Cystatin C influences the autoimmune but not inflammatory response to cartilage type II collagen leading to chronic arthritis development

Introduction: Collagen-induced arthritis (CIA) is a mouse model for rheumatoid arthritis (RA) and is induced after immunization with type II collagen (CII). CIA, like RA, is an autoimmune disease leading to destruction of cartilage and joints, and both the priming and inflammatory phases have been suggested to be dependent on proteases. In particular, the cysteine proteases have been proposed to be detrimental to the arthritic process and even immunomodulatory. A natural inhibitor of cysteine proteases is cystatin C. Methods: Cystatin C-deficient, sufficient and heterozygous mice were tested for onset, incidence and severity of CIA. The effect of cystatin C-deficiency was further dissected by testing the inflammatory effector phase of CIA; that is, collagen antibody-induced arthritis model and priming phase, that is, T cell response both in vivo and in vitro. In addition, in order to determine the importance of T cells and antigen-presenting cells (APCs), these cell populations were separated and in vitro T cell responses determined in a mixed co-culture system. Finally, flow cytometry was used in order to further characterize cell populations in cystatin C-deficient mice. Results: Here, we show that mice lacking cystatin C, develop arthritis at a higher incidence and an earlier onset than wild-type controls. Interestingly, when the inflammatory phase of CIA was examined independently from immune priming then cystatin C-deficiency did not enhance the arthritis profile. However, in line with the enhanced CIA, there was an increased T cell and B cell response as delayed-type hypersensitivity reaction and anti-CII antibody titers were elevated in the cystatin C-deficient mice after immunization. In addition, the ex vivo naive APCs from cystatin C-deficient mice had a greater capacity to stimulate T cells. Interestingly, dendritic cells had a more activated phenotype in naive cystatin C-deficient mice. Conclusions: The lack of cystatin C enhances CIA and primarily affects in vivo priming of the immune system. Although the mechanism of this is still unknown, we show evidence for a more activated APC compartment, which would elevate the autoimmune response towards CII, thus resulting in an enhanced development of chronic arthritis

Dendritic cell presentation of type II collagen

Type II collagen (CII) is the main protein component of cartilage and immune recognition of CII plays a critical role for the development of collagen induced arthritis (CIA), a widely used animal model for rheumatoid arthritis (RA). Antigen presentation is an important requirement for the immune response, the more efficient presentation of the antigen the better tolerance will be induced and thereby protect against arthritis. Out of different professional antigen presenting cells (APCs), Langerhans cells (LCs), a subtype of dendritic cells, have been shown to poorly present CII compared to other antigens tested, but also compared to other APCs like B cells and macrophages. The inability to present CII is an exception from the rule that dendritic cells (DCs) are efficient in antigen presentation and subsequently in priming of naive T cells. This incompetence could however be overcome by treating with cysteine protease inhibitors, inhibiting certain cathepsins important for antigen degradation of the CII peptide in endosomes. Cystatin C, a natural occurring inhibitor of cathepsin S, is expressed in immature DCs. Cystatin C deficient mice were back-crossed into arthritis susceptible B10.Q mouse strain, surprisingly, with no difference in CII presentation between different genotypes and APCs in vitro. However, upon CII immunization, homozygous cystatin C deficient mice had a more severe arthritis ans disease incidence compared to wild-type littermates. The anti-CII antibody titers and delayed type hypersensitivity response (DTH) were also increased in the cystatin C deficient mice compared to the wild-type controls. The interpretation of our data favours antigen presentation being the targe cells for cystatin C activity. The present observation that the NOD.Q strain, which is resistant ti CIA in spite of expressing the arthritis susceptibel MHC class II H2-Aq allele, in fact have LCs which do present CII provide possibility to genetically dissect the phenomena. The major loci located on chromosome 2 and 13 were found to control LC presentation of CII. Of these two loci ine of them strongly correlated with CIA, an observation confirmed with the help of the Cia2 congenic mouse. Thus within this limited region there are one or several genes that affect LC CII presentaion and possibly these could be same as the genes controlling CIA

Cysteine proteases in Langerhans cells limits presentation of cartilage derived type II collagen for autoreactive T cells.

Development of type-II collagen (CII)-induced arthritis (CIA) is dependent on activation of CII-reactive T cells. Dendritic cells (DCs) are believed to play a crucial role in antigen-specific priming of T cells but it is still unclear how the CII-reactive T cells are primed since Langerhans cells (LCs) are poor antigen-presenting cells for CII. In the present study we show that LCs, treated with cysteine protease inhibitors, are able to process and present CII to T-cell hybridomas specific for the immune-dominant glycosylated 259–270 peptide bound to the MHC class II molecule Aq. Interestingly, the self (mouse) CII peptide could also now be efficiently presented. The poor presentation by LCs is a peptide-specific effect, since both bovine CII (bCII) (presenting a different peptide on H-2r) and ovalbumin could be efficiently presented, and blockage of cysteine proteases did not enhance antigen presentation. The enhanced CII-presentation by cysteine protease inhibition is seen mainly in LCs and not in antigen-primed B cells or macrophages. B cell and macrophage presentation of CII occur even without protease inhibition and are only to a minor extent influenced by cysteine protease inhibition. These data suggest that a LC deficiency in processing of the immune-dominant CII epitope in both CIA and RA may limit the exposure of this self-antigen to T cells, but that presentation can be overcome by modulation of the peptide proteolysis during CII processing

Structure-Immune Response Relationships of Hapten-Modified Collagen II Peptides in a T-Cell Model of Allergic Contact Dermatitis.

Allergic contact dermatitis (ACD) is mediated by T cells that specifically recognize hapten-modified peptides. T cells are known to recognize antigens as short processed peptides bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APC). It has previously been demonstrated that T cells can specifically recognize carbohydrates on the lysine at position 264 of the immunodominant (256-273) sequence from type II collagen (CII) and that such recognition is critical for the development of arthritis in mice and may play a role in rheumatoid arthritis in humans. In the present study, we have used this approach in modeling ACD, but instead of the carbohydrate, the strong sensitizer 2,4-dinitrofluorobenzene (DNFB) is bound to the epsilon-amine of the lysine at position 264. Specific T-cell hybridomas of this antigenic peptide, with dinitrophenyl (Dnp) on the epsilon-amine of lysine at position 264 (CIILysDnp 3), were established from mice immunized with CIILysDnp 3. In an immune response assay, these T-cell hybridomas were tested with a series of new synthetic hapten-modified peptides, all chemically identical except for the stereochemimistry ( d, l) and the length of the position-264 amino acid side chain bonding the hapten. The T-cell hybridomas recognized the CIILysDnp 3 peptide used for immunization; interestingly, they also recognized the CII peptide with a one-carbon-longer side chain (homolysine), CIIhLysDnp 6, and CIIAlaPipDnp 11, having a ring structure analogous to that of lysine with the same number of carbons in the bonding chain as in the CIILysDnp 3 peptide used for immunization. Dnp-modified CII peptides with a shorter bonding chain produced no immune response. These data demonstrate that the T-cell recognition of the Dnp-modified peptides is highly specific and moreover dependent on the length of the amino acid side chain that bonds the Dnp

Mannan induces ROS-regulated, IL-17A-dependent psoriasis arthritis-like disease in mice.

Psoriasis (Ps) and psoriasis arthritis (PsA) are poorly understood common diseases, induced by unknown environmental factors, affecting skin and articular joints. A single i.p. exposure to mannan from Saccharomyces cerevisiae induced an acute inflammation in inbred mouse strains resembling human Ps and PsA-like disease, whereas multiple injections induced a relapsing disease. Exacerbation of disease severity was observed in mice deficient for generation of reactive oxygen species (ROS). Interestingly, restoration of ROS production, specifically in macrophages, ameliorated both skin and joint disease. Neutralization of IL-17A, mainly produced by γδ T cells, completely blocked disease symptoms. Furthermore, mice depleted of granulocytes were resistant to disease development. In contrast, certain acute inflammatory mediators (C5, Fcγ receptor III, mast cells, and histamine) and adaptive immune players (αβ T and B cells) were redundant in disease induction. Hence, we propose that mannan-induced activation of macrophages leads to TNF-α secretion and stimulation of local γδ T cells secreting IL-17A. The combined action of activated macrophages and IL-17A produced in situ drives neutrophil infiltration in the epidermis and dermis of the skin, leading to disease manifestations. Thus, our finding suggests a new mechanism triggered by exposure to exogenous microbial components, such as mannan, that can induce and exacerbate Ps and PsA