Original Contribution
Modification of lupus-associated 60-kDa Ro protein with the lipid oxidation product 4-hydroxy-2-nonenal increases antigenicity and facilitates epitope spreading

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Abstract

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. The Ro ribonucleoprotein particle, composed of a 60-kDa protein noncovalently associated with human cytoplasmic RNA, is the target of antibodies in 25–40% of lupus patients. Purified human 60-kDa Ro was found to be oxidatively modified. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore we hypothesized that oxidation by-products, such as 4-hydroxy-2-nonenal (HNE), could lead to neoantigens like HNE-modified 60-kDa Ro, which could in turn initiate autoimmunity or drive epitope spreading. To test this hypothesis we immunized rabbits with either HNE-modified 60-kDa Ro or the unmodified Ro. Intramolecular epitope spreading within the Ro molecule and intermolecular epitope spreading to La, double-stranded DNA, nRNP, and Sm occurred preferentially in HNE–Ro-immunized animals. Nonspecific anti-HNE antibody, generated by immunization with HNE–keyhole limpet hemocyanin conjugate, did not significantly bind to these autoantigens. These data may suggest a hitherto unappreciated mechanism by which oxidative stress facilitates epitope spreading in SLE.

Introduction

Systemic lupus erythematosus (SLE) is associated with the production of antibodies to self-constituents, particularly targeting certain specific ribonucleoprotein (RNP) particles [reviewed in 1]. Among these is the Ro RNP, composed of a 60-kDa protein (60-kDa Ro or SS-A) that is noncovalently associated with at least one of four short uridine-rich RNAs (the hY RNAs) [2], [3]. The hY RNAs are associated with the La or SS-B (48 kDa) autoantigen, at least transiently. Anti-Ro is found in up to 50% of patients with SLE, whereas anti-La is found in substantially fewer patients [4], [5]. Anti-Ro is associated with subacute cutaneous lupus, photosensitive skin rash, deficiency of early complement components, renal disease, neonatal lupus, lymphopenia, and neutropenia [6], [7], [8]. Anti-Ro is most likely the result of the interaction of HLA-DQα, HLA-DQβ, and the T cell receptor β [9], [10], [11], [12], [13], [14].

The mechanism by which tolerance is lost in patients with SLE, such that the Ro RNP complex becomes targeted, is unknown. Studies have shown that if tolerance is broken to one component of an in vivo complex, the immune response can then generalize and expand, so that an entire complex is no longer recognized as self by the immune system [15], [16], [17], [18]. This acquisition of new autoreactivity during the course of disease is known as epitope spreading. Intramolecular epitope spreading occurs when the antigen-specific autoimmune response spreads to different epitopes on the one protein. Intermolecular epitope spreading occurs when the response spreads to epitopes on other structural/functional proteins.

Tolerance to self is maintained by removal of self-reactive lymphocytes in the thymus during the maturation of the immune system [19], [20] and by making self-reactive T lymphocytes anergic in the periphery [21], [22]. Autoimmunity results when tolerance to self is broken. This results in the appearance of autoreactive lymphocytes. Three types of autoimmune responses have been described, namely B cell dominant (autoimmune hemolytic anemia and myasthenia gravis, for example), T cell dominant (experimental autoimmune encephalomyelitis, insulin-dependent diabetes mellitus, and collagen-induced arthritis are examples), and combinational types [23]. SLE arises from the emergence of both autoreactive T and B cells with an etiology that involves both genetic and environmental factors. Molecular mimicry of viral or bacterial antigens with self-determinants has been proposed as one of the pathogenic mechanisms for the appearance of autoreactive cells [24], [25]. The diversification and amplification of autoimmunity in an individual might result from epitope spreading, which has been described as an important factor. The concept of epitope spreading was first described in experimental autoimmune encephalomyelitis [26] and the concept has been extended to other autoimmune diseases [16], [17]. Several investigations based on immunization of nonautoimmune mice with self-peptides support the view that the highly diverse B and T cell autoimmune responses in SLE might originate from a single protein or even a single cryptic self-epitope without the need for foreign pathogens or molecular mimics [16], [27], [28], [29].

Free radical-mediated damage has been implicated in the pathogenesis of SLE and other diseases [30], [31], [32], [33], [34], [35]. Reactive lipid peroxidation products have been shown to form adducts with lysine, histidine, and cysteine targets [36], [37], [38], [39], [40]. One of the most common and reactive lipid oxidation products is 4-hydroxy-2-nonenal (HNE) [41]. Increased levels of HNE-modified proteins have been detected in the sera of children with autoimmune diseases [32]. HNE–protein adducts are potential neoantigens and so could be involved in the pathogenesis of autoimmune diseases.

We hypothesized that oxidative by-products, like HNE, would cross-link with 60-kDa Ro and help initiate autoimmunity. To test this hypothesis we immunized rabbits with either HNE-modified Ro or unmodified Ro and found that autoimmunity was established faster and more vigorously in the animals that were immunized with the modified Ro. Specific, rapid intra- and intermolecular epitope spreading occurred when animals were immunized with the HNE-modified Ro but not when immunized with either unmodified Ro or HNE–keyhole limpet hemocyanin (KLH).

Section snippets

Chemicals

4-Hydroxy-2-nonenal was from Cayman Scientific (Ann Arbor, MI, USA), immunofluorescent anti-nDNA test kits were from Helix Diagnostics (Sacramento, CA, USA) and ANA test kits were from Inova Diagnostics (San Diego, CA, USA). Fluorescein-conjugated anti-rabbit IgG was from Jackson Laboratories (Bar Harbor, ME, USA). All other chemicals were of reagent grade.

60-kDa Ro antigen

Human 60-kDa Ro [42], [43], [44] was purified from human liver and spleen obtained after autopsy from two patients as described for

Results

We have observed increased oxidative damage in lupus patients, in a previous study [50]. Building on these findings, we sought to determine whether purified 60-kDa Ro from a human source (tissue obtained at autopsy) was a target of oxidative modification in vivo. Human 60-kDa Ro was found to be HNE modified (Fig. 1), thus implying the potential for 60-kDa Ro to be a target for oxidative modification in SLE patients.

Therefore, we embarked on modifying purified bovine 60-kDa Ro with HNE in vitro

Discussion

This work describes a novel oxidative mechanism by which epitope spreading could occur. Oxidative modification of SLE-associated proteins such as 60-kDa Ro might result in the formation of chemical adducts which could serve as neoantigens that the immune system has probably not been exposed to. This modified Ro might be more readily internalized, on account of its neoconformation, than the unmodified Ro, by antigen-presenting cells, such as dendritic cells or macrophages. These in turn present

Acknowledgments

This work was supported by NIH Grant ARO1844 to R.H.S., funds from the Oklahoma Center for the Advancement of Science and Technology to R.H.S. and B.T.K., and HR02-149R to K.H.

References (59)

  • B.T. Kurien et al.

    Heat mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes

    J. Immunol. Methods

    (2002)
  • B.T. Kurien et al.

    Free radical mediated peroxidative damage in systemic lupus erythematosus

    Life Sci.

    (2003)
  • H. Itabe et al.

    Appearance of cross linked proteins in human atheroma and rat pre-fibrotic liver detected by a new monoclonal antibody

    Biochim. Biophys. Acta

    (1998)
  • E. Mottaran et al.

    Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease

    Free Radic. Biol. Med.

    (2002)
  • J.B. Harley et al.

    Systemic lupus erythematosus: RNA–protein autoantigens, models of disease heterogeneity, and theories of etiology

    J. Clin. Immunol.

    (1991)
  • C.A. O'Brien et al.

    A possible role for the 60-kDa Ro autoantigen in a discard pathway for defective 5S rRNA precursors

    Genes Dev.

    (1994)
  • H. Yamagata et al.

    Molecular properties of the Ro/SSA antigen and ELISA for quantitation of antibody

    J. Clin. Invest.

    (1984)
  • J.B. Harley et al.

    Anti-La/SSB antibody is present in some normals and is coincident with anti-Ro/SSA precipitins in systemic lupus erythematosus

    J. Rheumatol.

    (1984)
  • J.B. Harley et al.

    A model for disease heterogeneity in systemic lupus erythematosus: relationships between histocompatibility antigens, autoantibodies and lymphopenia or renal disease

    Arthritis Rheum.

    (1989)
  • B.T. Kurien et al.

    Anti-Ro mediates neutropenia in systemic lupus erythematosus by binding a 60,000 MW antigen on the surface of neutrophils

    Clin. Exp. Immunol.

    (2000)
  • A. Fujisaku et al.

    HLA-DQ gene complement and other histocompatibility relationships in man with the anti-Ro/SSA autoantibody response in systemic lupus erythematosus

    J. Clin. Invest.

    (1989)
  • M.B. Frank et al.

    Anti-Ro (SSA) autoantibodies are associated with T cell receptor beta genes in systemic lupus erythematosus patients

    J. Clin. Invest.

    (1990)
  • R.H. Scofield et al.

    Immunogenetics of epitopes of the carboxyl terminus of the human 60-kDa Ro autoantigen

    Clin. Exp. Immunol.

    (1995)
  • J.D. Reveille et al.

    Specific amino acid residues in the second hypervariable region of HLA-DQA1 and DQB1 chain genes promote the Ro (SS-A)/La (SS-B) autoantibody responses

    J. Immunol.

    (1991)
  • R.H. Scofield et al.

    Association of anti-Ro (SS-A) autoantibodies with glutamine in position 34 of DQA1 and leucine in position 26 of DQB1

    Arthritis Rheum.

    (1994)
  • R.H. Scofield et al.

    Immunization with short peptides derived from the systemic lupus erythematosus associated 60 kDa Ro autoantigen results in autoimmunity directed towards the Ro ribonucleoprotein

    J. Immunol.

    (1996)
  • R.H. Scofield et al.

    Immunization of mouse with 60 kDa Ro peptides results in epitope spreading if peptides are highly homologous between man and mouse

    Arthritis Rheum.

    (1999)
  • J.A. James et al.

    Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B′-derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity

    J. Exp. Med.

    (1995)
  • F. Topfer et al.

    Intra- and intermolecular spreading of autoimmunity involving the nuclear self antigens La (SS-B) and Ro (SS-A)

    Proc. Natl. Acad. Sci. USA

    (1995)
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