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Like rheumatoid arthritis (RA) in humans, collagen-induced arthritis (CIA) in mice is associated with not only MHC class II genetic polymorphism but also, to some extent, with other loci including genes encoding Fc gamma receptors (FCGRs) and complement C5. In this study, we used a cartilage antibody-induced arthritis (CAIA) model in which arthritis develops within a 12-h timeframe, to determine the relative importance of FCGRs and C5 (Hc). In CAIA, inhibiting or deleting FCGR3 substantially hindered arthritis development, underscoring the crucial role of this receptor. Blocking FCGR3 also reduced the levels of FCGR4, and vice versa. When employing an IgG1 arthritogenic cocktail that exclusively interacts with FCGR2B and FCGR3, joint inflammation was promptly initiated in Fcgr2b --  mice but not in Fcgr3 --  mice, suggesting that FCGR3 is sufficient for CAIA development. Regarding complement activation, Fcgr2b ++ .Hc ** mice with C5 mutated were fully resistant to CAIA, whereas Fcgr2b -- .Hc ** mice developed arthritis rapidly. We conclude that FCGR3 is essential and sufficient for CAIA development, particularly when induced by IgG1 antibodies. The human ortholog of mouse FCGR3, FCGR2A, may be associated with RA pathogenesis. FCGR2B deficiency allows for rapid arthritis progression and overrides the resistance conferred by C5 deficiency., (© 2024 The Authors. European Journal of Immunology published by Wiley‐VCH GmbH.)

A haplotype with tightly linked Fc gamma receptor (FcγR) genes is known as a major locus controlling immune responses and autoimmune diseases, including arthritis. Here, we split a congenic fragment derived from the NOD mouse (Cia9) to study its effect on immune response and arthritis in mice. We found that arthritis susceptibility was indeed controlled by the FcγR gene cluster and a recombination between the FcγR2b and FcγR3 loci gave us the opportunity to separately study their impact. We identified the NOD-derived FcγR2b and FcγR3 alleles as disease-promoting for arthritis development without impact on antibody secretion. We further found that macrophage-mediated phagocytosis was directly correlated to FcγR3 expression in the congenic mice. In conclusion, we positioned FcγR2b and FcγR3 alleles as disease regulatory and showed that their genetic polymorphisms independently and additively control innate immune cell activation and arthritis., (© 2020 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polymorphisms in the MHC class II (MHCII) genes are strongly associated with rheumatoid arthritis, supporting the importance of autoreactive T helper (Th) cells for the development of this disease. Here, we used pristane-induced arthritis (PIA), induced by the non-antigenic hydrocarbon pristane, to study the impact of different MHCII alleles on T-cell activation and differentiation. In MHCII-congenic rats with disease-promoting MHCII alleles, pristane primarily induced activation of Th1 cells, whereas activated T cells were Th17 biased in rats with protective MHCII alleles. Neutralization of IFN-γ during T-cell activation abrogated the development of disease, suggesting that Th1 immunity is important for disease induction. Neutralization of IL-17, by contrast, suppressed arthritis only when performed in rats with established disease. Adoptive T-cell transfers showed that T cells acquired arthritogenic capacity earlier in strains with a prevailing Th1 response. Moreover, upon pristane injection, these strains exhibited more Ag-primed OX40+ and proliferating T cells of polyclonal origin. These data show that T cells are polarized upon the first encounter with peptide-MHCII complexes in an allele-dependent fashion. In PIA, the polyclonal expansion of autoreactive Th1 cells was necessary for the onset of arthritis, while IL-17 mediated immunity contributed to the progression to chronic disease., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Autoantibody-mediated inflammation contributes to the development of rheumatoid arthritis (RA), and anti-type II collagen (CII) antibodies are present in the serum, synovial fluid, and cartilage of RA patients. We had previously generated and characterized knock-in mice expressing a germline-encoded, CII-specific IgH (B10Q.ACB), which demonstrated positive selection of self-reactive B cells. Here, we show that despite the spontaneous production of CII-specific autoantibodies, B10Q.ACB mice are protected from collagen-induced arthritis. Introducing a mutation in the Ncf1 gene, leading to ROS deficiency, breaks this strong arthritis resistance. Disease development in Ncf1-mutated B10Q.ACB mice is associated with an enhanced germinal center formation but without somatic mutations of the auto-reactive B cells, increased T-cell responses and intramolecular epitope-spreading. Thus, ROS-mediated B-cell tolerance to a self-antigen could operate by limiting the expansion of the auto-reactive B-cell repertoire, which has important implications for the understanding of epitope spreading phenomena in rheumatoid arthritis and other autoimmune diseases., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The NADPH oxidase 2 (NOX2) complex is responsible for the production of ROS in phagocytic cells. Genetic defects in NOX2 lead to opportunistic infections and inflammatory manifestations such as granulomas in humans, also known as chronic granulomatous disease (CGD). This condition is mirrored in mice with defective ROS production and interestingly both species are predisposed to autoimmune diseases. An unresolved question is whether the hyper-inflammation and tendency to develop autoimmunity are secondary to the increased infections, or whether these are parallel phenomena. We generated germ-free ROS deficient Ncf1 mutant mice that when reared in specific pathogen-free condition, are highly susceptible to collagen-induced arthritis compared with wild-type mice. Strikingly, arthritis incidence and severity was almost identical in germ-free and specific pathogen-free ROS-deficient mice. In addition, partial reduction of the microbial flora by antibiotics treatment did not alter the disease course. Taken together, this shows that ROS has a clear immune regulatory function that is decoupled from its function in host defence., (© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Extensive genome-wide association studies have recently shed some light on the causes of chronic autoimmune diseases and have confirmed a central role of the adaptive immune system. Moreover, better diagnostics using disease-associated autoantibodies have been developed, and treatment has improved through the development of biologicals with precise molecular targets. Here, we use rheumatoid arthritis (RA) as a prototype for chronic autoimmune disease to propose that the pathogenesis of autoimmune diseases could be divided into three discrete stages. First, yet unknown environmental challenges seem to activate innate immunity thereby providing an adjuvant signal for the induction of adaptive immune responses that lead to the production of autoantibodies and determine the subsequent disease development. Second, a joint-specific inflammatory reaction occurs. This inflammatory reaction might be clinically diagnosed as the earliest signs of the disease. Third, inflammation is converted to a chronic process leading to tissue destruction and remodeling. In this review, we discuss the stages involved in RA pathogenesis and the experimental approaches, mainly involving animal models that can be used to investigate each disease stage. Although we focus on RA, it is possible that a similar stepwise development of disease also occurs in other chronic autoimmune settings such as multiple sclerosis (MS), type 1 diabetes, and systemic lupus erythematosus., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Copy number variation, namely regions of the genome that can be either deleted or duplicated in a variable way, has emerged as an important source of genetic variance in the human genome. Genes with immunological functions are particularly prone to copy number variation, in part because this is a mechanism to expand the recognition repertoire; however, immunological genes not directly involved in immune recognition are also copy number variable but, despite the link between immunological function and copy number variation, very few copy number variants (CNVs) have been found to be associated with autoimmune diseases, even in recent large genome-wide CNV-association studies. Nonetheless, CNVs in FCGR3B, DEFB4, CCL3L1, C4A/B and NCF1 have been suggested to be associated with autoimmune diseases, although there is conflicting evidence in all cases. The reasons for the lack of definitive data on CNV-autoimmunity associations, as well as the technical challenges for the field are the focus of this review., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The dramatic increase in the amount of research data being produced necessarily leads to higher demands on statistical thresholds and on experimental planning. This is to avoid positive selection of multiple tested data. Here we would like to highlight the need for including littermate controls in animal experiments, in particular when genetically modified strains are analysed for quantitative phenotypes. Thus, this suggestion will have impact on most immunological experiments performed today., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The rat model is a widely used animal model in research, its popularity perhaps only surpassed by the mouse model. Rats are the preferred models for the study of transplantation and certain tumor and autoimmune diseases. In particular, the understanding of cardiovascular-related diseases and chronic inflammation has depended widely on the rat, from which models for both multiple sclerosis and rheumatoid arthritis have been derived. Until now, research in the rat has been hampered by a lack of precise gene-targeting technology in this model. This limitation, however, is rapidly changing; a recent example is the availability of B-cell-deficient rat strains obtained by the newly established zinc finger nucleases-targeting technology. As described in this issue of the European Journal of Immunology, genetic targeting of the rat, for example, using zinc finger nucleases-targeting technology, is likely to rapidly drive progress in the understanding of not only B-cell biology but also in the general understanding of rat disease models.

The calcium-dependent enzyme tissue transglutaminase (tTG) is associated with diverse biological functions, such as induction of apoptosis, modeling of the extracellular matrix, receptor-mediated endocytosis, cell growth and differentiation, cell adhesion and signal transduction. Also, it may deamidate glutamine residues to glutamic acid and catalyze cross-linking of proteins. In this study, we have investigated the impact of tTG for posttranslational modifications and cross-linking of the immunodominant T-cell epitope CII260-270 and their effects on the collagen-induced arthritis, an animal model for rheumatoid arthritis. By using mass spectrometry analysis and hybridoma assays, we have demonstrated that tTG could perform both types of modifications (deamidation and cross-link formation) on the immunodominant T-cell epitope CII259-273. Replacement of the glutamine at position 267 with glutamic acid leads to a decreased binding affinity to MHC II. T cells recognized both non-modfied (Q(267)) and modified (E(267)) CII259-273-peptides. We also show that administration of tTG leads to increased incidence, severity and histopathological manifestations of collagen-induced arthritis in mice. Moreover, we conclude that both processes, deamidation and cross-linking, are involved in the tTG-catalyzed reactions, and in vivo administration of tTG enhances arthritis severity and joint destruction in mice.

Collagen type II (CII) is a cartilage-specific target of pathologic humoral autoimmune responses in rheumatoid arthritis as well as in the collagen-induced arthritis model. The aim of the present study is to investigate the critical amino acid residues conferring CII epitope specificity of the prototypic arthritogenic murine mAb CIIC1. A homology model of the CIIC1 single-chain antibody fragment (CIIC1scFv) in complex with its triple helical epitope was established. In silico predictions based on extensive molecular dynamics simulations were experimentally tested by the recombinant expression and functional analysis of CIIC1scFv containing alanine replacements allowing the identification of crucial CII-binding sites in the CDR2 and CDR3 regions of both heavy and light chains. Since the conversion of the CIIC1scFv sequence into the respective germline at all 13 somatically mutated positions did not affect its CII binding, our data indicate that potentially harmful cartilage-specific humoral autoimmunity could be germline encoded. The molecular modeling further demonstrates that the rigid collagen triple helix restricts the likelihood of molecular interactions with the CDR regions of the antibody considerably compared with globular antigens. These sterical constraints provide an explanation as to why somatic mutations in the arthritogenic autoantibody have no obvious impact on CII recognition.

Previous data have indicated that IL-21 and/or IL-21R are crucial for the differentiation of naïve T cells into Th17 cells and also play a key role in the development of autoimmune disease. Given this, IL-21 and/or IL-21R are potential targets for therapy of such diseases; however, a study in this issue of the European Journal of Immunology, provides a new twist in the story as it is now shown that IL-21 and/or IL-21R play no role in Th17 development or autoimmune inflammatory disease. The reasons for these contradictory data are discussed in this Commentary.

The glycosylation status of IgG has been implicated in the pathology of rheumatoid arthritis. Earlier, we reported the identification of a novel secreted endo-beta-N-acetylglucosaminidase (EndoS), secreted by Streptococcus pyogenes that specifically hydrolyzes the beta-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 x 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 FcgammaR and disturbed the formation of stable immune complexes. Hence, the asparagine-linked glycan on IgG plays a crucial role in the development of arthritis.

Positional cloning of the underlying genes for the rare syndrome autoimmune polyendocrinopathy candidadiasis extrodermal dystrophy (APECED) opened a new venue of research on the role of central tolerance in autoimmunity. The associated autoimmune regulator gene (AIRE), was found to be expressed in medullary thymus epithelial cells (mTEC) in both man and mice, and to control promiscuous expression of sets of self antigens. The lack of AIRE in both mice and man led to the development of a quite specific, but also an inter-individual variable, set of autoimmune and infectious diseases. An article in this issue of the European Journal of Immunology demonstrates that several autoantigens controlled by AIRE are variably expressed in different human individuals. Most importantly it is shown that carriers of the type 1 diabetes (T1D) associated locus IDDM2 show lower expression of insulin in mTEC, as controlled by AIRE. The genetic variability of autoantigen expression in the thymus thus seems to determine the variable predisposition to autoimmunity.

Eosinophilia is a characteristic feature of many inflammatory diseases including inflammatory bowel disease and asthma. It also occurs in a subtype of rheumatoid arthritis but the role of eosinophils has been unclear and animal models have been lacking. Here, we introduce a new mouse model to study the role of eosinophilia in arthritis. Intraperitoneal injection of type II collagen alone, without any adjuvant, was sufficient to induce chronic arthritis in a mouse with transgenic T cells specific for type II collagen. The arthritis was accompanied by infiltration of eosinophils into the synovial tissue and the disease could be blocked with neutralizing anti-IL-5 antibodies. To our knowledge, this is the first description of an eosinophilic disease form of destructive arthritis.

It is widely believed that IL-4 exerts its influence by profiling the immune response during priming and expansion of immune cells, and thereby modulates the outcome of chronic inflammation. In the present investigation, collagen antibody-induced arthritis (CAIA) was used to delineate the role of IL-4 in a T cell-independent inflammatory phase. Mice predisposed to Th2 cytokines (BALB/c and STAT4-deficient mice) developed a more severe arthritis than mice biased towards Th1 cytokines (C57BL/6 and STAT6-deficient mice). Reduced incidence of CAIA was observed in IL-4-deficient mice compared to control littermates. Infiltrating cells in the paws of IL-4-sufficient mice had increased osteoclast activity and tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta secretion. Massive infiltration of granulocytes and joint and cartilage damage were present in arthritic paws. Depletion of IL-4 suppressed CAIA, which was abrogated by IFN-gamma neutralization. IL-1R- and IL-1RTNFR-deficient mice were completely resistant to CAIA. Thus, IL-4 promotes an antibody-mediated and TNF-alpha/IL-1beta-dependent inflammation in vivo.

Collagen type II (CII) is a relevant joint-specific autoantigen in the pathogenesis of rheumatoid arthritis (RA). Whereas the reasons for the breakage of self tolerance to this major cartilage component are still enigmatic, T cell responses to glycosylated CII determinants in RA patients indicate that post-translational modifications play a role. Since the conversion of arginine into citrulline by peptidylarginine deiminases (PAD) in some non-joint-specific antigens such as filaggrin or fibrin has been shown to give rise to RA-specific humoral immune responses, we investigated whether PAD modification of cartilage-specific CII might affect its recognition by circulating autoantibodies in early RA. In vitro treatment with purified PAD led to arginine deimination of native CII or of synthetic CII peptides as evidenced by amino acid analysis. The citrullination resulted in modified recognition of the immunodominant CII epitope C1(III) (amino acid residues 359-369) by murine and human antibodies. In a cohort of early RA patients (n=286), IgG antibodies directed toward a synthetic citrullinated C1(III) peptide (citC1(III)-P) were detectable with a prevalence of 40.4%. The partial autoantibody cross-reactivity between citC1(III)-P and citrullinated peptides mimicking epitopes of the cytoskeletal autoantigen filaggrin suggests that autoimmunity to cartilage-specific modified self might be a critical intermediate bridging recognition of PAD-modified extra-articular autoantigens with the disruption of tolerance to native cartilage constituents.

Among the arthritis-susceptible MHC (H-2)-congenic mouse strains, B10.RIII mice are highly susceptible to collagen-induced arthritis. Surprisingly, the B10.RIII strain was also more susceptible to the T cell independent model CAIA (collagen-antibody-induced arthritis). Through genome-wide genotyping, we found that the B10.RIII and B10.Q strains differed not only in chromosome 17 (MHC) but also in a region on chromosome 10, which contained a fragment from the MHC donor RIIIS/J. We isolated the chromosome 10 as well as the chromosome 17 segments on the B10.RIII and B10.Q backgrounds. Congenic mice containing the RIIIS/J-derived chromosome 10 segment showed significantly higher susceptibility and severity of arthritis with an enhanced autoimmune response to type II collagen. Furthermore, this chromosomal segment significantly promoted CAIA. Similarly, the RIIIS/J segment in chromosome 17 also promoted CAIA independently of other gene segments. These data show that other gene regions, apart from MHC class II, may explain effects both at the priming and effector level of arthritis observed in widely used MHC congenic strains. These new congenic fragments, on both chromosome 10 and 17, provide new mouse strains suitable for studies aiming at positional cloning of new genes associated with arthritis.

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.

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.

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 (FcgammaR) in anti-CII Ab-mediated arthritis was also investigated by comparing the clinical outcome in DBA/1 mice to those in FcgammaR-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 massive cellular 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 FcgammaR, as FcRgamma-deficient mice were completely resistant to Ab-mediated arthritis. The arthritis induced with the IgG1 and IgG2b Ab was also inhibited by FcgammaRIII 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 FcgammaRIIB. These results demonstrate that single IgG anti-CII mAb can induce erosive arthritis and that IgG anti-CII Ab mediate arthritis by engagement of FcgammaR.

Type II collagen (CII) is a candidate cartilage-specific autoantigen, which can become post-translationally modified by hydroxylation and glycosylation. T cell recognition of CII is essential for the development of murine collagen-induced arthritis (CIA) and also occurs in rheumatoid arthritis (RA). The common denominator of murine CIA and human RA is the presentation of an immunodominant CII-derived glycosylated peptide on murine Aq and human DR4 molecules, respectively. To investigate the importance of T cell recognition of glycosylated CII in CIA development after immunization with heterologous CII, we treated neonatal mice with different heterologous CII-peptides (non-modified, hydroxylated and galactosylated). Treatment with the galactosylated peptide (galactose at position 264) was superior in protecting mice from CIA. Protection was accompanied by a reduced antibody response to CII and by an impaired T cell response to the glycopeptide. To investigate the importance of glycopeptide recognition in an autologous CIA model, we treated MMC-transgenic mice, which express the heterologous CII epitope with a glutamic acid in position 266 in cartilage, with CII-peptides. Again, a strong vaccination potential of the glycopeptide was seen. Hence CII-glycopeptides may be the optimal choice of vaccination target in RA, since humans share the same epitope as the MMC mouse.

Rheumatoid arthritis as well as collagen-induced arthritis (CIA) is thought to involve T cell autoimmunity of the Th1 type and the Th2 cytokine IL-4 has been proposed to play a suppressive role. To exclude a possible skewing role of the mycobacteria used in the complete Freund's adjuvant (CFA) we induced CIA with type II collagen (CII) in incomplete Freund's adjuvant (IFA). Our results show that IL-4 deficiency leads to a lesser susceptibility to arthritis and lower B and T cell responses if induced with CII/IFA but not if induced with CII/CFA. In addition, IL-4-deficient mice were less susceptible to arthritis induced with monoclonal anti-CII antibodies. However, mice immunized with CII/IFA later developed a chronic relapsing disease, which was promoted by IL-4 deficiency. We conclude that IL-4 plays different roles depending on the type of adjuvant used and the phase (acute or chronic) of the clinical disease.

We have investigated the role of B cells in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) using B cell-deficient mice muMT) and mice bearing the X-linked immunodeficiency (xid). The mice were immunized with MOG(1-125 )in complete Freund's adjuvant but without use of pertussis toxin. B cell-deficient muMT mice on different genetic backgrounds (C57BL/10 and DBA/1 strains) developed EAE, although with a reduced clinical severity. Histological analyses revealed decreased demyelination in the central nervous system while the influx of inflammatory cells was similar or only slightly reduced as compared to B cell-sufficient control mice. Xid mice on the DBA/1 background also developed disease with a reduced disease severity. The anti-MOG antibody response in the xid mice was decreased, while the T cell response to MOG was unaffected. We thus demonstrate that B cells are not critical for the development of MOG-induced EAE but contribute to the severity. The contribution of B cells to pathogenesis appears to be mainly through demyelination rather than through inflammation.

Collagen-induced arthritis (CIA) is induced in H-2(q) mice after immunization with rat type II collagen (CII). The immunodominant T cell epitope on heterologous CII has been located to CII256-270. We have previously shown that TSC transgenic mice, which express the heterologous epitope in type I collagen (CI), e.g. in skin, are tolerized against rat CII and resistant to CIA. In this study we transplanted skin from TSC transgenic mice onto non-transgenic CIA-susceptible littermates to investigate whether introduction of this epitope to a naïve immune system would lead to T cell priming and graft rejection or instead to tolerance and arthritis protection. Interestingly, TSC grafts were accepted and not even immunization of recipient mice with CII in adjuvant induced graft rejection. Instead, TSC skin recipients displayed a reduced T and B cell response to CII and were also protected from arthritis. However, additional priming could break arthritis protection and was accompanied by an increased T cell response to the grafted epitope. Strikingly, despite the regained T cell response, development of arthritis was not accompanied by graft rejection, showing that these immune-mediated inflammatory responses involve different mechanisms.

The non-obese diabetic (NOD) mouse spontaneously develops diabetes and sialadenitis. The sialadenitis is characterized by histopathological changes in salivary glands and functional deficit similar to Sjögren's syndrome. In humans, Sjögren's syndrome could be associated with other connective tissue disorders, such as rheumatoid arthritis. In the present study the genetic control of sialadenitis in mice was compared to that of arthritis. We have previously reported a NOD locus, identified in an F2 cross with the H2(q) congenic NOD (NOD.Q) and C57BL/10.Q (B10.Q) strains, that promoted susceptibility to collagen-induced arthritis. The sialadenitis in NOD.Q showed a similar histological phenotype as in NOD, whereas no submandibular gland infiltration was found in B10.Q. The development of sialadenitis was independent of immunization with type II collagen and established arthritis. To identify the genetic control of sialadenitis, a gene segregation experiment was performed on an (NOD.QxB10.Q)F2 cross and genetic mapping of 353 F2 mice revealed one significant locus associated with sialadenitis on chromosome 4, LOD score 4.7. The NOD.Q allele-mediated susceptibility under a recessive inheritance pattern. The genetic control of sialadenitis seemed to be unique in comparison to diabetes and arthritis, as no loci associated with these diseases have been identified at the same location.

Cartilage-specific proteins are considered potential autoantigens that could continuously fuel autoimmune responses directed to the joints in rheumatoid arthritis (RA). Using recombinant chimeric collagen type II we have identified one major type II collagen (CII) epitope (denoted U1) recognized by RA sera. The U1 epitope is a triple helical structure formed by 11 amino acids (triple helical position 494-504) and colocalizes with the recently described alpha1beta1/alpha2beta1 integrin binding site. It is a major epitope, found in 14/22 RA sera positive for antibodies to CII. One individual could be followed for a long time and the results showed that IgG antibodies specific for the U1 epitope were maintained along the chronic disease course but suppressed during periods of cyclosporin A and anti-CD4 treatment. We also found that the U1 epitope was recognized in rats susceptible to collagen-induced arthritis. A monoclonal autoantibody (mAb 126.30) was raised from DA rats, which bound the same epitope. The antibodies bound the cartilage in vivo showing that the epitope is exposed to the immune system for immune complex formation in the intact joint.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune-mediated diseases such as diabetes and Sjögren's syndrome. To investigate whether NOD genes also promote autoimmune-mediated arthritis we established a NOD strain with an MHC class II fragment containing the A(q) class II gene predisposing for collagen induced arthritis (NOD.Q). However, this mouse was resistant to arthritis in contrast to other A(q) expressing strains such as B10.Q and DBA/1. To determine the major resistance factor/s, a genetic analysis was performed. (NOD.Q x B10.Q)F1 mice were resistant, whereas 27% of the (NOD.Q x B10.Q)F2 mice developed severe arthritis. Genetic mapping of 353 F2 mice revealed two loci associated with arthritis. One locus was found on chromosome 2 (LOD score 9.8), at the location of the complement factor 5 (C5) gene. The susceptibility allele was from B10.Q, which contains a productive C5 encoding gene in contrast to NOD.Q. The other significant locus was found on chromosome 1 (LOD score 5.6) close to the Fc-gamma receptor IIb gene, where NOD carried the susceptible allele. An interaction between the two loci was observed, indicating that they operate on the same or on interacting pathways. The genetic control of arthritis is unique in comparison to diabetes, since none of these loci have been identified in analysis of diabetes susceptibility.

Previous studies have established that NK cells express both inhibitory and activatory receptors. The inhibitory receptors have been shown to recognize major MHC class I molecules, but the physiological ligands for the activatory receptors have been only partly characterized. In this study we investigated whether NK cells could be activated by recognizing specific non-classical MHC class Ib molecules. NK cells from BN (RT1(n)) rats immunized in vivo with MHC-incompatible WF (RT1(u)) cells displayed cytolytic activity specific for product(s) of the MHC class Ib RT1-E(u) / C(u) region. These cells were shown to kill Rat2 fibroblast cells transfected with cDNA for RT1-E(u) but neither untransfected Rat2 nor a transfectant with the class Ia allele, RT1-A(u). Cytolysis of Rat2-RT1-E(u) was inhibited by the anti-RT1-E(u) antibody 70-3-C2. In addition, NK cells cytolytic against PVG (RT1(c)) targets, but not against WF (RT1(u)) or other allogeneic targets were activated after PVG immunization of BN rats. The generation of NK populations cytolytic for target cells of the same haplotype as the immunizing cells, but not for third-party targets, strongly suggests the existence of a selective NK-mediated response in vivo. We conclude that recognition of an allogeneic MHC class Ib RT1-E molecule activates NK cells and the specific cytolytic response could be regarded as adaptive.

The genetic susceptibility to collagen-induced arthritis (CIA) in mice, the most commonly used model for rheumatoid arthritis, has been analyzed. The highly susceptible B10.RIII strain was crossed with the resistant RIIIS/J strain and the F2 intercross mice were subjected to genomic screening using microsatellite markers. These strains share the MHC region on chromosome 17, known to be of importance in CIA (this locus is named Mcia1). The same cross has earlier been used to map the major genes outside the MHC controlling chronic relapsing experimental allergic encephalomyelitis (EAE). It was found that the major locus controlling CIA (Mcia2; lod 4.12) was located on chromosome 3 in the same region as one of the major loci controlling EAE (Eae3). The linkage was reproduced in a mouse strain in which the locus was isolated on the B10.RIII background at the N4I2 generation. A second putative locus was identified on chromosome 13 (lod 3.13). The present finding identifies new loci outside the MHC controlling CIA and provides evidence that mouse CIA is controlled by polymorphic genes.

Immunization of mice with type II collagen (CII) leads to collagen-induced arthritis (CIA), a model for rheumatoid arthritis. T cell recognition of CII is believed to be a critical step in CIA development. We have analyzed the T cell determinants on CII and the TCR used for their recognition, using twenty-nine T cell hybridomas derived from C3H.Q and DBA/1 mice immunized with rat CII. All hybridomas were specific for the CII(256-270) segment. However, posttranslational modifications (hydroxylation and variable O-linked glycosylation) of the lysine at position 264 generated five T cell determinants that were specifically recognized by different T cell hybridoma subsets. TCR sequencing indicated that each of the five T cell epitopes selected its own TCR repertoire. The physiological relevance of this observation was shown by in vivo antibody-driven depletion of TCR Valpha2-positive T cells, which resulted in an inhibition of the T cell proliferative response in vitro towards the non-modified CII(256-270), but not towards the glycosylated epitope. Most hybridomas (20/29) specifically recognized CII(256-270) glycosylated with a monosaccharide (beta-D-galactopyranose). We conclude that this glycopeptide is immunodominant in CIA and that posttranslational modifications of CII create new T cell determinants that generate a diverse TCR repertoire.

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 A(beta) 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 posttranslationally 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 myelin basic protein (MBP) peptide 63-88-induced experimental autoimmune encephalomyelitis (EAE) and its associated T cell cytokine profile are influenced by the rat major histocompatibility complex (MHC). There is an allele-specific protective influence of the MHC class I region, whereas the MHC class II region display either disease-protective or -promoting effects. To investigate if the MHC-associated protection is dependent on certain combinations of MBP peptide and MHC molecules, we have now used another peptide (MBP 89-101). A broader and different set of rat MHC alleles were associated with EAE induced with MBP 89-101 as compared to MBP 63-88. All EAE-susceptible strains mounted peptide-specific strong T helper (Th) 1-like immune responses in vitro. Immunization of rats with an extended peptide (MBP 87-110) induced EAE associated with the same MHC haplotypes as the 89-101 peptide, except in LEW.1N (RT1 pi) rats which were relatively resistant. Only this strain responded with additional Th2-like and transforming growth factor-beta responses to the peptide in vitro. In vivo depletion of CD8+ cells aggravated the disease in this strain. We conclude that both MHC-controlled promoting and protective influences on EAE are dependent on certain MHC/MBP peptide combinations, and that the 87-110 region of MBP contains a major MHC-associated encephalitogenic epitope in the rat.

It is well known that T cells recognize antigen as processed peptides bound to major histocompatibility complex molecules on the surface of antigen-presenting cells. Recently, it has been shown that T cells can specifically recognize synthetic glycopeptides. However, whether glycopeptides are selected for presentation during antigen processing of glycoproteins and eventually elicit carbohydrate-specific T cells is still an open question. In this study, we utilized synthetic glycopeptides to analyze T cell recognition of the naturally glycosylated immunodominant peptide representing type II collagen (CII) residues 256-270. In this peptide, lysines at positions 264 and 270 may be post-translationally modified by hydroxylation and subsequent O-linked glycosylation with beta-galactosyl or alpha-glucosyl-(1-->2)-beta-galactosyl residues. T cell hybridomas established from type II collagen-immunized mice specifically recognized CII 256-270 with either galactose or glucosyl-galactose at position 264. The T cell hybridoma recognizing glucosyl-galactose displayed no cross-reactivity either to galactose or to the structurally different alpha-galactosyl-(1-->4)-beta-galactose. Furthermore, the T cell hybridoma recognizing galactose did not cross-react to glucosyl-galactose or galactosyl-galactose, indicating that the antigen-presenting cells (bulk spleen cells, lipopolysaccharide-stimulated spleen cells, anti-CD40-stimulated spleen cells, peritoneal exudate cells or CFA-primed lymph node cells) inefficiently processed carbohydrates when the antigen was given as a glycopeptide.

Dendritic cells, such as epidermal Langerhans cells, play a crucial role for the antigen-specific priming of T cells. We have addressed the question whether dendritic cells present collagen, a major protein component in tissues through which dendritic cells migrate, i.e. the basement membrane, dermis, and synovial tissue. Langerhans cells, spleen cells and peritoneal macrophages were compared for antigen-presenting capacity using a panel of mouse T cell hybridomas reactive with different determinants on type II collagen, myelin basic protein, ovalbumin and pepsin. Langerhans cells did not present any of the type II collagen determinants, unless the antigen was administered as a 15-mer peptide, but did present myelin basic protein, ovalbumin and pepsin. Spleen cells and peritoneal macrophages, in contrast, presented all type II collagen determinants. This biased antigen presentation was also observed when Langerhans cells were pulsed with antigen in vivo. The inability to present type II collagen is related to the collagen sequence as such, since both native type II collagen, type II collagen alpha chains, as well as a type II collagen determinant incorporated in type I collagen, were not presented by Langerhans cells. In addition, granulocyte/macrophage colony-stimulating factor-expanded blood dendritic cells displayed the same biased antigen presentation, suggesting that the inability to present collagen is not restricted to dendritic cells localized in epidermis. B cell-deficient mice could prime a type II collagen-reactive T cell response, thus excluding B cells as obligatory antigen-presenting cells for the priming of collagen-reactive T cells. We suggest that neither Langerhans cells nor B cells, but macrophages are the primary antigen-presenting cells in the immune response towards type II collagen.

B10.RIII mice develop chronic and relapsing experimental autoimmune encephalomyelitis (EAE) after immunization with the myelin basic protein (MBP) peptide 89-101 (VHFFKNIVTPRTP). To investigate the basis for the chronicity of the disease, the subsequent development of an immune responses to other parts of the MBP protein were investigated. Onset of disease occurs 9-25 days after immunization with MBP89-101. T cell responses towards a series of MBP peptides were assessed in an enzyme-linked immunospot assay detecting single cells secreting IFN-gamma. There were responses not only to MBP89-101, but also towards peptides derived from sequences outside of MBP89-101. These peptides were of two kinds: those with sequences completely outside the 89-101 stretch of MBP; and those sharing a short sequence with MBP89-101 depending on alternative splicing of MBP mRNA. Immunization with these peptides also produced chronic EAE and a spreading of the immune response to other MBP peptides. Immunization with stepped peptides around the relevant region (MBP87-110) showed that peptides sharing a 6-amino-acid motif induced EAE after immunization. After MBP89-101 peptide immunization, T cells isolated from lymph nodes did not cross-react in vitro to the other peptides sharing this motif. We suggest that one mechanism for the development of relapses during the disease course is the recruitment of new T cells with specificity for MBP peptides not derived from the peptide used for immunization. This is the first time such a mechanism has been demonstrated in a chronic autoimmune disease model.

Antigen-specific IgE up-regulates the specific IgM, IgG1, IgG2a and IgE response in vivo when given to mice together with antigen. The enhancement is mediated by the low-affinity receptor for IgE, Fc epsilon RII or CD23, as demonstrated both in CD23-deficient mice and by blocking CD23 with anti-CD23 monoclonal antibodies. A possible mechanism behind the regulatory effects of CD23 is that the IgE/CD23/antigen complex is endocytosed by B cells, leading to increased antigen processing and presentation on major histocompatibility complex (MHC) class II molecules to T helper cells. In the present study we have found that the expression of CD23 is reduced fivefold on splenic B cells in mice genetically deficient for IL-4. When IL-4-deficient mice and normal littermates were immunized with 2,4,6-trinitrophenyl (TNP)-specific IgE followed by bovine serum albumin (BSA)-TNP or with BSA-TNP alone, the BSA-specific IgG1 and IgG2a responses were equally well augmented by IgE in all mice. In addition, a low but significant IgE response was seen even in the IL-4-deficient mice. Thus, enhancement of the antibody response through IgE and CD23 occur in the absence of IL-4 and is not dependent on CD23 up-regulation.

The major histocompatibility complex (MHC) class II region is assumed to influence autoimmune diseases such as rheumatoid arthritis. In the mouse, the H-2q haplotype is associated with susceptibility to collagen-induced arthritis, while the H-2p haplotype is not. The class II A molecules of these haplotypes differ by only four amino acids in the first domain of the beta chain. To test if this difference accounts for the MHC influence on susceptibility to collagen-induced arthritis, H-2p mice were made transgenic with an Abp gene altered to resemble the Abq gene. The transgenic A beta chain hybridized with the A alpha p chain and was shown to be physiologically expressed by testing antigen-presentation capacity to Aq-restricted T cell hybridomas and with FACS analyses. These transgenic mice developed an autoimmune response to type II collagen and also collagen-induced arthritis. The data unequivocally suggest the Ab gene as a major genetic susceptibility locus for autoimmune collagen-induced arthritis.

Erosive arthritis is a common and feared complication of staphylococcal infection. The reason(s) for the progressive course of the arthritis is unknown. It has been recently established that enterotoxins produced by Staphylococcus aureus display superantigen properties leading to stimulation of T cells carrying distinct T cell receptor V beta elements. This finding provides a potential connection between staphylococcal exoproteins and endogenous immune mechanisms participating in the infectious process. We have recently describe successful induction of infections arthritis in mice after intravenous inoculation of a toxic shock syndrome toxin-1 (TSST-1)-producing S. aureus LS-1 strain. Using this model we have now found a clonal expansion of T cells expressing V beta 11+ T cell receptor in the synovial tissue of arthritic mice. The role of TSST-1 as a superantigen inducing oligoclonal expansion was confirmed in an in vitro culture system. The expansion of V beta 11+ T cells proved to be of arthritogenic significance since mice genomically deleted of the V beta 11+ T cells did not develop arthritis and since pretreatment of healthy mice with anti-CD4 or anti-V beta 11 monoclonal antibodies inhibited arthritis. In addition, CD4+ and V beta 11+ T cells showed themselves to be of pathogenic significance in staphylococcal-induced mortality, since mice depleted of such populations showed increased survival. We propose that in hematogenously spread S. aureus-induced arthritis the TSST-1-dependent clonal expansion of CD4+ V beta 11+ T cells is a driving pathogenic force.

The Y-linked autoimmune accelerating gene mutation (yaa), first discovered in the BXSB mouse strain, is known to accelerate spontaneous autoantibody production and subsequent development of lupus disease. We have investigated the role of the yaa gene in the development of the type II collagen (CII)-induced arthritis (CIA), which is used as a model for rheumatoid arthritis. In contrast to the accelerating effects on development of lupus autoimmunity we can show that the presence of BXSB Y chromosome carrying the yaa gene block development of CIA in F1 crosses with three normally CIA-susceptible strains, DBA/1, C3H.Q and B10.Q. Backcross experiments showed an additional modulatory effect from other BXSB genes or possibly from DBA/1 X chromosome. To evaluate the effect mediated by the yaa gene alone, the BXSB Y chromosome was bred into the DBA/1 gene background. The DBA/1 congenic DBA/1.yaa male mice were less susceptible to arthritis development than their DBA/1 counterparts. (B10.QxDBA/1.yaa)F1 acquired resistance to arthritis development similar to that of DBA/1.yaa, indicating a role for the yaa gene alone. The serum levels of autoantibodies to CII were significantly suppressed in all strains carrying yaa. In DBA/1.yaa mice a reduced number of T cells were found to produce interferon-gamma after in vitro stimulation with CII. Thus, although autoreactive B cells are important in both diseases they play different roles in murine lupus and in CIA.

Polymorphism of the major histocompatibility complex (MHC) influences susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP) in rats. Current concepts relate such influences to the capacity of class II molecules to present relevant peptides to autoreactive T cells. We have here analyzed the MHC influence on the immune response and the development of EAE after immunization with the immunodominant peptide MBP-63-88. Analysis of MHC-congenic LEWIS strains showed that RT1a, RT1c and RT1(1) haplotypes are permissive for disease induction, whereas RT1d and RT1u are resistant. All EAE responding strains showed peptide-specific proliferation and interferon (IFN)-gamma secretion, but no early significant tendency to express interleukin (IL-4) or transforming growth factor (TGF)-beta mRNA in lymphocytes in response to the MBP 63-88, 7 days post immunization (p.i.). Later, 14 days p.i., peptide-specific induction of IL-4 and TGF-beta occurred in RT1(1) rats. Among the EAE non-responders strains, only the RT1u rats showed an immune response to MBP 63-88. This response, however, was qualitatively different from the immune response in the EAE-susceptible strains. Thus, there was no proliferation and only moderate IFN-gamma production in response to peptide, but in contrast, a significant and early peptide-induced IL-4 and TGF-beta response was observed. The data suggest that the MHC-associated susceptibility to EAE is partly related to the ability to mount a TH1-like immune response while the MHC-associated EAE resistance may either be related to MBP peptide non-responsiveness or to peptide recognition and induction of a qualitatively different and disease down-regulatory immune response.

Immunization with type II collagen (CII) induces collagen-induced arthritis (CIA) in animals, and B cells reactive with CII are involved in the induction and manifestation of the disease. In this study, B cell hybridomas producing IgG antibodies specific for a major epitope on mouse CII (the "C1" epitope, amino acid 316-333), were isolated 11 days after immunization from draining lymph nodes in DBA/1 mice. Injection into neonatal mice of purified and biotinylated monoclonal antibodies binding the C1 epitope led to a specific binding to joint cartilage, demonstrating that the antibodies interact with native antigen in vivo. cDNA sequencing of the B cell clones revealed that they all expressed the same combination of a variable heavy chain (VH J558 family) and light chain (V kappa 21 family) germ-line gene, apparently lacking somatic mutations. The presence of isotype-switched B cells expressing a certain combination of V genes encoding antibodies that bind epitopes in vivo, indicates that this B cell population has been peripherally selected.

The T cell recognition of type-II collagen (CII) in H-2q mice, susceptible to CII-induced arthritis, was analyzed. With the use of T cell hybridomas derived from rat CII-immunized mice, a peptide corresponding to amino acids 245-270 on chick CII was found to harbor a T cell epitope which is present on heterologous CII (chick, rat, human, and bovine CII) but not on autologous CII. It was shown that this epitope was located within amino acids 260-270, although flanking regions in either direction were necessary for proper recognition. A peptide corresponding to human CII (256-270) was used for further studies. A single amino acid difference at position 266 between mouse CII (aspartic acid) and heterologous CII (glutamic acid) strongly influenced recognition of this peptide. No response towards the mouse peptide was seen with any of the T cell hybridomas. Inhibition studies revealed that the mouse peptide did not bind as well to major histocompatibility complex as the corresponding heterologous peptide. Both peptides gave rise to a T cell response after immunization. However, immunization with the heterologous peptide resulted in a response strictly directed to rat CII and the immunogen while immunization with the autologous peptide elicited T cells which reacted in a heteroclitic fashion, with a stronger response to the heterologous peptide than to the autologous peptide, and did respond to rat CII but not to mouse CII. We suggest that aspartic acid in position 266 results in a cryptic determinant in mouse CII which is neither recognized after CII immunization nor capable of tolerance induction. A glutamic acid at position 266, however, gives rise to an immunodominant epitope which is recognized by a large proportion of the T cells activated after immunization with heterologous CII.

It is well documented that antigen recognition by T cells requires small peptides which are generated by protein cleavage in antigen-presenting cells. These peptides have to associate with major histocompatibility complex (MHC) molecules in order to be recognized. An inhibitory peptide may bind to the same site of the MHC-encoded protein but is not recognized by the T cell. Here we describe a stimulatory and an inhibitory peptide sequence within human collagen type II (CII) as defined by means of the same autoreactive human T cell clone. Most interestingly, the inhibitory peptide is not generated by regular processing in peripheral blood mononuclear cells but only in the presence of an antibody that binds to the same domain and thereby seems to protect the inhibitory sequence. This finding may indicate that certain autoantibodies have the potential to block autoreactive T cells with specificity for a distinct epitope on the same antigen.

Collagen-induced arthritis (CIA) in rats, induced with homologous type II collagen (CII), is a genetically more restricted disease and has better resemblance to rheumatoid arthritis by its chronic disease course, than CIA induced with heterologous CII. The DA strain is highly susceptible to CIA induced with homologous CII, while the Lewis strain is resistant. (DAxLew)F1 is susceptible and backcrossing to Lewis reveals a close, but not complete, association of both arthritis and CII responsiveness to the RT1a haplotype. Analyses of congenic strains on DA and Lewis backgrounds suggest that expression of a major histocompatibility complex class II Ba molecule, encoded from the RT1Ba locus, is associated with arthritis susceptibility and CII responsiveness. The second exons coding for the first domains of the alpha and beta chains of both the RT1a and RT1l haplotypes were sequenced and the deduced amino acid sequences compared with the corresponding molecule associated with susceptibility to CIA in the mouse (H-2 Aq). The sequences of the respective alleles revealed no obvious structural homology explaining the extensive similarities in the development of chronic autoimmune arthritis. Instead, this finding implies that different trimolecular constituents (i.e. class II, T cell receptor, and CII peptides) may yield an antigen presentation event that is able to trigger a similar autoaggressiveness in the two rodent species.

Activation of T cells is critical for the development of type II collagen (CII)-induced arthritis (CIA). However, the relative importance of T cells in their delivery of help to B cells, promoting autoantibody formation or acting as inflammatory initiating cells, is unclear. The effect of a monoclonal antibody directed to the alpha/beta T cell receptor (TcR) on the development of autologous CIA was studied. Two weeks after immunization with autologous CII the onset of severe arthritis occurred, followed by a chronic arthritis activity in the peripheral joints. Anti-TcR treatment before immunization suppressed the incidence of arthritis and the autoantibody response to CII. Treatment given immediately before the expected onset delayed the appearance of arthritis. Treatment given to already arthritic rats reduced the severity. In the latter two groups the serum levels of anti-CII autoantibodies were not affected. The duration of the ameliorating effect was limited and with the return of arthritis a concomitant antibody response towards the injected mouse anti-TcR antibody was observed. These results show that the role of T cells in both the induction and perpetuation of CIA is essential and not limited to the triggering of production of pathogenic anti-CII autoantibodies.

Development of experimental allergic encephalomyelitis (EAE) in the SJL (H-2s) mice is associated with a T cell-dependent autoimmune response to the C-terminal part of the myelin basic protein (MBP). In this study the influence of both H-2 and non-H-2 genetic background on EAE induced with the MBP89-101 peptide is described. Analysis of different H-2q haplotype strains, B10G, B10Q, SWR and NFR/N, showed that the B10 background is relatively resistant to disease induction. Both SWR and NFR/N were susceptible to EAE showing that the H-2q haplotype is permissive for EAE development induced with MBP89-101 and that the T cell receptor (TcR) haplotype or complement C5 deficiency exert no significant influence on disease susceptibility. In a series of H-2-congenic strains on the B10 background only B10RIII (H-2r) mice were susceptible to EAE. The B10RIII mice developed a severe EAE with early onset and chronic progressive or relapsing course of disease. In addition, B10RIII mice treated with Freund's complete adjuvant and pertussis toxin alone showed an early monophasic disease. The clinical observations were confirmed by immunohistopathologic analysis of the central nervous system. In these studies, we also applied antibodies to different TcR V beta elements which showed no specific limitation of the used TcR among infiltrating T cells in the target tissue in any of the strains. It is concluded that an MBP peptide-specific disease can be induced in three different haplotypes and it is possible that shared structures between the As, Aq and Ar molecules are of importance for the trigger of encephalitogenic T cells with different TcR V elements. The presently described chronic EAE model induced in the B10RIII mice will be of value as a model for multiple sclerosis.