The Ncf1 gene was recently identified as a strong regulator of severe arthritis in rat. This finding was surprising, because the disease-promoting allele mediated a lower level of reactive oxygen species in NADPH oxidase-expressing cells. We have now investigated a splice mutation of the Ncf1 gene in B10.Q mice, causing a truncated and nonfunctional Ncf1 protein. We found that the mutated Ncf1 led to a more severe and chronic relapsing collageninduced arthritis. Enhanced IgG and delayed-type hypersensitivity responses against type II collagen were seen, indicating increased activity of autoreactive T cells. Interestingly, female Ncf1-mutated mice spontaneously developed severe arthritis during the postpartum period. The arthritis was accompanied by an increased antibody response to type II collagen, with the same fine specificity as in collagen-induced arthritis. The enhancing effect of the mutated Ncf1 could also be shown to be more general in that it enhanced myelin oligodendrocyte glycoprotein protein-induced experimental autoimmune encephalomyelitis, a model for multiple sclerosis. These results show that Ncf1, a gene important for oxidative burst, regulates the susceptibility and severity of both arthritis and encephalomyelitis and modulates, directly or indirectly, the level of T cell-dependent autoimmune responses. rodent ͉ T lymphocytes ͉ NADPH

The identification of genes underlying quantitative-trait loci (QTL) for complex diseases, such as rheumatoid arthritis, is a challenging and difficult task for the human genome project. Through positional cloning of the Pia4 QTL in rats, we found that a naturally occurring polymorphism of Ncf1 (encoding neutrophil cytosolic factor 1, a component of the NADPH oxidase complex) regulates arthritis severity. The disease-related allele of Ncf1 has reduced oxidative burst response and promotes activation of arthritogenic T cells. Pharmacological treatment with substances that activate the NADPH oxidase complex is shown to ameliorate arthritis. Hence, Ncf1 is associated with a new autoimmune mechanism leading to severe destructive arthritis, notably similar to rheumatoid arthritis in humans.

Rats and mice with a lower capacity to produce reactive oxygen species (ROS) because of allelic polymorphisms in the Ncf1 gene (which encodes neutrophil cytosolic factor 1) are more susceptible to develop severe arthritis. These data suggest that ROS are involved in regulating the immune response. We now show that the lower capacity to produce ROS is associated with an increased number of reduced thiol groups (؊SH) on T cell membrane surfaces. Artificially increasing the number of reduced thiols on T cells from animals with arthritis-protective Ncf1 alleles by glutathione treatment lowered the threshold for T cell reactivity and enhanced proliferative responses in vitro and in vivo. Importantly, T cells from immunized congenic rats with an E3-derived Ncf1 allele (DA.Ncf1 E3 rats) that cannot transfer arthritis to rats with an arthritis-associated Dark Agouti (DA)-derived mutated Ncf1 allele (DA.Ncf1 DA rats) became arthritogenic after increasing cell surface thiol levels. This finding was confirmed by the reverse experiment, in which oxidized T cells from DA.Ncf1 DA rats induced less severe arthritis compared with controls. Therefore, we conclude that ROS production as controlled by Ncf1 is important in regulating surface redox levels of T cells and thereby suppresses autoreactivity and arthritis development.Ncf1 ͉ reactive oxidative species ͉ p47 phox ͉ NADPH ͉ glutathione R eactive oxygen species (ROS) are generally thought to be harmful and to play a disease-enhancing role in autoimmune diseases such as arthritis (1, 2). However, we have found that a decreased capacity to produce ROS because of polymorphisms in Ncf1 increases susceptibility for autoimmunity and arthritis (3, 4). Ncf1 encodes neutrophil cytosolic factor 1 (Ncf1, also known as p47phox), the activating protein in the NADPH oxidase complex that produces ROS upon activation. In the rat, a SNP in the Ncf1 gene was identified by positional cloning to be one of the strongest genes in regulating both oxidative burst and arthritis (3). In the mouse, a spontaneous mutation was identified that affects splicing and results in expression of truncated, less functional Ncf1 protein (5), which also resulted in increased arthritis and autoimmunity (4). Hence, it was clear that the Ncf1 gene that controls oxidative burst also controlled the autoimmune response and severity of arthritis in both rats and mice.It has been shown that arthritis as induced by immunization with pristane in rats and collagen in mice expressing polymorphic Ncf1 is T cell dependent. In the rat model, only T cells from DA.Ncf1 DA rats [Dark Agouti (DA) rats with the mutated Ncf1 DA allele from the DA rat] can transfer disease to naïve DA.Ncf1 DA recipients, whereas T cells from the congenic DA.Ncf1 E3 rats (DA rats with the WT Ncf1 E3 allele from the E3 rat) cannot (3, 6). In the mouse model, a mutation in Ncf1 results in an increased delayed-type hypersensitivity response and serum levels of anti-collagen type II (CII) IgG antibodies (4), indicating enhanced activation of autoreactive T ...

AcknowledgementsWe wish to thank Holger Luthman, Liselotte Bäckdahl and Ulrica Ribbhammar for critical reading of review text.

Rheumatoid arthritis (RA) is a chronic and genetically complex inflammatory disorder that leads to erosive destruction of peripheral joints. The use of animal models mimicking RA, such as pristane-induced arthritis (PIA) in rats, should facilitate its genetic analysis. Pristane is a non-immunogenic synthetic oil that, after a single subcutaneous injection into DA rats, induces arthritis restricted to peripheral joints with a chronic relapsing disease course. To identify genes involved in the control of chronic arthritis, we made crosses between susceptible DA rats and resistant E3 rats and analysed the progeny with microsatellite markers covering the entire rat genome. Our results show that different arthritis phenotypes are associated with different chromosomal loci. Loci on chromosomes 4 and 6 (Pia2 and Pia3) influence arthritis onset, whereas a locus on chromosome 12 (Pia4) is associated with severity and joint erosion. We found that chronicity is associated with a different set of loci, one on chromosome 4 and the other on chromosome 14 (Pia5, Pia6). These findings demonstrate for the first time that different phases of a chronic self-perpetuative disease which mimics RA are associated with distinct sets of genes.

We conclude that the lack of phagocyte-derived oxidative burst is associated with spontaneous autoimmunity and linked with type I IFN signature in both mice and humans.

No abstract

Pristane-induced arthritis (PIA) in rats, a model for rheumatoid arthritis (RA), is a T cell-dependent disease. However, pristane itself is a lipid and unable to form a stable complex with a MHC class II molecule. Therefore, the specificity and function of the T cells in PIA are as unclear as in rheumatoid arthritis. In this study, we show that activated CD4+ αβT cells, which target peripheral joints, transfer PIA. The pristane-primed T cells are of oligo or polyclonal origin as determined by their arthritogenicity after stimulation with several mitogenic anti-TCRVβ and anti-TCRVα mAbs. Arthritogenic cells secreted IFN-γ and TNF-α (but not IL-4) when stimulated with Con A in vitro, and pretreatments of recipient rats with either anti-IFN-γ or a recombinant TNF-α receptor before transfer ameliorated arthritis development. Most importantly, we show that these T cells are MHC class II restricted, because treatment with Abs against either DQ or DR molecules ameliorates arthritis development. The MHC class II restriction was confirmed by transferring donor T cells to irradiated recipients that were syngenic, semiallogenic, or allogenic to MHC class II molecules, in which only syngenic and semiallogenic recipients developed arthritis. These data suggest that the in vivo administration of a non-antigenic adjuvant, like pristane, activates CD4+ αβT cells that are MHC class II restricted and arthritogenic.