF-ß by the Th3 subset of CD4⁺ T cells, leading to cross-regulation of Th1 cell-mediated effector functions (37, 38). TGF-ß is associated with negative immunoregulatory functions, suppressing the growth of hemopoietic progenitor cells (39), T cells (40), and B cells (41). Lymphokine secretion by T cells is also inhibited by TGF-ß (42). The inhibition of hemopoietic cell growth and differentiation by TGF-ß may explain the lower thrombocytopenia found in the ß₂GPI-fed tolerant mice. On the other hand, TGFß has costimulatory roles in both the growth and maturation of CD8⁺ T cells (43), which points to the contribution of CD8⁺ cells, and not CD4⁺ cells, in the adoptive transfer of ß₂GPI tolerance to naive mice.

Therefore, it is possible that the down-regulation of the cellular and humoral responses following oral administration of ß₂GPI reflects the up-regulation of TGF-ß production by the CD8⁺ T cells from the ß₂GPI-tolerized mice.

The tolerance to ß₂GPI could be adoptively transferred by CD8⁺ cells, and not by CD4⁺ T cells, into naive mice. The CD8⁺ cells from the ß2GPI-tolerized mice showed: 1) ß₂GPI specificity of those cells, since addition of those cells to ß₂GPI-specific proliferation response reduced significantly and in a dose-dependent manner the [³H]thymidine uptake, but failed to do so in OVA-specific proliferation assay; 2) the CD8⁺ were TGF-ß secretors and weak proliferators; 3) the CD8⁺ cells did not have a direct CTL activity, leading to the conclusion that oral administration of ß₂GPI did not activate CTL precursors; moreover, oral feeding with ß₂GPI resulted in suppression of CTL CD8⁺ T cells priming upon immunization with ß₂GPI; 4) the CD8⁺ cells were found to be MHC class I restricted.

We postulate that induction of active suppression following low dose feeding with ß₂GPI occurs primarily in gut lymphoid tissue. Induction of CD8⁺ suppressor T cells by oral ß₂GPI suggests that APCs (dendritic cells) in the gut may process ß₂GPI via the class I pathway (exposing cryptic epitopes to the immune system). Soluble proteins and peptides are known to be taken up by villous enterocytes (epithelial cells) in the small intestine for transport to the circulation, which suggests that they might also be relevant APC. Villous enterocytes in the small intestine express both MHC class I and class II proteins (44) and are reported to present soluble Ags to T cells in vitro (45). It was reported that such Ag-pulsed enterocytes preferentially stimulated CD8⁺ T cells in rats and human (46). Thus, the enterocytes and CD8⁺ suppressor T cells, in concert with TGF-ß, may constitute a system that prevents humoral and cell-mediated responses to ß₂GPI.

Oral administration of ß₂GPI at early and late stages of APS development revealed that induction of tolerance could be efficient mainly when the ß₂GPI was given at an early time point after immunization. When it was given at a late stage of the disease (70 days postpriming), no significant improvement in the clinical picture of the disease was documented. The basis of this inability lies in the fact that, while B cell responses may ultimately be affected by the induction of oral tolerance, such induction is almost exclusively a T cell phenomenon, with only secondary effects on B cells. Recently, we have shown preferential Th1 cell response at an early stage of experimental APS development, resulting in enhanced expression of IL-2 and IFN, while at a later stage of the disease there is a shift into Th2 cell response resulting in high levels of IL-4- and IL-6-producing cells accompanied by a preferential B cell (or humoral) response. Treatment by anti-idiotypic Ab caused a shift from Th2 into Th1 response and amelioration of the clinical manifestations of the disease.⁴ One of the major proposed mechanisms of oral tolerance is that it leads to more impairment of Th1 cells then Th2 cells and that the former are more critical to cell-mediated immunity, while the latter are more critical to humoral immunity. This possibility was supported by evidence that Th1 cells are more susceptible to apoptotic cell death and to active suppression mediated by suppressive cytokines (47). An example of the greater effect of oral tolerance on T cell than B cell responses is the profound effect of oral tolerance on IgE (allergic) responses (48). IgE responses are exquisitely dependent on T cell-B cell interaction (analogous to antiphospholipid Abs in experimental APS), so that any impairment of T cell function would be likely to have a profound effect on the IgE response. Additional relevant examples of the unresponsiveness of the humoral response at the late stage of ongoing experimental APS came from the fact that a large number of substance members, such as polysaccharide Ags, which interact directly with B cells, fail to elicit oral tolerance (49).

Our data support the notion that ß₂GPI may be the target autoantigen in APS and that the response is Ag driven, since immunization with ß₂GPI causes induction of anti-ß₂GPI and aCL production, in some cases associated with findings of APS (11, 12, 13, 14, 15). It is not surprising, therefore, that APS syndrome was lately reported as "the antiphospholipid/cofactor syndrome," which consists of all the characteristics of APS associated with anti-ß₂GPI Abs only (50). Oral administration of ß₂GPI might be considered as a treatment in patients with APS, mainly at an early stage of disease.

	Acknowledgments

 
We thank Dr. G. Bercke, Department of Immunology, Weizmman Institute of Science, for help in characterizing the CTL activity of our CD8⁺ cells; Dr. L. Weiner, Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, for very helpful discussions; and Rachlin Ludmila for excellent technical assistance.

	Footnotes

 
¹ This study was supported by the Japanese-Israeli Binational Grant for Research No. 8928197 and the Israeli Ministry of Sciences.

² Address correspondence and reprint requests to Dr. Y. Shoenfeld, Department of Medicine B, Sheba Medical Center, Tel-Hashomer, 52621, Israel. E-mail address:

³ Abbreviations used in this paper: APS, antiphospholipid syndrome; aPTT, activated partial thromboplastin time; ß₂GPI, ß-₂-glycoprotein I; MAC, macrophage; LNC, lymph node cell; HEL, hen egg lysozyme.

⁴ Krause, I., M. Blank, Y. Levy, V. Barak, T. Koike, and Y. Shoenfield. Anti-idiotype immunomodulation of experimental antiphospholipid syndrome via reversal effect on Th1/Th2 expression. Submitted for publication.

Received for publication March 17, 1997. Accepted for publication July 14, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Harris, E. N., A. E. Gharavi, M. L. Boey, B. M. Patel, C. G. Mackworth-Young, S. Loizou, G. R. V. Hughes. 1983. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 2:1211.[Medline]
2. Asherson, R. A.. 1989. A "primary" anti-phospholipid syndrome. J. Rheumatol. 15:1742.
3. Alarcon-Segovia, D., J. Sanchez-Guerrero. 1986. Primary anti-phospholipid syndrome. J. Rheumatol. 16:482.
4. Hughes, G. R. V., E. N. Harris, A. E. Gharavi. 1986. The anti-cardiolipin syndrome. J. Rheumatol. 13:486.[Medline]
5. Lockshin, M. D.. 1987. Anti-cardiolipin antibody. Arthritis Rheum. 30:471.[Medline]
6. McNeil, H. P., C. N. Chesterman, S. A. Krilis. 1991. Immunological and clinical importance of anti-phospholipid antibodies. Adv. Immunol. 49:193.[Medline]
7. Tsutsumi, A., E. Matsuura, K. Ichikawa, A. Fujisaku, M. Mukai, S. Kobayashi, T. Koike. 1996. Antibodies to ß₂-glycoprotein I and clinical manifestations in patients with systemic lupus erythematosus. Arthritis Rheum. 39:1466.[Medline]
8. McNeil, H. P., R. J. Simpson, C. N. Chesterman, S. A. Krilis. 1990. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: ß₂-glycoprotein I (apolipoprotein H. Proc. Natl. Acad. Sci. USA 87:4120.[Abstract/Free Full Text]
9. Kandiah, D. A., S. A. Krilis. 1994. Beta₂-glycoprotein I. Lupus 3:207.[Free Full Text]
10. Igarashi, M., E. Matsuura, Y. Igarashi, H. Nagae, K. Ichikawa, D. A. Triplett, T. Koike. 1996. Human ß₂-glycoprotein I as an anticardiolipin cofactor determined using deleted mutants expressed by a baculovirus system. Blood 87:3262.[Abstract/Free Full Text]
11. Gharavi, A. E., L. R. Summaritano, J. Wen, E. B. Elkon. 1992. Induction of antiphospholipid antibodies by immunization with ß₂-glycoprotein I (apolipoprotein H). J. Clin. Invest. 90:1105.
12. Pierangeli, S. S., E. N. Harris. 1993. Induction of phospholipid-binding antibodies in mice and rabbits by immunization with human ß₂-glycoprotein I or anticardiolipin antibodies alone. Clin. Exp. Immunol. 93:269.[Medline]
13. Blank, M., D. Faden, A. Tincani, J. Kopolovic, I. Goldberg, B. Gilburd, G. Balestrieri, G. Valesini, Y. Shoenfeld. 1994. Immunization with anticardiolipin cofactor (ß₂-glycoprotein-I) induces experimental APS in naive mice. J. Autoimmun. 7:441.[Medline]
14. Garcia, C., A. Kanbour-Shakir, H. Tang, L. R. Espinoza, A. E. Gharavi. 1996. Induction of experimental antiphospholipid syndrome in PL/J mice following immunization with ß₂-glycoprotein I. J.Invest. Med. 44:69A.
15. Aron, A. L., R. L. Cuellar, R. L. Brey, S. Meceown, L. R. Espinoza, Y. Shoenfeld. 1995. Early onset of autoimmunity in MRL/++ mice following immunization with ß₂-glycoprotein I. Clin. Exp. Immunol. 101:78.[Medline]
16. Mowat, A. M.. 1987. The regulation of immune responses to dietary protein antigens. Immunol. Today 8:93.
17. Higgins, P., H. L. Weiner. 1988. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein and its fragments. J. Immunol. 140:440.[Abstract]
18. Rizzo, L. V., N. E. Miller-Rivero, C. C. Chan, B. Wiggert, R. B. Nussenblatt, R. R. Caspi. 1994. Interleukin-2 treatment potentiates induction of oral tolerance in a murine model of autoimmunity. J. Clin. Invest. 94:1668.
19. Weiner, H. L., A. Friedman, A. Miller, S. J. Khoury, A. Al-Sabbach, L. M. B. Santos, M. Sayegh, R. B. Nussenblatt, D. E. Trenthan, D. A. Hafler. 1994. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu. Rev. Immunol. 12:809.[Medline]
20. Weiner, H. L., G. A. Mackin, M. Matsui, E. J. Orav, S. J. Khoury, D. M. Dawson, D. A. Hafler. 1993. Double-blind pilot trial of oral tolerization with myelin antigens in multiple sclerosis. Science 259:1321.[Abstract/Free Full Text]
21. Trentham, D. E., R. A. Dynesius-Trentham, E. J. Orav, D. Combitchi, C. Lorenzo, K. L. Sewell, D. A. Hafler, H. L. Weiner. 1993. Effects of oral administration of type II collagen on rheumatoid arthritis. Science 261:1727.[Abstract/Free Full Text]
22. Whitacre, C. C., I. E. Gienapp, C. G. Orosz, D. Bitar. 1991. Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy. J. Immunol. 147:2155.[Abstract]
23. Chen, Y., J. I. Inobe, R. Marks, P. Gonnella, V. K. Kuchroo, H. L. Weiner. 1995. Peripheral deletion of antigen-reactive T cells following oral tolerance. Nature 376:177.[Medline]
24. Ke, Y., J. A. Kapp. 1996. Oral antigen inhibits priming of CD8⁺ CTL, CD4⁺ T cells, and antibody responses while activating CD8⁺ suppressor T cells. J. Immunol. 156:916.[Abstract]
25. Chen, Y., J. Inobe, H. L. Weiner. 1995. Induction of oral tolerance to myelin basic protein in CD8-depleted mice: both CD4⁺ and CD8⁺ cells mediate active suppression. J.Immunol. 155:910.[Abstract]
26. Miller, A., O. Lider, A. B. Roberts, M. B. Sporn, H. L. Weiner. 1992. Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of TGF-ß following antigen specific triggering. Proc. Natl. Acad. Sci. USA 89:421.[Abstract/Free Full Text]
27. Son, C. O., M. Tomasi, M. T. Dertzbauelgh, G. Thaggard, R. Hunter, C. Weaver. 1996. Oral-antigen delivery by way of a multiple emulsion system enhances oral tolerance. Ann. NY Acad. Sci. 778:156.[Medline]
28. Khoury, S. J., O. Lider, A. Al-Sabbach, H. L. Weiner. 1990. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. III. Synergistic effect of lipopolysaccharide. Cell. Immunol. 131:302.[Medline]
29. Sun, J. B., J. Homgren, C. Czerkinsky. 1994. Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. Proc. Natl. Acad. Sci. USA 91:10795.[Abstract/Free Full Text]
30. Ponz, E., H. Wurm, G. M. Kostner. 1979. Investigations on ß₂-glycoprotein I in the rat: isolation from serum and demonstration in lipoprotein density fractions. Int. J. Biochem. 11:265.
31. Garside, P., M. Steel, E. A. Worthey, A. Satoskar, J. B. Alexander, H. Luethmann, F. Y. Liew, A. M. Mowat. 1995. T helper 2 cells are subject to high dose oral tolerance and are not essential for its induction. J.Immunol. 154:5649.[Abstract]
32. Kim, J. H, M. Ohsawa. 1995. Oral tolerance to ovalbumin in mice as a model for detecting modulators of the immunologic tolerance to a specific antigen. Biol. Pharm. Bull. 18:854.[Medline]
33. Ma, C. G., G.-X. Zhang, B.-G. Xiao, Z.-Y. Wang, J. Link, T. Olsson, H. Link. 1996. Mucosal tolerance to experimental autoimmune myasthenia gravis is associated with down regulation of AChR-specific IFN- expressing Th1-like cells and upregulation of TGFß mRNA in mononuclear cells. Ann. NY Acad. Sci. 778:273.[Medline]
34. Aramaki, Y., Y. Fuijii, H. Suda, I. Suzuki, T. Yadoma, S. Tsuchiya. 1994. Induction of oral tolerance after feeding with ragweed pollen extract in mice. Immunol. Lett. 40:21.[Medline]
35. Fredman, A., 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.[Abstract/Free Full Text]
36. Friedman, A.. 1996. Induction of anergy in Th1 lymphocytes by oral tolerance: importance of antigen dosage and frequency of feeding. Ann. NY Acad. Sci. 778:103.[Medline]
37. Moore, M. W., F. R. Carbone, M. J. Bevan. 1988. Introduction of soluble protein into class I pathway of antigen processing and presentation. Cell 54:777.[Medline]
38. Chen, Y., V. K. Kuchro, J. I. Inobe, D. A. Hafler, H. L. Weiner. 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265:1237.[Abstract/Free Full Text]
39. Keller, J. R., C. Mantel, G. K. Sing, L. R. Ellingsworth, S. K. Ruscetti, F. W. Ruscetti. 1988. Transforming growth factor ß selectively regulates early murine hematopoietic progenitors and inhibits the growth of IL-3-dependent myeloid leukemia cell lines. J. Exp. Med. 168:737.[Abstract/Free Full Text]
40. Kehrl, J. H., L. M. Wakefield, A. B. Roerts, S. Jakowlew, M. Alvarez-Mon, R. Derynck, M. B. Sporn, A. S. Fauci. 1986. Production of transforming growth factor-ß by human T lymphocytes and its potential role in the regulation of T cell growth. J. Exp. Med. 163:1037.[Abstract/Free Full Text]
41. Lee, G., L. R. Ellingsworth, S. Gillis, R. Wall, P. W. Kincade. 1987. ß Transforming growth factors are potential regulators of B lymphocytes. J. Exp. Med. 166:1290.[Abstract/Free Full Text]
42. Espevik, T., I. Figari, M. Shalaby, G. Lackides, G. Lewis, M. Shepard, M. Palladino. 1987. Inhibition of cytokine production by cyclosporin A and transforming growth factor-ß. J. Exp. Med. 166:571.[Abstract/Free Full Text]
43. Lee, H.-M., S. Rich. 1993. Differential activation of CD8⁺ T cells by transforming growth factor-_ß1. J. Immunol. 151:668.[Abstract]
44. Panja, A., L. Mayer. 1994. Diversity and function of antigen presenting cells in mucosal tissues. P. L. Ogra, and J. Mestecky, and M. E. Lamm, and W. Strober, and J. R. McGhee, and J. Bienenstock, eds. Handbook of Mucosal Immunology 177. Academic Press, San Diego, CA.
45. Bland, P. W., D. M. Kambarage. 1991. Antigen handing by the epithelium and lamina propria macrophages. Gastroenterol. Clin. North Am. 20:577.[Medline]
46. Mayer, L., R. Shilien. 1987. Evidence for function of Ia molecules on gut epithelial cells in man. J. Exp. Med. 166:1471.[Abstract/Free Full Text]
47. Zhang, X., T. Brunner, L. Carter, R. Dutton, P. Rogers, L. Bradley, T. Sato, J. Reed, D. Green, S. Swain. 1997. Unequal death in T helper cell Th1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185:1837.[Abstract/Free Full Text]
48. Ngan, J., S. L. Kind. 1978. Suppressor T cells for IgE and IgG in Peyer’s patches of mice made tolerance by the oral administration of ovalbumin. J. Immunol. 120:861.[Abstract/Free Full Text]
49. Stokes, C. R., T. J. Newby, J. H. Huntley, D. Patel, F. J. Bourne. 1979. The immune response of mice to bacterial antigens given by mouth. Immunology 38:497.[Medline]
50. Alarcon-Segovia, D., A. R. Cabral. 1996. The antiphospholipid/cofactor syndromes. J. Rheumatol. 23:8.

This article has been cited by other articles:

				Y Shoenfeld and M Blank Autoantibodies associated with reproductive failure Lupus, September 1, 2004; 13(9): 643 - 648. [Abstract] [PDF]

				J. George, N. Yacov, E. Breitbart, L. Bangio, A. Shaish, B. Gilburd, Y. Shoenfeld, and D. Harats Suppression of early atherosclerosis in LDL-receptor deficient mice by oral tolerance with {beta}2-glycoprotein I Cardiovasc Res, June 1, 2004; 62(3): 603 - 609. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blank, M. Articles by Shoenfeld, Y. Search for Related Content PubMed PubMed Citation Articles by Blank, M. Articles by Shoenfeld, Y.