CD68-expressing cells can prime T cells and initiate autoimmune arthritis in the absence of reactive oxygen species.

It is widely believed that DC, but not macrophages, prime naïve T cells in vivo. Here, we investigated the ability of CD68-expressing cells (commonly defined as macrophages) in priming autoreactive T cells and initiating collagen-induced arthritis (CIA) in the mouse. For this purpose, a transgenic mouse was developed (MBQ mouse) where macrophages exclusively expressed the MHC class II H2-A(q) (A(q)) on an H2-A(p) (A(p)) background. A(q), but not A(p) expression mediates susceptibility to CIA through presentation of type II collagen (CII) to T cells. CIA severity is enhanced by a mutation in the Ncf1 gene, impairing reactive oxygen species (ROS) production by the phagocyte NADPH oxidase (NOX2) complex. Expression of functional Ncf1 on macrophages was previously shown to protect from severe CIA. To study the effect of ROS on macrophage-mediated priming of T cells, the Ncf1 mutation was introduced in the MBQ mouse. Upon CII immunization, Ncf1-mutated MBQ mice, but not Ncf1 wild-type MBQ mice nor Ncf1-mutated A(p) mice, activated autoreactive T cells and developed CIA. These findings demonstrate for the first time that macrophages can initiate arthritis and that the process is negatively regulated by ROS produced via the NOX2 complex.

Cartilage oligomeric matrix protein induction of chronic arthritis in mice.

OBJECTIVE: To develop a new mouse model for arthritis using cartilage oligomeric matrix protein (COMP) and to study the role of major histocompatibility complex (MHC) and Ncf1 genes in COMP-induced arthritis (COMPIA). METHODS: Native (pentameric) and denatured (monomeric) COMP purified from a rat chondrosarcoma was injected into mice with Freund's adjuvant to induce arthritis. C3H.NB, C3H.Q, B10.P, B10.Q, (B10.Q x DBA/1)F1, (BALB/c x B10.Q)F1, Ncf1 mutated, H-2Aq, H-2Ap, and human DR4+-transgenic mice were used. Anti-COMP antibodies and COMP levels in the immune sera were analyzed, and passive transfer of arthritis with purified immune sera was tested. RESULTS: Immunization with rat COMP induced a severe, chronic, relapsing arthritis, with a female preponderance, in the mice. The disease developed in C3H.NB mice, but not in B10.P mice, although they share the same MHC haplotype. Both H-2q and H-2p MHC haplotypes allowed the initiation of COMPIA. Using H-2Aq-transgenic and H-2Ap-transgenic mice, we demonstrated a role of both the Aq and Ep class II molecules in this model. Interestingly, the introduction of a mutation in the Ncf1 gene, which is responsible for the reduced oxidative burst phenotype, into the COMPIA-resistant B10.Q mouse strain rendered them highly susceptible to arthritis. In addition, the transfer of anti-COMP serum was found to induce arthritis in naive mice. Mice transgenic for the rheumatoid arthritis (RA)-associated DR4 molecule were found to be highly susceptible to COMPIA. CONCLUSION: Using rat COMP, we have developed a new and unique mouse model of chronic arthritis that resembles RA. This model will be useful as an appropriate and alternative model for studying the pathogenesis of RA.

Therapeutic vaccination of active arthritis with a glycosylated collagen type II peptide in complex with MHC class II molecules.

In both collagen-induced arthritis (CIA) and rheumatoid arthritis, T cells recognize a galactosylated peptide from type II collagen (CII). In this study, we demonstrate that the CII259-273 peptide, galactosylated at lysine 264, in complex with Aq molecules prevented development of CIA in mice and ameliorated chronic relapsing disease. In contrast, nonglycosylated CII259-273/Aq complexes had no such effect. CIA dependent on other MHC class II molecules (Ar/Er) was also down-regulated, indicating a bystander vaccination effect. T cells could transfer the amelioration of CIA, showing that the protection is an active process. Thus, a complex between MHC class II molecules and a posttranslationally modified peptide offers a new possibility for treatment of chronically active autoimmune inflammation such as rheumatoid arthritis.

The major T cell epitope on type II collagen is glycosylated in normal cartilage but modified by arthritis in both rats and humans.

Type II collagen (CII) is a target for autoreactive T cells in both rheumatoid arthritis and the murine model collagen-induced arthritis. The determinant core of CII has been identified as CII260-270, and the alteration of this T cell epitope by posttranslational modifications is known to be critical for development of arthritis in mice. Using CII-specific T cell hybridomas we have now shown that the immunodominant T cell epitope in the normal (healthy) human and rat joint cartilage is O-glycosylated at the critical T cell receptor recognition position 264 with a mono- or di-saccharide attached to a hydroxylysine. In contrast, in the arthritic human and rat joint cartilage there are both glycosylated and non-glycosylated CII forms. Glycosylated CII from normal cartilage could not be recognized by T cells reactive to peptides having only lysine or hydroxylysine at position 264, showing that antigen-presenting cells could not degrade the O-linked carbohydrate. Thus, the variable forms of the glycosylated epitope are determined by the structures present in cartilage, and these vary during the disease course. We conclude that the chondrocyte determines the structures presented to the immune system and that these structures are different in normal versus arthritic states.

Collagen type II (CII)-specific antibodies induce arthritis in the absence of T or B cells but the arthritis progression is enhanced by CII-reactive T cells.

Antibodies against type II collagen (anti-CII) are arthritogenic and have a crucial role in the initiation of collagen-induced arthritis. Here, we have determined the dependence of T and B cells in collagen-antibody-induced arthritis (CAIA) during different phases of arthritis. Mice deficient for B and/or T cells were susceptible to the CAIA, showing that the antibodies induce arthritis even in the absence of an adaptive immune system. To determine whether CII-reactive T cells could have a role in enhancing arthritis development at the effector level of arthritis pathogenesis, we established a T cell line reactive with CII. This T cell line was oligoclonal and responded to different post-translational forms of the major CII epitope at position 260-270 bound to the Aq class II molecule. Importantly, it cross-reacted with the mouse peptide although it is bound with lower affinity to the Aq molecule than the corresponding rat peptide. The T cell line could not induce clinical arthritis per se in Aq-expressing mice even if these mice expressed the major heterologous CII epitope in cartilage, as in the transgenic MMC (mutated mouse collagen) mouse. However, a combined treatment with anti-CII monoclonal antibodies and CII-reactive T cells enhanced the progression of severe arthritis.

Analysis of autoreactive T cells associated with murine collagen-induced arthritis using peptide-MHC multimers.

CD4(+) T cells that recognize residues 256-270 of type II collagen (CII) associated with the I-A(q) (A(q)) molecule play a central role in disease pathogenesis in murine collagen-induced arthritis (CIA). Disease is most efficiently induced by immunization with heterologous CII, which elicits heterologous, e.g. bovine, CII256-270:I-A(q)-specific T cells that only poorly cross-react with mouse CII. The self-epitope differs from heterologous CII256-270 by a conservative change of glutamic acid (heterologous) to aspartic acid (mouse) at position 266 which confers a lower affinity for binding to the I-A(q) molecule. To date, characterization of the nature of T cell recognition in this model has been hindered by the lack of suitable, labeled multimeric peptide-MHC class II complexes. Here, we describe the biochemical properties of both recombinant bovine CII256-270:I-A(q) (bCII256-270:I-A(q)) and mouse CII256-270:I-A(q) (mCII256-270:I-A(q)) complexes, and use these as fluorescently labeled multimers (tetramers) to characterize the specificity of CII-reactive T cells. Our analyses show that an unexpectedly high percentage of bCII256-270:I-A(q)-specific T cells are cross-reactive with mCII256-270:I-A(q). Interestingly, one T cell clone which has a relatively high avidity for binding to self-CII256-270:I-A(q) shows a marked increase in binding avidity at physiological temperature, indicating that this TCR has unusual thermodynamic properties. Taken together, our analyses suggest that the low affinity of mCII256-270 for I-A(q) may lead to a state of ignorance which can be overcome by priming CII-specific T cells with heterologous CII. This has relevance to understanding the mechanism by which CIA is induced and provides an explanation for the low arthritogenicity of mouse CII.

Basal metabolic rate and the evolution of the adaptive immune system.

Vertebrates have evolved an adaptive immune system in addition to the ancestral innate immune system. It is often assumed that a trade-off between costs and benefits of defence governs the evolution of immunological defence, but the costs and benefits specific to the adaptive immune system are poorly known. We used genetically engineered mice lacking lymphocytes (i.e. mice without adaptive, but with innate, immunity) as a model of the ancestral state in the evolution of the vertebrate immune system. To investigate if the magnitude of adaptive defence is constrained by the energetic costs of producing lymphocytes etc., we compared the basal metabolic rate of normal and lymphocyte-deficient mice. We found that lymphocyte-deficient mice had a higher basal metabolic rate than normal mice with both innate and adaptive immune defence. This suggests that the evolution of the adaptive immune system has not been constrained by energetic costs. Rather, it should have been favoured by the energy savings associated with a combination of innate and adaptive immune defence.