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Keywords:

  • autoantibody;
  • epitope spreading;
  • Grp78;
  • Ro (SS-A)

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Patterns of autoantibody production are diagnostic of many autoimmune disorders; the recent observation of additional autospecificities towards stress-induced proteins may also provide insight into the mechanisms by which such responses arise. Grp78 (also known as BiP) is a target of autoaggressive B and T cell responses in our murine model of anti-Ro (SS-A) autoimmunity and also in rheumatoid arthritis. In this report we demonstrate reciprocal intermolecular spreading occurs between Ro52 and Grp78 in immunized mice, reflecting physiological association of these molecules in vivo. Moreover, we provide direct biochemical evidence that Grp78 associates with the clinically relevant autoantigen, Ro52 (SS-A). Due to the discrete compartmentalization of Ro52 (nucleocytoplasmic) and Grp78 (endoplasmic reticulum; ER) we propose that association of these molecules occurs either in apoptotic cells, where they have been demonstrated indirectly to co-localize in discrete apoptotic bodies, or in B cells themselves where both Ro52 and Grp78 are known to bind to immunoglobulin heavy chains. Tagging of molecules by association with Grp78 may facilitate receptor mediated phagocytotsis of the complex; we show evidence that exogenous Grp78 can associate with cell surface receptors on a subpopulation of murine splenocytes. Given the likelihood that Grp78 will associate with viral glycoproteins in the ER it is possible that it may become a bystander target of the spreading antiviral immune response. Thus, we propose a model whereby immunity elicited towards Grp78 leads to the selection of responses towards the Ro polypeptides and the subsequent cascade of responses observed in human disease.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Autoimmunity towards heat shock proteins (HSP) has been observed in a number of experimental models of autoimmunity and in human autoimmune disease [1–6]. These responses may be manifested as either T cell proliferative responses or as autoantibodies towards the endogenous HSP. For many years these responses were thought to be the consequence of cross-reactivity between endogenous HSP and pathogen-derived HSP, which have been observed to be immunodominant following infection. This substantiated immunological dogma that autoreactivity follows from the immune system being activated towards bona-fide pathogen-derived antigen that leads subsequently to cross-recognition of the self-protein. More recently HSP have been found to have an abundance of immunological features, including functioning as carriers of antigenic peptides, immunopotentiators and immunomodulators.

Pathogen-derived HSP are effective carriers of peptide and protein immunogens and possess the ability to enhance immunity towards the bound antigen [7], as well as eliciting HSP-specific immunity. In addition, there is mounting evidence that endogenous HSP also have this immunopotentiating effect. For example, there is a strong correlation between HSP expression and the immunogenicity and ultimate clearance of tumours [8,9]. Srivastava and co-workers [9,10] have demonstrated that HSP isolated from tumour cells can elicit immune responses towards intact tumours. This protective antitumour immunity was associated with the induction of CD8+ antitumour cytotoxic T lymphocytes (CTL) that protected mice immunized with tumour-derived HSP from fatal tumour loads. The mechanism whereby tumour-derived HSPs elicit such potent immunity is implicit in the observation that HSPs chaperone peptides derived from cytosolic proteins [11,12], including tumour antigen-derived peptides. Not only does this protect the antigen against extracellular proteolysis, cell surface receptors present on professional antigen-presenting cells (APC) exist that bind in a specific and saturable manner to HSP–Ag complexes facilitating receptor-mediated phagocytosis and antigen presentation [13]. In addition to evidence that HSPs function in class I-restricted Ag processing events, there is strong evidence that HSP70, including Grp78 and Hsc70, are involved in Ag processing in the class II compartment [14,15]. Thus, HSPs appear to associate with antigenic peptides generated within the cytoplasm of cells as well as self-antigens in non-stressed cells, and may play a role in modulating ensuing immunity towards the bound species.

Intermolecular spreading of humoral autoimmunity to different components of the La/Ro ribonucleoprotein (RNP) complex has been reported following active immunization with individual components La, 52-kDa Ro (Ro52), 60-kDa Ro (Ro60) and poorly tolerized subdominant determinants of La [16–19]. This murine model of linked anti-La/Ro responses supports the physical association of these proteins in RNP complexes and/or co-localization in surface membrane blebs in apoptotic cells [18]. B cell epitope spreading appears to be driven by intermolecular help from Ro- or La-specific T cells to B cells with autospecificity for La and Ro epitopes. These B cells are capable of capturing La/Ro RNP complexes and presenting determinants from different antigens in the complex to autoreactive T cells of a single specificity [18]. In addition, studies of epitope spreading may provide a lead to exploring and identifying components that may interact directly or indirectly with La/Ro RNP complexes and thereby influence their immunogenicity. In a recent study we demonstrated spreading of the immune response from Ro52 and Ro60 (but not La) to calreticulin (CR) in murine experimental autoimmunity, consistent with the notion that CR may associate with the subpopulation of Ro particles from which La has already dissociated [20]. Because CR is an HSP and molecular chaperone [21,22], we have extended these studies to include members of the hsp70 and hsp90 families. The secondary recruitment of autoantibodies to Grp78 and Hsp72 in Ro-immunized mice provides evidence for their physical association and co-localization with Ro52 and Ro60 [23]. In this report, we demonstrate reciprocity in the intermolecular spreading of B cell immunity between Grp78 and Ro autoantigens and provide biochemical evidence for an association between Ro52 and Grp78. We propose a model whereby immunity elicited towards Grp78 leads to the selection of responses towards the Ro polypeptides and the subsequent cascade of responses observed in murine experimental autoimmunity and human disease.

MATERIALS AND METHODS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Proteins

Recombinant murine Ro52 was produced as hexa-histidine fusion proteins (6xHis) and purified under denaturing conditions as described [23]. Recombinant 6xHis-mLa was purified as a soluble protein using Ni-NTA metal affinity chromatography [24]. A soluble mRo60 lacking the NH2-terminal 82 amino acids (MBP-mRo60) and soluble full-length mouse calreticulin (MBP-mCR) were expressed as maltose binding protein (MBP) fusion proteins and were purified by maltose affinity chromatography, as described previously [20]. Purified recombinant human Hsp72 (>99% amino acid identity to murine Hsp72), and recombinant hamster glucose-regulated protein Grp78 (>99% amino acid identity to murine Grp78) were obtained from StressGen Biochemicals (Victoria, BC, Canada).

Animals and immunization

Groups of three to six C3H/HeJ mice were immunized and boosted twice subcutaneously with 100 µg of 6xHis-mRo52, 6xHis-mla, MBP-mRo60, MBP, Hsp72 or Grp78 and sera collected at different time-points, as described previously [20]. Initial immunizations were given in Freund's complete adjuvant (FCA; Gibco BRL, Grand Island, NY, USA) and boosts in Freund's incomplete adjuvant (FIA; Gibco BRL).

Enzyme-linked immunosorbent assays (ELISAs)

The recombinant fusion proteins and different HSPs were coated onto Polysorp microtitre plates (Nunc, Roskilde, Denmark) at a saturating concentration of 2 µg/ml in 0·03 m carbonate buffer pH 9·6 overnight at 4°C. Blocking and washing steps were performed as described previously [20], with mouse sera screened at a 1 : 500 dilution and bound antibodies detected with an alkaline phosphatase-labelled antimouse IgG (Sigma, St Louis, MO, USA).

Immunoblots

Recombinant proteins, HSPs (1 µg/lane) or mouse cell lysates prepared after heat shock for 30 min at 45°C (2 × 105 cells/lane) were electrophoresed in 10% SDS-PAGE gels under reducing conditions, electroblotted to nitrocellulose membranes and probed with sera (diluted 1 : 1000) from the different groups of mice. Bound antibody was detected by enhanced chemiluminescence (ECL), as described previously [20] (Amersham, Aylesbury, UK). Antibodies to Grp78 (SPA-826 a rabbit polyclonal antiserum raised against rat Grp78 amino acids 645–654 and a murine monoclonal antibody SPA-827 raised against the C-terminus of rat Grp78 (amino acids 648–654)) and Hsp72 (murine monoclonal antibody SPA-810 raised against purified intact human Hsp72) were obtained from StressGen.

Grp78 binding assays

Competition binding assays that measure the ability of synthetic peptides to compete the binding of reduced and carboxy-methylated α-lactalbumin (RCMLA) to Grp78 were performed as described [25,26]. After electrophoretic separation of the free Grp78 from Grp78-RCMLA or Grp78-peptide complexes under native PAGE conditions, Grp78 and was visualized by immunoblotting with rabbit polyclonal antibodies raised against recombinant murine Grp78 [27] and detected using ECL. A capture ELISA was also utilized to examine the ability of Grp78 to associate with a peptide derived from amino acids 378–391 of mRo52 (ENGFWTIWLWQDSY) relative to a control peptide. In order to improve the capture a multimeric peptide (MAP) construct consisting of a branched lysine backbone and four copies of the monomer (both monomer and MAP Ro52378−391 were synthesized and supplied by Mimotopes Pty Ltd, Clayton, Australia) was incorporated into the ELISA assay. Such multimeric constructs provide improved accessibility to the capture molecule and thus enhanced detection of binding in ELISA based assays [28].

Flow cytometric analysis of association of HSP with populations of splenocytes

The ability of HSPs to associate with surface of splenocytes was examined by indirect immunofluorescence. Single cell suspensions of splenocytes prepared from the spleens of C3H/HeJ mice were incubated for 30 min with 0·5, 1·0 or 5 µg/ml of Grp78, Hsp70 or Hsp90 (StressGen) that had been biotinylated using standard succinimide ester-based chemistries [29] using EZ-link long-chain biotin-NHS (Pierce Biotechnology, Rockford, IL, USA). After three washes, the cells were incubated with extravidin–phycoerythrin (Sigma) conjugate and cells examined by flow cytometry. Staining with 1·0 µg of biotinylated HSP was demonstrated to be saturating and only data using this concentration of HSP are shown.

RESULTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Intermolecular spreading of the autoantibody response from Ro (SS-A) autoantigen to Grp78 is reciprocal

Normal mice immunized with Ro52 or Ro60 demonstrate kinetically distinct, non-cross-reactive antibody responses towards the endogenous molecular chaperones murine calreticulin (mCR), Grp78 and Hsp72 [23]. The recruited responses towards mGrp78 and mHsp72 occur with similar kinetics and we have shown previously that they represent shared reactivity towards conserved determinants present in both HSPs [23]. To investigate whether immunity towards Grp78 or Hsp72 elicited reciprocal reactivities towards mRo52 or mRo60, groups of mice were immunized with Grp78 or Hsp72 and boosted twice at days 14 and 28 postimmunization. Sera were collected at 7-day intervals and reactivity towards a panel of recombinant autoantigens assessed by ELISA (Fig. 1a,b) and immunoblot (Fig. 1c,d). Mice immunized with Hsp72, an inducible form of cytoplasmic Hsp70, demonstrated responses only towards the immunogen with no recruited specificities towards Ro52, Ro60, La, CR or Grp78 evident. In contrast, mice immunized with Grp78 demonstrated a robust response against the immunogen, followed by kinetically distinct responses to mRo52, mRo60, mCR but not mla. Interestingly, these mice also developed reactivity towards Hsp72, suggesting that only immunization with Grp78 resulted in cross-reactive antibodies against the other HSP70 family member. The specificity of these responses was also examined by immunoblot using recombinant material and shown to recognize recombinant autoantigens of the correct apparent molecular weight based on their migration in SDS-PAGE. No response was observed to the mRo60 in immunoblot; this is due to the requirement of conformational determinants for the detection of recruited Ro60 specific antibody responses [30,31]. Importantly, Grp78 and Hsp72 immunized mice also recognized endogenous murine HSPs in the heat-shocked murine cell line EL-4, reflecting the ability of these antibodies to recognize lysates containing the native murine proteins (Fig. 2). The migration positions of murine Grp78 and Hsp72 were confirmed by immunoblot with a panel of defined antibodies with specificity for the two HSPs.

image

Figure 1. Reciprocal intermolecular spreading is observed only between Grp78 and Ro (SS-A) antigens. (a) Kinetics of autoantibody production in C3H/HeJ mice immunized with 100 µg Grp78 (a) or Hsp72 (b) in Freund's complete adjuvant (day 0) and boosted twice subcutaneously with 100 µg Grp78 (a) or Hsp72 (b) in Freund's incomplete adjuvant (days 14 and 28). Sera were taken at weekly intervals and assessed for reactivity towards the immunogen (Grp78 (□)), mRo52 (○), Hsp72 (◊), mRo60 (▪), mCR (◆), mla (•) and the control protein MBP (▵) by ELISA. Immunoblot of Grp78 (c) and Hsp72 (d) immune sera against recombinant autoantigens (1 µg/lane) electrophoresed on a 10% SDS-PAGE gel.

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image

Figure 2. Immunoblots of Grp78- and Hsp70-immunized mice with murine lysates reveal reactivity with endogenous HSPs. Murine EL-4 cell lysates were prepared after heat shock for 30 min at 45°C and electrophoresed in 10% SDS–PAGE. The lysates were probed with Grp78 and Hsp70 immune sera as well as with commercially available anti-Grp78 monoclonal and polyclonal antibodies and an anti-Hsp70 monoclonal antibody.

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Grp78 binds to Ro52 378–391 in competitive ELISA and gel shift assays

The reciprocal nature of the Grp78–Ro52/Ro60 determinant spreading suggests that these molecules are able to associate non-covalently in vivo. We have shown previously that Ro52 possesses a putative Grp78 binding motif [18,23] comprising a clustered series of dominant binding sequences (amino acids 378–391). A synthetic peptide corresponding to this region of the Ro52 molecule was synthesized and examined for association with Grp78 in a gel shift assay [25] and a capture ELISA (Fig. 3a–c). In both cases, the Ro52378−391 peptide was observed to associate with Grp78 as indicated by either disruption of a Grp78-RCMLA (reduced and carboxy-methylated α-lactalbumin) complex via competitive binding of the monomeric Ro52378−391 peptide to Grp78 (Fig. 3a,b) or via solid phase capture of Grp78 via an immobilized Ro52378−391 MAP peptide construct (tetravalent) followed by development with a Grp78 specific MoAb (Fig. 3c). The use of the MAP construct enhances the accessibility of the adsorbed peptide and was found to produce superior capture of Grp78 compared to the immobilized monomer (data not shown). In both cases the binding was specific to the Ro52378−391 peptide, as addition of La25–44 or La287–301 peptides (both monomer or MAP) failed to produce competitive binding in the gel shift assay or capture Grp78. Of interest, all three autoantigen-derived peptides were capable of binding to Hsp72 in a capture ELISA format (data not shown). An IC50 value of approximately 60 µm was observed in the gel shift assay, a value that falls within the range of peptides found to bind to Grp78 in previous studies [25,26].

image

Figure 3. Region of mRo52 associates with Grp78. A region of mRo52 (amino acids 378–391) was examined in a competition gel shift assay for the ability to dissociate Grp78 from a complex with a model substrate molecule [reduced and carboxy-methylated α-lactalbumin (RCMLA)]. Complexes of Grp78–RCMLA were incubated with graded concentrations of the competitor peptide (0–200 µm) for 2 h at 37°C and electrophoresed under native PAGE conditions. Grp78 was detected by immunoblot using a specific monoclonal antibody. (b) The intensity of bands in the immunoblot was assessed by laser scanning densitometry and the percentage competition plotted as a function of peptide concentration. An IC50 of 60 µm was derived for the mRo52 378–391 peptide, indicative of moderate to strong binding. (c) A capture ELISA showing specific capture of Grp78 by a multimeric Ro52 MAP 378–391 peptide construct (◆) compared to an irrelevant MAP from La 25–44 ( bsl00165) or 287–301 (not shown).

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Grp78 and Hsp70 but not Hsp90 stain a population of murine splenocytes

We hypothesized that the association of autoantigens with HSPs and subsequent induction of reciprocal spread immunity may reflect a generic function of HSPs in protecting the antigen from proteolysis and potentially targeting the antigen to APC. To test this hypothesis we examined whether Grp78, Hsp70 (a chaperone known to bind to APC) and Hsp90 (the constitutive form of Hsp90 which was not anticipated to bind to splenocytes) were capable of binding a population of murine splenocytes. In preliminary experiments, both Grp78 and Hsp70 bound to subpopulations of murine splenocytes under saturating concentrations of biotinylated HSP when visualized by extravidin–PE conjugate. Results are shown as contour plots with fluorescence associated with bound HSP on the y-axis and forward scatter on the x-axis (Fig. 4). Both forward scatter-high and a proportion of forward scatter-low splenocytes were stained by Hsp70 and Grp78. Further experiments aimed at characterizing these populations of splenocytes are currently under way.

image

Figure 4. Preliminary analysis of Grp78 surface binding to a population of murine splenocytes. Single cell suspensions of splenocytes prepared from the spleens of C3H/HeJ mice were incubated for 30 min with PBS (a) or 1 µg/ml of biotinylated Grp78 (b), Hsp70 (c) or Hsp90 (d). After three washes, the cells were incubated with streptavidin–phycoerythrin conjugate and cells examined by flow cytometry. Results are shown as PE fluorescence on the y-axis and forward scatter on the x-axis.

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DISCUSSION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Our previous studies have suggested a role for Grp78 immunity in the induction or maintenance of Ro autoimmunity. Here we provide further data that show biochemical evidence of an association between Grp78 and Ro52 and reciprocity in linked immunity observed between these two self-proteins. Ro52, Ro60 and Grp78 have been demonstrated previously to co-localize to small apoptotic blebs in UV irradiated cells [32]. Monoclonal antibodies specific for the Ro polypeptides and Grp78 stained these apoptotic bodies intensely, while La-specific antibodies did not. We have hypothesized previously that other endoplasmic reticulum (ER)-resident proteins such as calreticulin may also co-localize to similar structures, given our observation that Ro-immunized mice demonstrate immune spreading to calreticulin [20], and other ER-derived glycoproteins are also detectable in these apoptotic blebs. Grp78 and related polypeptides are expressed on the surface of apoptotic and tumour cells, suggesting potential for these proteins to also be translocated and co-localize with the Ro autoantigens [33,34]. Thus, we hypothesize that Grp78 and Ro polypeptides may co-localize during apoptosis or under other relevant physiological conditions. In addition to apoptosis as a source of co-localized Ro and Grp78 complexes we hypothesize that certain immunoglobulin (Ig) molecules may bridge Ro52 and Grp78. Ro52 has been shown to interact with certain subtypes of human Ig [35,36] both in vivo and in vitro. Grp78 was first characterized as an immunoglobulin heavy-chain binding protein [37], suggesting another potential association of Grp78 and Ro52 via bridging Ig molecules. This fits well with our model of determinant spreading in which Ig-producing B cells are the driving force behind recruitment of secondary autoantibody responses.

Consistent with the spreading data, Ro52 contains a dominant Grp78 binding motif, which when examined for association with Grp78 in vitro demonstrated good binding in both capture ELISA or competitive gel shift assays. This suggests that Ro52 can associate with Grp78 when these molecules co-localize to the same intracellular compartment [23]. Ro60 did not contain a dominant Grp78 binding motif but did contain several potential binding sequences, while La did not contain any potential Grp78 binding sequences [23]. Thus, it is possible that Grp78 also binds to Ro60 either directly or indirectly by binding to Ro60 associated Ro52 molecules. It is worthwhile noting that the Ro52 378–391 peptide contains a core of three tryptophan residues (ENGFWTIWLWQDSY), an amino acid residue particularly favoured for binding to Grp78 [25].

The potential association of Grp78 with the Ro polypeptides may provide an insight into the immunogenicity of these low abundance proteins. Studies pioneered by Srivastava and colleagues [38] have demonstrated that HSP-associated antigen is up to 400 times more immunogenic than antigen alone in the presentation of CD8-restricted T cell antigens to specific CTL. It is tempting to speculate that Grp78 may also enhance the immunogenicity of bound Ro polypeptides leading to preferential uptake and presentation of class II-restricted epitopes to CD4+ T-helper cells.

The mechanism by which Grp78 autoimmunity is triggered or initiated remains unclear. Grp78 is a highly abundant protein sequestered in ER. It shares very high amino acid identity to bacterial and other pathogen-derived homologues, and thus there exists the potential for cross-reactivity between responses towards immunodominant pathogenic HSP and endogenous Grp78. Triggering mechanisms such as viral infection or physiological stresses may change the expression and distribution of Grp78 and allow for the association of this molecule with both pathogen-derived molecules or self-proteins that normally do not intersect the ER-Golgi compartment. Our preliminary studies reveal a population of murine splenocytes that were bound by both Hsp70 and Grp78, suggesting that Grp78-associated material may be taken up preferentially by APC via an opsonin-like activity of Grp78. A number of candidate cell surface receptors could be involved in Grp78 binding, including CD91 [13,39] or surface Ig molecules.

We therefore propose a model (Fig. 5) whereby the intra- and extracellular pool of Grp78 can modulate immunity towards bound antigen. We postulate four pools of Grp78 complexes (quadrants of the inner square of Fig. 5), representing Grp78 bound to normal physiological ligands in the ER (I), free Grp78 which by virtue of its high homology to foreign Hsp 70 may be immunogenic (II), and Grp78 bound to non-physiological ligands in virally infected cells (III) or in apoptotic bodies (IV). In this model shaded quadrants represent potentially immunogenic Grp78 or Grp78 complexes. In our hypothetical model, ligands bound to Grp78 under normal physiological conditions in the ER do not produce an immune outcome due to sequestration of the complexes in the ER (I). The high level of homology between pathogen-derived and autologous HSPs suggests that a pool of cross-reactive T cell reactivity may exist (II), fuelling Grp78-mediated intermolecular help towards Ro-specific B cells, as we have elaborated in more detail elsewhere [18,23]. Similarly, the interaction of Grp78 with viral and other pathogen-derived glycoproteins in the ER may not only tag such molecules for immune responses but also, via determinant spreading mechanisms, expose Grp78 to an autoimmune cascade (III). For example, Grp78 is known to associate with measles virus glycoproteins and infection up-regulates a number or ER-resident chaperones [40,41]. Similar observations have been made for hepatitis B and C viral infections [42,43], herpes simplex virus type 1 infection [44] and rotavirus infection [45,46]. Many viral infections are also associated with apoptosis of the infected cell, thus one possible means of HSP–viral antigen complex exposure may occur during of cellular apoptosis (IV), where Grp78–antigen complexes may be concentrated in apoptotic blebs and taken up by APC [47–52] or released complexes may be taken up by receptor-mediated phagocytosis, as described recently for other HSPs [13].

image

Figure 5. Model of Grp78-mediated immunity. The intra- and extracellular pool of Grp78 can modulate immunity towards bound antigen. I. Under normal physiological conditions Grp78 is sequestered in the ER and does not become a target of CD4+ T cells. II. The high level of homology between Gp78 and pathogen-derived HSPs may fuel Grp78-mediated intermolecular help towards Ro-specific B cells. III. Grp78 interacts with viral and other pathogen-derived glycoproteins in the ER and becomes exposed to autoreactive environments. IV. Many of these infections are also associated with apoptosis of the infected cell. Grp78-Ro complexes may concentrate in apoptotic blebs and either the blebs taken up by directly by APC or the released complexes may be taken up by receptor mediated phagocytosis, facilitating the ensuing autoreactivity.

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Recent studies have highlighted a role for Grp78-specific autoimmunity in the pathogenesis of rheumatoid arthritis and related disorders. Between 30 and 60% of RA patients exhibited anti-Grp78 autoantibodies with similar levels of T cell autoreactivity [3,4]. In addition, autoreactivity towards calreticulin is also observed in RA [3]. Grp78 expression is also elevated in the synovium of RA patients [3], perhaps explaining the tissue-specific nature of the RA episodes. These studies validate our experimental approach in mice as a methodology to map targets of primary autoimmunity and recruited autoimmune responses and may provide clues to potential triggers of autoimmune disease.

ACKNOWLEDGEMENTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

This work was supported by grants from the NHMRC Australia and the Arthritis Foundation of Australia. A.W. Purcell is a C.R. Roper Fellow of the Faculty of Medicine, Dentistry and Health Science at the University of Melbourne.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
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