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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. REFERENCES

The key immunological event in the pathogenesis of the autoimmune liver disease primary biliary cirrhosis is breakdown of T-cell self-tolerance to pyruvate dehydrogenase complex (PDC). The mechanism resulting in this breakdown of tolerance remains unclear. Mice exposed to self-PDC mount no immune response; however, animals coexposed to self-PDC and PDC of foreign origin (which in isolation induces a cross-reactive antibody but not an autoreactive T-cell response) show breakdown of T-cell as well as B-cell tolerance. This observation raises the possibility that a cross-reactive antibody response to self-PDC can promote breakdown of T-cell tolerance. The aim of this study was to address the hypothesis that breakdown of T-cell tolerance to PDC can be driven by the presence of B cells and/or antibodies cross-reactive with this self-antigen. Naive female SJL/J mice were exposed to self-PDC alone and in the presence of purified splenic B cells from animals primed with foreign PDC (or controls) or purified immunoglobulin (Ig) G from the same animals. Breakdown of T-cell tolerance was assessed by splenic T-cell proliferative response to antigen at 5 weeks. CD4+ T-cell proliferative responses indicative of breakdown of T-cell tolerance to self-PDC were seen in the majority (7 of 9, 78%) of animals receiving self-PDC together with purified PDC-reactive B cells. Tolerance breakdown was not seen in animals receiving self-PDC with purified anti-PDC IgG or with B cells from animals sensitized with an irrelevant antigen. In conclusion, breakdown of T-cell tolerance to the highly conserved self-antigen PDC may be mediated by high-level presentation of self-derived epitopes by activated cross-reactive B cells. (HEPATOLOGY 2005.)

The autoimmune liver disease primary biliary cirrhosis (PBC) is characterized by a breakdown in immune self-tolerance to the highly conserved mitochondrial self-antigen pyruvate dehydrogenase complex (PDC).1 High-titer serum antibodies reactive with self-PDC are present in over 95% of PBC patients and represent an important serological marker for disease diagnosis.2–4 The principal B-cell epitope within PDC has been localized to a lipoic acid–binding domain within the dihydrolipoamide acetyltransferase (E2) component of PDC,5, 6 with the lipoic acid moiety forming a key part of the epitope.7 PDC-E2 specific autoantibodies cross-react fully with the E3-binding protein (E3BP, previously known as protein X) component of the complex.8

Despite their almost ubiquitous presence in PBC, the role played by PDC-E2–specific antibodies in the pathogenesis of PBC remains unclear. In particular, there are no strong data to implicate them in the damage to the biliary epithelial cells lining the small intrahepatic bile ducts that represent the cardinal histological lesion of PBC. Of particular note is the fact that anti-PDC antibody responses can occur in the setting of bacterial infection in the apparent absence of any damage to biliary epithelial cells.9, 10

There is an increasing body of evidence to suggest that the effector mechanism responsible for biliary epithelial cell damage in PBC may be apoptosis induced by self-PDC specific cytotoxic effector T cells known to be present at relatively high levels in the peribiliary portal tract infiltrate stages in the disease process when biliary epithelial cell damage is known to be occurring.11–13 The apparent importance of the increasingly well-characterized autoreactive CD4+ and CD8+ PDC-specific T-cell response in the pathogenesis of PBC has led to the obvious question as to how T-cell tolerance breaks down to as highly conserved and ubiquitously expressed a self-antigen as PDC.14 Exposure to cross-reactive bacterial PDC, microorganism-derived proteins showing structural cross-reactivity with PDC, and xenobiotics mimicking the lipoate-containing structure of PDC have been proposed as mechanisms for the induction of PDC tolerance breakdown in PBC. However, animal modeling studies suggest that simple exposure to homologues of self-PDC results in breakdown of B-cell but not T-cell tolerance. A reformulation of the key question regarding the pathogenesis of PBC would therefore be: How does breakdown of B-cell tolerance to PDC (an apparently relatively easily achievable state with limited pathological sequelae) result in the much more restricted (and seemingly pathologically significant) state of T-cell tolerance breakdown?

We have previously demonstrated that female SJL/J mice sensitized with foreign PDC rapidly develop high titre autoantibody responses reactive with self-PDC.15 The development of anti-PDC autoantibodies is not, in the short term, accompanied by breakdown of T-cell tolerance. However, when animals are cosensitized with foreign PDC and self-PDC (to which, following exposure in isolation, the animals are fully tolerant), autoreactive T-cell responses to self-PDC are seen. One interpretation of this observation would be that whereas self-PDC in isolation is nonimmunogenic, exposure to self-antigen in the context of an antibody response reactive with it renders it immunogenic. In this study, we set out to address the hypothesis that breakdown of T-cell tolerance to self-PDC can be driven by an autoreactive B-cell response using an in vivo active and passive transfer approach.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. REFERENCES

Experimental Design

We have previously demonstrated that female SJL/J mice, which are normally tolerant of self-PDC, break T-cell tolerance when simultaneously exposed to foreign PDC (which in isolation induces an antibody response reactive with self-PDC but does not break T-cell tolerance) and self-PDC. The hypothesis being addressed in the current study is that the antibody response cross-reactive with self-PDC that is induced following coexposure with foreign PDC drives the breakdown of T-cell tolerance to self-PDC. In this study, animals were initially sensitized with foreign PDC (of bovine origin) in complete Freund's adjuvant (CFA) or with CFA alone (study phase 1). At 8 weeks following sensitization, serum and splenic tissue were obtained and the immunoglobulin (Ig) G and splenic B-cell fractions, respectively, were derived. In the experimental phase of the study, groups of naive animals received antigen-primed or control serum in the presence and absence of self-PDC or antigen-primed or control B-cells in the presence and absence of self-PDC (study phase 2). The outcome measure of the study was breakdown of CD4+ T-cell tolerance to self-PDC in the recipients at 5 weeks after transfer.

Antigens

Bovine PDC was isolated from heart muscle as previously described.16 Mouse PDC (self-PDC) was also isolated from heart muscle using a scaled-down method that omitted the final separation step of 2-oxoglutarate dehydrogenase complex from PDC as previously described.17 Purity of the PDC preparations was assessed via SDS-PAGE and was found to be more than 95% pure. All protein concentrations used for sensitizations were based on the concentration of PDC alone.

Sensitizations and Transfers

Female SJL/J mice were obtained from Harlan Olac (Bicester, Oxfordshire, United Kingdom). Sensitizations and/or transfers were performed when the animals were 12 to 16 weeks of age.

Study Phase 1.

Mice were sensitized as previously described17, 18 with 500 μg bovine PDC (bPDC) emulsified 1:1 (vol/vol) with CFA, consisting of 10 mg/mL Mycobacterium tuberculosis strain H37Ra (Difco, Detroit, MI) in incomplete Freund's adjuvant (Sigma, Poole, Dorset, United Kingdom) (bPDC donor). Age-matched control mice received CFA homogenized with isotonic saline 1:1 (vol/vol) (control donor). All sensitizations were performed intraperitoneally in a final volume of 100 μL. All mice were subjected to a single sensitization and were killed at 8 weeks after sensitization. The study was performed to appropriate ethical standards under UK Home Office Licence.

Study Phase 2.

Naive recipients received either B cells or purified IgG fraction from bPDC or control donors, in the presence or absence of mouse PDC (mPDC).

  • 1
    B-cell recipients. Animals received 1 × 106 B cells from bPDC donors (isolated as outlined below) resuspended in 50 μL isotonic mixed with an equal volume of isotonic saline containing 100 μg mPDC via the intraperitoneal route (group 1). Control animals received B cells from control donors pulsed with mPDC (group 2), B cells from control donors pulsed with saline (group 3), B cells from bPDC donors pulsed with saline (group 4), or mPDC in the absence of B cells (group 5).
  • 2
    Antibody recipients. Animals received either 500 μg purified IgG fraction from bPDC donors (isolated as outlined below) resuspended in 50 μL isotonic saline mixed with an equal volume of isotonic saline containing 100 μg mPDC (group 6) or isotonic saline alone (group 7) or 500 μg purified IgG fraction from control donors mixed with mPDC (group 8) or saline (group 9). In each case, the antigen/antibody mixture was added to CFA containing 1 mg Mycobacterium tuberculosis strain H37Ra (Difco) mixed 1:1 (vol/vol) with incomplete Freund's adjuvant (Sigma) and administered intraperitoneally.

The sensitization combinations used for the transfer experiments are summarized in Table 1. Positive control animals received cosensitization with mPDC and bPDC in CFA using a previously described sensitization regime.15

Table 1. Summary of Transfer Study Design
Study GroupbPDC Donor B-CellControl Donor B-CellbPDC Donor IgControl Donor IgmPDC
1++
2++
3+
4+
5+
6++
7+
8++
9+

Splenic Mononuclear Cell Preparation

Spleens were removed intact and washed in RPMI 1640 (Sigma). The tissues were disaggregated via passage through a 70-μm nylon mesh (Becton Dickinson, Oxford, United Kingdom), and mononuclear cells were purified via centrifugation on Histopaque 1.083 (Sigma) at 400g for 20 minutes. The interfacial cells were recovered, washed three times in complete medium (RPMI 1640 supplemented with L-glutamine [2 mmol/L], 5% fetal calf serum, penicillin [100 U/mL], and streptomycin [0.1 mg/mL]; all Sigma) and counted.

CD4+ T-Cell Proliferation Assay

The proliferation of antigen-specific CD4+ T cells was assayed via [3H]thymidine incorporation into DNA. Mononuclear cells were cultured in 96-well U-bottomed plates (Sarstedt, Leicester, United Kingdom) at a concentration of 2 × 105 cells/100 μL in complete medium. Filter-sterilized bPDC or mPDC was added to cells at a final concentration of 10 μg/mL in a volume of 100 μL (optimized in earlier studies). Pentuplicate wells were used for each antigen and for control wells without added antigen. After 96 hours in culture at 37°C under 5% CO2, each well was pulsed with 1 μCi of [3H]thymidine (2.0 Ci/mmol; ICN, Hampshire, United Kingdom) in 30 μL of complete medium. After a further 18 hours in culture, the cells were harvested and the DNA-incorporated radiation was assessed via liquid scintillation counting.

Splenic B-Cell Isolation

Splenic mononuclear cells were isolated from bPDC mice and control donors in study phase 1 at 8 weeks after sensitization. The mononuclear cells were further cell fractioned via magnetic cell sorting (MACS; Miltenyi Biotech, Bisley, Surrey, United Kingdom) using anti-CD19–labeled beads according to the manufacturer's protocol. Briefly, mononuclear cells were washed via centrifugation at 400g, then resuspended in buffer (0.5% [vol/vol] fetal calf serum in phosphate-buffered saline [PBS]) at a concentration of 90 μL/107 total cells. MACS CD19+ Microbeads were added at a concentration of 10 μL/107 total cells. After mixing, cells were incubated at 10°C for 15 minutes. Cells were washed by adding 20 times the volume of labeling buffer and centrifuged at 400g for 10 minutes. The pellet was resuspended in 500 μL buffer and applied to a positive separation column (MS+) attached to a MACS magnet. The unbound, negative cells were washed through three times with buffer. The CD19+ B cells were isolated by removing the column from the magnet and flushing them out with buffer. The number of cells in the positive fraction was counted and resuspended in isotonic saline at a concentration of 5 × 106 cells/250 μL.

Sample purity with regard to B-cell preparation was confirmed via immunofluorescent labeling and flow cytometry using a FACScan flow cytometer (Becton Dickinson) and the Lysis II software package. Cells were stained with optimal concentrations of anti-CD19 FITC antibody (clone ID3; Pharmingen, San Diego, CA) for 20 minutes at 4°C, then washed via centrifugation at 400g for 5 minutes and resuspended in PBS with 2.5% fetal calf serum; 104 cells were counted per sample. Flow cytometer acquisition characteristics were set up following the manufacturer's recommendation.

Affinity Purification of IgG Fraction

Serum was prepared from pooled terminal bleeds of either bPDC or control donors. In each case the serum was diluted 1:1 with PBS (pH 7.0) and passed through a 0.45-μmol/L filter before loading on PBS-equilibrated, serially connected 1-mL HiTrap Protein G/Protein A columns (Amersham Biosciences, Bucks, United Kingdom). Following extensive washing in PBS, the bound IgG fraction was eluted in 5 column volumes of elution buffer (0.1 mol/L glycine, 0.15 mol/L NaCl [pH 2.6]). Samples were immediately neutralized with 1 mol/L Tris/HCl (pH 8.0), desalted, and concentrated into PBS using Vivaspin 6 centrifugal ultrafiltration units (Vivascience, Hannover, Germany). Protein concentration was assessed and samples were stored at −20°C until use.

Statistical Analysis

Comparisons of mean incorporated cpm for control and antigen-containing wells both between subject groups and on an individual animal basis were made using a paired t test. Frequencies of positive response in different groups were compared using a χ2 test.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. REFERENCES

As previously demonstrated, cosensitization of animals (n = 5) with mPDC and bPDC resulted in a significant breakdown of T-cell tolerance to self-PDC (Fig. 1). This breakdown in T-cell tolerance occurred, again as previously demonstrated, in the context of an antibody response showing reactivity between self and foreign forms of PDC (data not shown).

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Figure 1. T-cell proliferative response to mPDC and in background wells without antigen (control) of splenocytes from SJL/J mice (n = 5) cosensitized with bPDC and mPDC. mPDC, mouse pyruvate dehydrogenase complex, bPDC, bovine pyruvate dehydrogenase complex.

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To study the role played by the B-cell response in the breakdown of T-cell tolerance to self-PDC, naive animals were exposed to mPDC in the context of either purified splenic B cells from PDC-sensitized animals or purified Ig fraction from the same animals. Animals adoptively transferred with the splenic B-cell fraction purified from bPDC-sensitized donors (Fig. 2A) pulsed with mPDC (group 1) demonstrated a significant in vitro splenic T-cell proliferative response to self-PDC (Fig. 2B). This significant response was absent from animals receiving purified B cells from donors sensitized with CFA alone in either the presence (Fig. 2C, group 2) or absence (group 3, data not shown) of mPDC. The individual well [3H]thymidine incorporation data for splenocytes in control wells and in wells pulsed with mPDC, together with individual mouse statistical analyses for mice in group 1 and group 2, are presented in Figs. 3 and 4, respectively.

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Figure 2. Breakdown of tolerance to self-PDC in animals receiving B cells from PDC-sensitized donors mixed with mPDC. (A) FACS analysis of the donor B-cell population confirming purity of the transferred cell population. Proliferative response of splenic mononuclear cells from animals in (B) group 1 and (C) group 2 to mPDC and bPDC compared with proliferation in control wells not containing antigen. Con, control; mPDC, mouse pyruvate dehydrogenase complex; bPDC, bovine pyruvate dehydrogenase complex.

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Figure 3. Individual well counts for responses to mPDC of splenocytes from mice in group 1. Each graph denotes an individual mouse. Horizontal lines denote mean values. Comparisons are with control well values. Statistical analysis is by paired t test versus control (nonantigen-pulsed) wells. ns, not significant; Con, control; mPDC, mouse pyruvate dehydrogenase complex.

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thumbnail image

Figure 4. Individual well counts for responses to mPDC of splenocytes from mice in group 2. Each graph denotes an individual mouse. Horizontal lines denote mean values. Comparisons are with control well values. Statistical analysis is by paired t test versus control (nonantigen-pulsed) wells. ns, not significant; Con, control; mPDC, mouse pyruvate dehydrogenase complex.

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Within the individual subject groups breakdown of tolerance to mPDC (defined as a statistically significant difference in the proliferative response to mPDC compared with that seen in control wells) was seen in 7 of 9 (78%) of animals in group 1 (Fig. 3). In comparison, breakdown of T-cell tolerance to self-PDC was seen in 0 of 5 animals in group 2 (Fig. 4; P = .005 vs. group 1), 1 (20%) of 5 animals in group 4 (P < .05 vs. group 1), and 0 of 5 animals in group 5 (P = .005 vs. group 1) (Fig. 5).

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Figure 5. Percentages of animals showing breakdown of T-cell tolerance to mPDC in group 1 (n = 9), group 2 (n = 5), group 4 (n = 5), and group 5 (n = 5) *P < .05 vs. group 1. **P < .005.

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In contrast to the animals receiving purified B cells from bPDC-sensitized donors, animals receiving purified IgG from bPDC-sensitized donors (confirmed to show anti-PDC reactivity by immunoblot [Fig. 6A]) mixed with mPDC and CFA showed, as a group (group 6), no breakdown of T-cell tolerance to mPDC (Fig. 6B–C). Within the groups, tolerance breakdown (as previously defined) was seen in 1 (16%) of 6 animals in group 6 (P < .05 vs. group 1), 0 of 6 animals in group 7 (P < .005 vs. group 1), 1 of 6 animals in group 8 (P < .05 vs. group 1), and 0 of 6 animals in group 9 (P < .005 vs. group 1).

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Figure 6. Lack of breakdown of tolerance to self-PDC in animals receiving purified IgG from PDC-sensitized donors mixed with mPDC and CFA. (A) Immunoblot of serum from representative bPDC donor against bPDC (protein stain lane 1, immunoblot lane 3) and mPDC (protein stain lane 2, immunoblot lane 4). Proliferative response of splenic mononuclear cells from animals in (B) group 6 and (C) group 9 to mPDC and bPDC compared with proliferation in control wells not containing antigen. ns, not significant; Con, control; mPDC, mouse pyruvate dehydrogenase complex; bPDC, bovine pyruvate dehydrogenase complex.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. REFERENCES

In this study, we have demonstrated that naive female SJL/J animals break T-cell tolerance to self-PDC when exposed to self-PDC mixed with B cells expressing surface Ig reactive with self-PDC. Such tolerance breakdown was absent from animals exposed to either self-PDC or self-PDC reactive B cells in isolation, or from animals exposed to self-PDC mixed with control B cells. Our observations suggest that the B-cell response to self-PDC so characteristic of PBC may play a critical role in driving the PDC-specific autoreactive T-cell response implicated in target cell damage.

There are two potential mechanisms for antigen-specific B-cell promoted breakdown of T-cell tolerance to self-PDC. The first potential mechanism is through binding of self-PDC to reactive Ig B-cell surface receptors increasing uptake, processing, and presentation of self-derived epitopes.19 Critically for this model, it has been demonstrated that B cells can indeed act as professional antigen-presenting cell (APC) breaking T-cell tolerance, provided they have an activated phenotype and express surface receptor reactive with the antigen to which T-cell tolerance is broken (the precise circumstances encountered in our model).20 The second potential mechanism is through self-PDC reactive antibody–promoting uptake and presentation of self-antigen by other professional APCs through the formation of immune complexes taken up and processed at an increased rate by APCs.21 In the current study, we found that whereas autoreactive B-cells were able to promote tolerance breakdown in vivo, the addition of self-PDC reactive antibodies was not. Our findings would therefore appear to favor the direct B-cell model in the in vivo in setting. Studies in other animal models of T-cell autoimmunity, most notably diabetes in the nonobese diabetic mouse and in experimental allergic encephalomyelitis, have suggested that B-cell–deficient mice are resistant to the development of autoimmune disease.22–27 However, the protocols adopted in these studies (either B-cell depletion through the use of anti-μ antibody from birth or the use of congenitally B-cell–deficient animals) fail to distinguish between a B-cell– and an antibody-driven process. The demonstration that B-cell–deficient JH-MRL lpr+ mice lack the significant increase in T-cell numbers in lymphoid organs characteristic of wild-type MRL lpr+ animals,28 but that animals expressing a novel IgM transgene leading to expression of B-cell surface Ig but not Ig secretion express the “wild-type” T-cell rich phenotype,28 would argue for a direct role for B-cell function in at least this alternative autoimmune model.

In contrast to our observation supporting a direct role for autoreactive B cells—but not antimitochondrial antibody—in the breakdown of T-cell tolerance to self-PDC, in vitro studies in the human setting have suggested that the presence of anti-PDC antibodies can augment the propagation of CD8+ T-cell lines from peripheral blood mononuclear cell precursors after in vitro stimulation by dendritic cells pulsed with recombinant PDC-E2.12 Specifically, pulsing with PDC-E2 containing immune complexes as opposed to naked antigen significantly reduced the concentration of pulsing antigen required for PDC-specific cytotoxic T lymphocyte (CTL) induction. Further studies will therefore be required (including characterization of the CTL response) before finally excluding an in vivo role for autoreactive antibody to go along with the clearly demonstrated B-cell effect in our model. Taken together, however, the murine and human studies strongly suggest that one key role played by the well-characterized anti-PDC autoantibody response in PBC is in driving T-cell tolerance breakdown (with strong direct and indirect evidence to implicate this autoreactive T-cell response in target cell damage). These observations would be compatible with a two-stage model for disease pathogenesis. In the first stage, an antibody response reactive with self-PDC would be induced. The induction of this response would be necessary, but in isolation insufficient for the induction of PBC. In the second stage, this B-cell response would drive the breakdown of T-cell tolerance seemingly associated with target cell damage.

Important questions remain regarding the second stage of this model. These include the source of the self-PDC needed to drive T-cell tolerance breakdown in the context of cross-reactive B cells and the stimuli required to maintain a state of B-cell activation. Intercurrent localized infection resulting in cell necrosis and PDC release in the context of toll-like receptor (TLR) ligand expression would be one potential mechanism (of particular possible importance given the potential for stimulation via TLR9 (a key TLR for B-cell activation29) to promote T-cell autoreactivity in both the PDC-tolerance breakdown and other autoimmune disease models.30–32 Another potential mechanism for exposure to self-PDC at least could be through the recently demonstrated expression of PDC on the surface membrane by cells undergoing apoptosis,33 with localized infection inducing focal apoptosis representing a potential mechanism for the tissue tropism of the process. Further work is required in this area. With regard to the first stage of the model, human and murine studies have suggested that the induction of an antibody response reactive with self-PDC may result from a number of different priming events. Among the events demonstrated to induce an antibody response cross-reactive with self-PDC are exposure to bacterial PDC9, 10 or other microbial structural mimics34 and exposure to chemically modified PDC or mimicking xenobiotics.35, 36 Other potential events currently lacking demonstration of direct in vivo priming are exposure to PDC components modified during caspase cleavage in cells undergoing apoptosis37 and exposure to cross-reactive retroviral proteins.38 The diversity of the potential events giving rise to antibody responses cross-reactive with PDC, which could promote subsequent T-cell tolerance breakdown, suggests the intriguing possibility that PBC could represent a condition with a common final pathway (T-cell tolerance breakdown and target cell damage) but with multiple triggers (each able to induce a B-cell response cross-reactive with self-PDC).

REFERENCES

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. REFERENCES