Kutty Selva Nandakumar

Collagen type II-specific monoclonal antibody-induced arthritis in mice: description of the disease and the influence of age, sex, and genes.

Transfer of collagen type II (CII)-specific monoclonal antibodies induces an acute form of arthritis (collagen type II antibody-induced arthritis, CAIA) in naïve mice. Arthritis was induced using a pair of monoclonal antibodies M2139 and CIIC1, binding to J1 and C1(I) epitopes of CII, respectively. Thereafter, lipopolysaccharide injection was used to increase the incidence and severity of the disease. This model was used to investigate the effect of genes, age, and sex as well as effector cells in the end-stage effector phase of arthritis pathogenesis. Injection of a single monoclonal antibody induced arthritis only after lipopolysaccharide stimulation. CAIA showed differences in disease penetration among the susceptible strains indicating the importance of non-major histocompatibility complex genes on the antibody effector pathway. B-cell-deficient mice were susceptible to CAIA and in some genetic backgrounds B-cell deficiency leads to enhanced arthritis. Histology of the affected paws revealed massive infiltrations of neutrophils along with bone and cartilage erosion, pannus formation, and fibrin deposition. Depletion of neutrophils significantly reduced the incidence and severity of the disease. CAIA susceptibility increased with age. Males were more susceptible than females and estrogen treatment decreased the development of arthritis. We conclude that CAIA is an acute arthritis triggered by antibody binding and neutrophils bypassing immune activation but with many characteristics in common with collagen-induced arthritis.

Macrophages suppress T cell responses and arthritis development in mice by producing reactive oxygen species

Reduced capacity to produce ROS increases the severity of T cell-dependent arthritis in both mice and rats with polymorphisms in neutrophil cytosolic factor 1 (Ncf1) (p47phox). Since T cells cannot exert oxidative burst, we hypothesized that T cell responsiveness is downregulated by ROS produced by APCs. Macrophages have the highest burst capacity among APCs, so to study the effect of macrophage ROS on T cell activation, we developed transgenic mice expressing functional Ncf1 restricted to macrophages. Macrophage-restricted expression of functional Ncf1 restored arthritis resistance to the level of that of wild-type mice in a collagen-induced arthritis model but not in a T cell-independent anti-collagen antibody-induced arthritis model. T cell activation was downregulated and skewed toward Th2 in transgenic mice. In vitro, IL-2 production and T cell proliferation were suppressed by macrophage ROS, irrespective of T cell origin. IFN-gamma production, however, was independent of macrophage ROS but dependent on T cell origin. These effects were antigen dependent but not restricted to collagen type II. In conclusion, macrophage-derived ROS play a role in T cell selection, maturation, and differentiation, and also a suppressive role in T cell activation, and thereby mediate protection against autoimmune diseases like arthritis.

Induction of arthritis by single monoclonal IgG anti-collagen type II antibodies and enhancement of arthritis in mice lacking inhibitory FcγRIIB

IgG anti‐collagen type II (CII) antibodies (Ab) can induce arthritis in healthy mice. Here we have investigated if single monoclonal IgG anti‐CII Ab can induce arthritis in CIA‐susceptible DBA/1 mice and if there is an IgG subclass dependency. The involvement of Fc receptors for IgG (FcγR) in anti‐CII Ab‐mediated arthritis was also investigated by comparing the clinical outcome in DBA/1 mice to those in FcγR‐deficient mice. We demonstrate for the first time that single mAb to naive DBA/1 mice can induce persistent arthritis. Histology of the inflamed joints revealed massivecellular infiltrate and cartilage and bone destruction. All IgG subclasses tested (IgG1, IgG2a and IgG2b) were arthritogenic, with the IgG1 and IgG2b isotypes as the dominating arthritogenic Ab. Pathogenicity was dependent on engagement of activating FcγR, as FcRγ‐deficient mice were completely resistant to Ab‐mediated arthritis. The arthritis induced with the IgG1 and IgG2b Ab was also inhibited by FcγRIII disruption, whereas arthritis mediated by the IgG2a Ab was not substantially affected. The arthritic response of the IgG1 and IgG2b isotypes, but not of the IgG2a Ab, was further enhanced in mice lacking the inhibitory FcγRIIB. These results demonstrate that single IgG anti‐CII mAb can induce erosive arthritis and that IgG anti‐CII Ab mediate arthritis by engagement of FcγR.

Antibody-induced arthritis: disease mechanisms and genes involved at the effector phase of arthritis

During the development of rheumatoid arthritis (RA) autoantibodies to IgG-Fc, citrullinated proteins, collagen type II (CII), glucose 6 phosphoisomerase (G6PI) and some other self-antigens appear. Of these, a pathogenic effect of the anti-CII and anti-G6PI antibodies is well demonstrated using animal models. These new antibody mediated arthritis models have proven to be very useful for studies involved in understanding the molecular pathways of the induction of arthritis in joints. Both the complement and FcγR systems have been found to play essential roles. Neutrophils and macrophages are important inflammatory cells and the secretion of tumour necrosis factor-α and IL-1β is pathogenic. The identification of the genetic polymorphisms predisposing to arthritis is important for understanding the complexity of arthritis. Disease mechanisms and gene regions studied using the two antibody-induced arthritis mouse models (collagen antibody-induced arthritis and serum transfer-induced arthritis) are compared and discussed for their relevance in RA pathogenesis.

Complement activation by both classical and alternative pathways is critical for the effector phase of arthritis

To analyze the role of the classical and alternative pathways of complement activation in the effector phase of arthritis, we have induced arthritis in C3‐ and factor B (FB)‐deficient (C3–/– and FB–/–) DBA/1J mice using well‐defined monoclonal IgG2b and IgG2a antibodies to type II collagen. In control DBA/1J mice, severe swelling of the joints, destruction of cartilage and erosion of bone developed very rapidly with a 100% incidence and a peak on days 7–10. Although 75% of C3–/– mice developed arthritis, the clinical severity was very mild and the onset was delayed. Severity of arthritis in FB–/– mice ranked intermediate in comparison with C3–/– and control mice with an incidence of 100%. Immunohistochemical analysis of the inflamed joints demonstrated substantial reduction in macrophage and neutrophilic leukocyte infiltration in both C3–/– and FB–/– mice, thereby confirming the clinical findings. We conclude that both the classical and the alternative pathways of complement activation are involved in the effector phase of arthritis.

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.

IL-10-Deficient B10.Q Mice Develop More Severe Collagen-Induced Arthritis, but Are Protected from Arthritis Induced with Anti-Type II Collagen Antibodies

IL-10 is a pleiotropic cytokine with stimulatory and inhibitory properties, and is thought to have a protective role in rheumatoid arthritis and collagen-induced arthritis (CIA). In this study, we investigated how IL-10 deficiency affects CIA and anti-collagen type II (CII) Ab-transferred arthritis in C57BL/10.Q (B10.Q) mice. The B10.Q.IL-10(-/-) mice had an 8-cM 129/Ola fragment around the IL-10 gene. The mice were treated with antibiotics, appeared healthy, and had no colitis. T cells from IL-10(-/-) mice expressed similar levels of IFN-gamma, IL-2, and IL-4 after mitogen stimulation; however, macrophages showed a reduced TNF-alpha production compared with IL-10(+/-) littermates. IL-10(-/-) mice had an increased incidence, and a more severe CIA disease than the IL-10(+/-) littermates. To study the role of IL-10 in T cell tolerance, IL-10(-/-) were crossed into mice carrying the immunodominant epitope, CII(256-270), in cartilage (MMC) or in skin (TSC). Both IL-10(-/-) and IL-10(+/-) MMC and TSC mice were completely tolerized against CIA, indicating that lack of IL-10 in this context did not break tolerance. To investigate whether IL-10 was important in the effector phase of CIA, arthritis was induced with anti-CII Abs. Surprisingly, IL-10(-/-) were less susceptible to Ab-transferred arthritis, as only 30% showed signs of disease compared with 90% of the littermates. Therefore, IL-10 seemed to have a protective role in CIA, but seemed to exacerbate the arthritogenicity of anti-CII Abs. These data emphasize the importance of studying IL-10 in a defined genetic context in vivo, to understand its role in a complex disease like arthritis.

Endoglycosidase treatment abrogates IgG arthritogenicity: Importance of IgG glycosylation in arthritis.

The glycosylation status of IgG has been implicated in the pathology of rheumatoid arthritis. Earlier, we reported the identification of a novel secreted endo‐β‐N‐acetylglucosaminidase (EndoS), secreted by Streptococcus pyogenes that specifically hydrolyzes the β‐1,4‐di‐N‐acetylchitobiose core of the asparagine‐linked glycan of human IgG. Here, we analyzed the arthritogenicity of EndoS‐treated collagen type II (CII)‐specific mouse mAb in vivo. Endoglycosidase treatment of the antibodies inhibited the induction of arthritis in (BALB/c × B10.Q) F1 mice and induced a milder arthritis in B10.RIII mice as compared with the severe arthritis induced by non‐treated antibodies. Furthermore, EndoS treatment did not affect the binding of IgG to CII and their ability to activate complement, but it resulted in reduced IgG binding to FcγR and disturbed the formation of stable immune complexes. Hence, the asparagine‐linked glycan on IgG plays a crucial role in the development of arthritis.

Ethanol prevents development of destructive arthritis

Environmental factors are thought to play a major role in the development of rheumatoid arthritis. Because the use of ethanol is widespread, we assessed the role of ethanol intake on the propensity to develop chronic arthritis. Collagen type II-immunized mice were given water or water containing 10% (vol/vol) ethanol or its metabolite acetaldehyde. Their development of arthritis was assessed, as well as the impact of ethanol on leukocyte migration and activation of intracellular transcription factors. Mice exposed daily to this dose of ethanol did not display any liver toxicity, and the development of erosive arthritis was almost totally abrogated. In contrast, the antibody-mediated effector phase of collagen-induced arthritis was not influenced by ethanol exposure. Also, the major ethanol metabolite, acetaldehyde, prevented the development of arthritis. This antiinflammatory and antidestructive property of ethanol was mediated by (i) down-regulation of leukocyte migration and (ii) up-regulation of testosterone secretion, with the latter leading to decreased NF-κB activation. We conclude that low but persistent ethanol consumption delays the onset and halts the progression of collagen-induced arthritis by interaction with innate immune responsiveness.

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.