Natural selection does not operate on single genes but rather on molecular pathways. Thus, the discovery of a genetic polymorphism, like in Ncfl, regulating a disease trait, opens up the possibility for the identification of a new pathway leading to arthritis. The pathways are likely to be shared between different species; however, the exact nucleotide variant in the rat Ncfl gene is probably specific. Thus, for further studies it can be assumed that the pathway will operate similarly in rats, mice, and humans.
To investigate this possibility we looked to the mouse. In the mouse there is an arthritis QTL identified that contains the Ncfl gene. However, we found that Ncfl was not polymorphic and we did not find an effect on the oxidative burst using a congenic strain with a fragment derived from C3H on a B10.Q background (P. Olofsson et al., unpublished data). Another mouse that was made deficient for Ncfl was not possible to use for our purpose, as the mutation results in a complete destruction of the gene. Moreover, the Ncfl-targeted mutation also contains a genetically linked 129-derived fragment ,known to contain other polymorphic genes of importance for arthritis . However, a worldwide search through available mouse strains identified a variant of a C57B1/6 strain with mutations in both the leptin receptor and the Ncfl gene . The point mutation in the Ncfl gene is located at position -2 in exon 8 and results in an aberrant splicing of the Ncf1 mRNA transcripts. One of the transcripts resulting from this mutation gives rise to low levels of a protein that is truncated, lacking eight amino acids in the second SH3 domain that are important for interaction with the Ncf2 protein, thus resulting in absence of detectable NADPH oxidase activity. Consequently, we now had another mutation that presumably should affect the same pathway as was discovered in the rat. The C57Bl/6 mouse with the mutation in the Ncf1 gene was therefore backcrossed to the arthritis-susceptible B10.Q strain. The mice were tested for susceptibility to CIA, where arthritis is induced by an intradermal injection of rat collagen type II (CII) emulsified in complete Freund's adjuvant. The B10.Q mice homozygous for the mutation in Ncf1 developed severe arthritis with chronic development, i.e., similar to the low oxidative burst responder DA rat strain . Mice heterozygous for the mutation were found to develop a less severe arthritis than Ncf1 -mutated mice resembling the situation in rats where one functional Ncf1 allele is enough to correct both oxidative burst and disease severity. Interestingly, both the delayed type hypersensitivity and antibody response to CII were higher in the Ncf1 mutated mice, indicating an increased activity of CII reactive T cells. We also found that some female Ncf1 mutated mice in the breeding colony spontaneously developed arthritis. In an experiment, 3 out of 12 female mice developed severe arthritis a few days after partus, confirming earlier findings that there is a high risk for arthritis susceptibility during the postpartum period . The arthritis was severe and chronic, as in CIA, and the mice with spontaneous arthritis also had a higher level of antibodies against CII with the same fine-specificity as in CIA .
The overall data clearly suggest another role of NADPH oxidase-derived ROS than just elimination of pathogens, adding a complexity to the redox status of the cell. ROS response has been reported not only in phagocytes but also in antigen-presenting cells (APCs), such as dendritic cells (DCs) . Also, T cells have been reported to express Ncf1 during antigen presentation and activation , adding further complexity to the process of finding the mechanisms behind the phenotype.
The immune regulatory effect of ROS could be involved in one or more of the different stages of the interactions of the immune system. There are a number of pathways that are proposed to be affected by free radicals and state of oxidation. On the antigen-presenting level, it has been shown that by inhibiting ROS production, the proliferation of T cells in response to antigen presentation of DCs is prevented , suggesting that the burst response in an APC could modulate the antigen presentation capacity of that cell. The level of radicals could also directly act on T cells. ROS could possibly be transferred over the synapse from APC to T cell and thereby alter the oxidation state of the recipient cell. It has been suggested that the level of oxidation on membranes of cells, influenced by interacting cells, are important for function . Extracellular ROS has also been shown to act as an immune regulator through its interaction with the T cell proliferation regulator NO . The oxidation could also possibly act by affecting other important structures on the cell surface and thereby alter their status. It has been shown that oxidation on T cell receptor signaling pathways, such as the linker for activation of T cells (LAT) molecule, modulates the function of T cells . It is also possible that ROS by itself acts as a modulator of the system. It is known that ROS in low concentrations serves as a second messenger in the initiation and amplification of signaling at the antigen receptor in lymphocyte activation (reviewed in ). Another possibility is that ROS is involved in feedback mechanisms. Direct cell-cell contact between an activated T cell and human PMN has been shown to induce intracellular ROS production in the PMN, which then could act as an intracellular messenger . Further studies are needed in order to clarify which effect is of importance for the development of arthritis.
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