Mikael Vestberg

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.

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.

The structural basis of MHC control of collagen-induced arthritis; binding of the immunodominant type II collagen 256 – 270 glycopeptide to H-2Aq and H-2Ap molecules

The Aq major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen‐induced arthritis (CIA), whereas the closely related H‐2Ap molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H‐2Aq and H‐2Ap molecules (referred to as Aq and Ap) to bind and present collagen type II (CII)‐derived glycosylated and non‐glycosylated peptides. T cell clones specific for the immunodominant CII 256 – 270 peptide and restricted to both Aq and Ap molecules were identified. When these clones were incubated with CII protein and either Aq‐ or Ap‐expressing antigen‐presenting cells (APC), only Aq‐expressing APC were able to induce stimulation. With the use of Aβ transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC‐ or non‐MHC genes. Peptide binding studies were performed using affinity‐purified MHC class II molecules. The CII 256 – 270 peptide bound with lower affinity to the Ap molecule than to the Aq molecule. Using a set of alanine‐substituted CII 256 – 270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the Aq and Ap molecule, i.  e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslotionally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256 – 270 peptide did not change the anchor positions used for binding to the Aq or Ap molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the Aq molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256 – 270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II‐associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.

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.

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.

Influence of CD4 or CD8 deficiency on collagen-induced arthritis

The role of T cells in the mouse collagen‐induced arthritis (CIA) model for rheumatoid arthritis is not clarified, and different results have been reported concerning the role of CD4 and CD8 T cells. To address this issue, we have investigated B10.Q mice deficient for CD4 or CD8. The mice lacking CD4 were found to be less susceptible to disease, but not completely resistant, whereas the CD8 deficiency had no significant impact on the disease. No difference in the development of late occurring relapses was noted. Interestingly, the CD4‐deficient mice had a severely reduced response to the glycosylated form of the immunodominant type II collagen (CII) 256–270 peptide whereas the response to the non‐glycosylated peptide was not significantly different. Furthermore, CD4‐deficient mice had lower antibody responses to CII, explaining the lower disease susceptibility. In comparison with previously reported results, it is apparent that the lack of CD4 molecules has a different impact on CIA if present on different genetic backgrounds, findings that could possibly be related to the occurrence of different disease pathways of CIA in different mouse strains.

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 Freunds 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.

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‐Aq (Aq) on an H2‐Ap (Ap) background. Aq, but not Ap 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 Ap 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.

The H2-Ab gene influences the severity of experimental allergic encephalomyelitis induced by proteolipoprotein peptide 103-116

Immunization of H2(p) and H2(q) congenic C3H mouse strains with the PLP 103-116 peptide elicited two distinct experimental allergic encephalomyelitis (EAE) disease courses. C3H.Q (H2(q)) mice developed an acute-phase disease with classical ascending paralytic signs whereas C3H.NB (H2(p)) developed a highly variable disease course with symptoms originating from CNS above the spinal chord. C3H.Q lacks functional H2-E molecules and share H2-Aalpha with C3H.NB. To examine if the differences found at positions 85, 86, 88, and 89 in the Abeta-chains account for disease susceptibility, H2(q) mice were made transgenic with the Ab(p) gene. The Ab(p)-transgenic mice on the C3H.Q background developed a more severe disease course, demonstrating the importance of class II. However, the onset was not affected and the disease showed a classical ascending paralysis similar to the C3H.Q suggesting that the observed brain symptoms were related to nonclass II genes. Inhibition studies performed on affinity purified MHC class II molecules indicated that the PLP 103-116 peptide bound to A(p) with slightly higher affinity than to A(q). Both A(q) and A(p) formed long-lived stable complexes (t(1/2)>24 h) with the PLP 103-116 peptide, but a higher amount of the peptide was loaded on to A(p) compared with A(q). An F2 gene segregation experiment, in which the low PLP 103-116 binding A(r) molecule and the high binding A(p) molecule could be compared for the influence on the disease susceptibility, indicated a role for both peptide binding affinity and non-MHC genes. Based on our results, we conclude that the H2-Ab gene controls severity of EAE but not necessarily the onset or type of disease course and that affinity of the disease-promoting peptide for the class II molecule is a critical pathogenic factor.

Primed B cells present type-II collagen to T cells.

Development of type‐II collagen (CII)‐induced arthritis (CIA) is dependent on a T‐cell mediated activation of autoreactive B cells. However, it is still unclear if B cells can present CII to T cells. To investigate the role of B cells as antigen‐presenting cells (APCs) for CII, we purified B cells from lymph nodes of immunized and nonimmunized mice. These B cells were used as APC for antigen‐specific T‐cell hybridomas. B cells from naïve mice did present native, triple‐helical, CII (nCII) but also ovalbumin (OVA) and denatured CII (dCII) to antigen‐specific T‐cell hybridomas. In addition, B cells primed with nCII or OVA, but not dCII, activated the antigen‐specific T‐cell hybridomas two to three times better than naïve B cells. We conclude that antigen‐primed B cells have the capacity to process and present CII to primed T cells, and antigen‐primed antigen‐specific B cells are more efficient as APC than naïve B cells. We further conclude that B cells have the potential to play an important role as APC in the development of CIA.