Nucleic acid-stimulated antigen-presenting cells trigger T cells to induce disease in a rat transfer model of inflammatory arthritis

https://doi.org/10.1016/j.jaut.2011.02.007Get rights and content

Abstract

Autoimmune responses to heterogeneous nuclear ribonucleproteins (hnRNP) occur in many systemic autoimmune diseases, particularly in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus. In RA, humoral and/or cellular autoimmunity to hnRNP-A2/B1 is the most prominent anti-nuclear reactivity, being detectable in more than 50% of patients. However, its pathogenic role has not been fully elucidated yet. Here, we report that splenocytes from rats with pristane-induced arthritis transfer disease after in vitro restimulation with hnRNP-A/B antigens. Remarkably, disease transfer can be blocked by nuclease treatment of hnRNPs and is also achieved with splenocytes stimulated with hnRNP-A/B associated DNA or RNA oligonucleotides (ON) alone. Induction of proinflammatory cytokines in splenocytes stimulated with hnRNP-A/Bs or ONs involves Toll-like receptors (TLR) 7 and 9 but not TLR3. Furthermore, although T cells are the main mediators of disease transfer they require restimulation with TLR-activated antigen-presenting cells such as macrophages in order to become arthritogenic. Thus, the autoantigenic properties of hnRNPs appear to be mediated by their associated nucleic acids binding to TLR7 and 9. Our data explain the specific selection of hnRNP-A2/B1 as autoantigen in RA and reveal the requirement of interaction between innate and adaptive immunity to initiate and drive inflammation in autoimmune arthritis.

Introduction

Rheumatoid arthritis (RA) is a chronic autoinflammatory condition affecting approximately 1% of the world’s population and is characterized by a destructive inflammation of the joints, leading to progressive disability and reduced life expectancy. The RA synovial membrane is infiltrated by immune cells, predominantly macrophages, neutrophils and T cells, resulting in the chronic overproduction of proinflammatory cytokines and proteolytic enzymes that cause cartilage and bone degradation [1].

Genetic and serologic evidence in both RA and experimental models of arthritis suggests a pathogenic role for both innate and adaptive autoimmune processes. Toll-like receptors (TLRs), a family of evolutionarily conserved pattern recognition receptors, form a bridge between the innate and adaptive arms of the immune system, and have been suggested to be important factors in the development of RA. The 10 human TLRs identified to date can be classified into two distinct groups based on cellular distribution and ligand repertoire. Cell surface-expressed TLRs 1, 2, 4, 5, and 6 recognize ligands of bacterial and fungal origin, whereas TLRs 3, 7, 8, and 9 are expressed predominantly in the endosomal compartment and detect mainly viral nucleic acids. In addition to microbial products, TLRs are activated by a number of endogenous molecules that can be produced during tissue damage and have been detected in inflamed joints [2]. Examples of such molecules include heat shock proteins, hyaluronan, and high-mobility group box protein-1, but also endogenous RNA derived from necrotic cells that may activate cultured RA synovial fibroblasts (RASF) via TLR3 [3]. Since the expression of TLRs 2, 3, 4, and 7 has been reported in human RA tissue [4], [5], [6] the concept of endogenous ligand-driven activation of TLR signalling has raised interest in these receptors as potential candidates in the induction and/or maintenance of chronic inflammatory conditions such as RA.

Autoimmune reactivity to antigens that are associated with nucleic acids is a hallmark of many autoimmune disorders and is commonly found in diseases such as systemic lupus erythematosus (SLE), progressive systemic sclerosis, primary Sjögren's syndrome, and, though to a lesser extent, also RA. In RA, the most specific anti-nuclear reactivity is directed towards heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1, an abundant RNA- and DNA- binding protein that shows a predominantly nuclear localization and exerts multiple functions including the processing, transport and translation of mRNA [7], [8]. HnRNP-A2/B1 is targeted by autoantibodies and T cells of patients with RA, SLE, and mixed connective tissue disease [9], [10], [11], [12], [13]. Interestingly however, B cell epitope recognition was found to differ among the diseases [14], [15] and remarkable differences were observed between RA and SLE patients with respect to T cell reactivity [10], [11].

In a recent study we discovered hnRNP-A2/B1 to be an early B- and T cell-autoantigen in pristane-induced arthritis (PIA), a rat model of inflammatory arthritis. PIA closely mimics RA as it fulfills many of the clinical criteria of RA, including a symmetrical involvement of peripheral joints and the presence of rheumatoid factor, the destruction of cartilage and bone, and a chronic disease course [16]. Pronounced T cell reactivity against hnRNP-A2/B1 was seen already one week before disease onset while autoantibodies were detectable around onset with titres increasing during the acute phase, returning to normal levels thereafter. T cells reactive to hnRNP-A2/B1 were CD4 positive and responded to stimulation by hnRNP-A2/B1 with the expression of high amounts of IFN-γ and IL-17 [17], [18]. Unexpectedly, hnRNP-A2/B1 was also able to stimulate secretion of inflammatory cytokines in monocytes and macrophages derived from naïve (non pristane-primed) animals, that was dependent on the TLR adaptor molecule MyD88 [17].

In the current study we demonstrate that splenocytes from pristane-primed rats restimulated with hnRNP-A2/B1 or other closely related hnRNP proteins induce a highly inflammatory and erosive arthritis in naïve recipient rats that clinically and histologically resembles arthritis triggered by direct injection of pristane. Importantly, disease transfer can be inhibited by treatment of hnRNP-A/B with nucleases or chloroquine, an inhibitor of endosome acidification, and can also be achieved by stimulation of splenocytes with certain hnRNP-associated RNA- or DNA sequences alone, revealing that activation of endosomal TLRs is essential for disease transfer. Although arthritis is induced by T cells, transfer requires coincubation with TLR7 and/or TLR9-expressing antigen-presenting cells (APC). Thus, the arthritogenic properties of hnRNPs are based on cooperation between innate and adaptive immunity. Interestingly, macrophages appear to have higher capacities than dendritic cells for the induction of arthritogenicity in T cells. Our data significantly support the hypothesis of a pathogenic role of hnRNP-A/B proteins in erosive arthritis and suggest that autoimmunity to nucleic acid-associated autoantigens has the potential to contribute crucially to the development of diseases such as PIA and RA.

Section snippets

Animals

The immune response to hnRNP-A2/B1 has been previously shown to be particularly associated with the RT1f (MHC) haplotype on the DA background (DA.1F) [17]. All animals were therefore conducted with congenic DA.1F rats (originating from Zentralinstitut für Versuchstierzucht, Hannover, Germany). Rats were bred and maintained under Specific Pathogen Free conditions in the animal facility of the Institute of Biomedical Research, Medical University of Vienna, Austria. Rats were housed in groups of

Cells from pristane-primed rats restimulated with hnRNP-A2/B1 or other members of the hnRNP-A/B family transfer arthritis to naïve rats

HnRNP-A2 and its splice variant -B1 (hnRNP-A2/B1), also known as the RA33 autoantigen in RA and SLE [8] has recently been shown to be targeted by autoantibodies and Th1 cells of pristane-injected DA.1F rats [17]. The early response of the rats’ immune system already before the onset of clinical arthritis and the prominent early overexpression of the antigen in the inflamed joint argue for a direct involvement of hnRNP-A2/B1 in the pathogenesis of PIA. To further address this issue we used a CD4+

Discussion

Activation of TLRs by extracellularly released host-derived nucleic acids has been suggested to contribute to the pathogenesis of autoimmune or autoinflammatory diseases and is now considered a prime candidate target for new therapeutic interventions [29]. While there is already ample evidence about a direct involvement of these nucleic acids in lupus [23], [30], [31], [32], [33] and psoriasis [34], [35], their role has not been fully elucidated in inflammatory arthritis yet. Indirect evidence

Conclusion

In summary, our data provide new and exciting evidence for a pathogenic role of nucleic acids in pristane-induced arthritis, an animal model of arthritis very closely resembling human disease. Specific selection of hnRNP-A/B proteins as autoantigens in this model could therefore be due to the internal adjuvant properties of the associated RNAs and DNAs activating endosomal TLRs. Since approximately 50% of patients with RA and SLE also show autoreactivity to hnRNPs similar mechanisms may also be

Acknowledgements

We thank Elisabeth Höfler, Ruth Byrne, and Edith Pfeiffer for excellent technical assistance with ELISA, confocal microscopy, and irradiation of animals, respectively. We are grateful to Prof. Josef Smolen for helpful discussions and general support of the project. Isolation of cell populations by FACS was performed at the Core Unit Cell Sorting of the Medical University of Vienna. This project was supported from the EU Framework 6 Integrated Project AUTOCURE (LSHB-CT-2006-018661) and the EU

References (60)

  • M. Iwahashi et al.

    Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis

    Arthritis & Rheumatism

    (2004)
  • T.R. Radstake et al.

    Expression of toll-like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin-12 and interleukin-18 via interferon-gamma

    Arthritis & Rheumatism

    (2004)
  • A. Mayeda et al.

    Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins

    EMBO Journal

    (1994)
  • G. Steiner et al.

    Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 p.otein of the heterogeneous nuclear ribonucleoprotein complex

    Journal of Clinical Investigation

    (1992)
  • E.L. Greidinger et al.

    Human T cell clones specific for heterogeneous nuclear ribonucleoprotein A2 autoantigen from connective tissue disease patients assist in autoantibody production

    Arthritis & Rheumatism

    (2004)
  • R. Fritsch et al.

    Characterization of autoreactive T cells to the autoantigens heterogeneous nuclear ribonucleoprotein A2 (RA33) and filaggrin in patients with rheumatoid arthritis

    Journal of Immunology

    (2002)
  • R. Fritsch-Stork et al.

    The spliceosomal autoantigen heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) is a major T cell autoantigen in patients with systemic lupus erythematosus

    Arthritis Research & Therapy

    (2006)
  • G. Steiner et al.

    Autoantibodies to the A/B proteins of the heterogeneous nuclear ribonucleoprotein complex: novel tools for the diagnosis of rheumatic diseases

    International Archives of Allergy & Immunology

    (1996)
  • S. Trembleau et al.

    Immunodominant T-cell epitopes of hnRNP-A2 associated with disease activity in patients with rheumatoid arthritis

    European Journal of Immunology

    (2010)
  • K. Skriner et al.

    Anti-A2/RA33 autoantibodies are directed to the RNA binding region of the A2 p.otein of the heterogeneous nuclear ribonucleoprotein complex. Differential epitope recognition in rheumatoid arthritis, systemic lupus erythematosus, and mixed connective tissue disease

    Journal of Clinical Investigation

    (1997)
  • G. Schett et al.

    B cell epitopes of the heterogeneous nuclear ribonucleoprotein A2: identification of a new specific antibody marker for active lupus disease

    Annals of the Rheumatic Diseases

    (2009)
  • C. Vingsbo et al.

    Pristane-induced arthritis in rats: a new model for rheumatoid arthritis with a chronic disease course influenced by both major histocompatibility complex and non-major histocompatibility complex genes

    American Journal of Pathology

    (1996)
  • M.H. Hoffmann et al.

    The rheumatoid arthritis-associated autoantigen hnRNP-A2 (RA33) is a major stimulator of autoimmunity in rats with pristane-induced arthritis

    Journal of Immunology

    (2007)
  • M.H. Hoffmann et al.

    Immmunopathogenesis of rheumatoid arthritis. Induction of arthritogenic autoimmune responses by proinflammatory stimuli

    Annals of the New York Academy of Sciences

    (2009)
  • C. Ospelt et al.

    Overexpression of toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: toll-like receptor expression in early and longstanding arthritis

    Arthritis & Rheumatism

    (2008)
  • F.C. Arnett et al.

    The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis

    Arthritis & Rheumatism

    (1988)
  • M.H. Hoffmann et al.

    Gait changes precede overt arthritis and strongly correlate with symptoms and histopathological events in pristane-induced arthritis

    Arthritis Research & Therapy

    (2010)
  • J. Holmberg et al.

    Pristane, a non-antigenic adjuvant, induces MHC class II-restricted, arthritogenic T cells in the rat

    Journal of Immunology

    (2006)
  • C.M. Lau et al.

    RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement

    Journal of Experimental Medicine

    (2005)
  • J. Vollmer et al.

    Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8

    Journal of Experimental Medicine

    (2005)
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    Present address: Division of Medical Inflammation Research, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-171 77 Stockholm, Sweden. Tel.: +46 8 52487715; fax: +46 8 52487750.

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