The main findings in the present study are that immune complexes formed by CSF autoantibodies were potent inducers of IFN-α as well as IP-10 (CXCL10), IL-8, and MCP-1, all of which have been reported to be elevated in CSF from NPSLE patients (6
). Furthermore, the strong correlation between CSF IFN-α and IP-10 induction, as well as the ability of a neutralizing anti-IFN-α to inhibit IP-10 production, suggests that the presence of IP-10 in CSF could be explained by stimulation by IFN-α. A second important observation was that normal serum, but not normal CSF, could attenuate IFN-α stimulation by immune complexes, which was, in part, explained by the inhibitory effect of normal IgG (see below).
Markers of neuronal and astrocyte damage are markedly elevated in the CSF of NPSLE patients (34
). Therefore, patients with NPSLE have both Ags and autoantibodies to form immune complexes to stimulate IM in the CNS. There are many potential sources of apoptotic or necrotic cells in NPSLE, such as cell damage induced by neurocytotoxic autoantibodies that now includes anti-ribosome P (35
) and anti-NMDAR Abs (36
) discussed above. Additionally, injury to endothelial cells by as yet poorly defined mechanisms or ischemic thrombosis associated with anticardiolipin Abs may release cellular Ags. Regardless of the source of Ag, the presence of CSF autoantibodies that bind to cellular Ags serves as a potentially powerful amplifier of inflammation in the brain. While low CSF IgG concentrations and limited volumes of CSF precluded identification of specific autoantibodies that stimulate IFN-α in CSF, we observed that the IFG activity in CSF correlated with serum anti-RNP, but not with other known autoantibody specificities. Additionally, four sera that tested negative for anti-dsDNA had among the highest IFG activity in CSF (), and IFN-α induction was RNase sensitive (). These observations suggest that autoantibodies targeted to RNA-containing Ags, rather than DNA-containing Ags, may be especially relevant to the IFG activity detected in NPSLE CSF, although studies of larger numbers of NPSLE patients and direct analysis of CSF will be needed to validate this observation. This finding is of interest since plasma containing Abs targeting RNA-containing protein Ags have previously been associated with the ability to induce high levels of IFN response genes in WISH cells (37
), and targeted deletion of TLR7 that interacts with RNA, but not deletion of TLR9 that interacts with DNA, attenuated disease in a mouse model of SLE (38
IFN-α was originally detected in the CSF by Winfield et al. (4
) and has been directly implicated as a causative factor in NPSLE by Shiozawa et al. (6
). In the latter study, IFN-α was detected in the CSF of five of six NPSLE patients and in the microglia and neurons following autopsy analysis of a patient who died from CNS lupus (6
). Although a lower frequency of CSF IFN-α detection has been reported in other studies, we used an ultrasensitive chemiluminescence method and detected >2 pg/ml IFN-α in most of the NPSLE+
CSF tested, whereas IFN-α was below the level of detection (0.8 pg/ml) in three normal CSF. The concentrations of IP-10 were highly significantly correlated with IFN-α in the NPSLE CSF, strongly supporting the experimental evidence presented that IP-10 in CSF is stimulated by IFN-α. Although pDCs have not been studied in the brains of NPSLE patients, elevated numbers of pDCs have been detected in the CSF of all neuroinflammatory diseases examined (39
). Alternatively, IM could be produced by CNS resident cells, and it is interesting that astrocytes are capable of IFN-α and IP-10 production in Aicardi-Goutières syndrome (40
The mechanisms responsible for IFN-mediated neuropsychiatric disease following systemic administration of IFN-α therapy are uncertain, but there is considerable evidence that this effect could be direct. Type I IFN receptors are found in the glia as well as neurons (41
). It has been suggested that IFNs alter brain function through alterations in serotonin, noadrenalin, the hypothalamic-pituitory-adrenal access or by generating toxic metabolites through the kynurenine pathway (17
). It is also possible that the neuronal effects are secondary to release of another factor or factors by nonneuronal cells since most cell types express type 1 receptors and there are several hundred IFN response genes. Finally, since only about a third of patients develop CNS manifestations on pharmacologic IFN therapy, additional host factors must determine susceptibility to the CNS effects.
The IFG activity of CSF obtained from NPSLE+ patients was much higher than that observed in other disease controls, suggesting that induction of IFN-α as well as other cytokines contribute to the pathogenesis of disease. In fact, the results reported herein show that almost all cytokines previously implicated in NPSLE could be induced by CSF autoantibodies in the presence of cell debris and appropriate responding cells. Some (IP-10 and IL-6), but not all, of these inflammatory mediators could be directly induced by type I IFNs depending on the responder cell type. This study therefore provides a strong mechanistic link between the subset of CSF autoantibodies directed against nucleoproteins and the inflammatory mediators described previously in NPSLE. Rather than excluding a role for direct Ab-mediated cytotoxic effects, these results suggest a two-step model of NPSLE where certain cytotoxic autoantibodies release nucleoproteins and other autoantibodies bind to form immune complexes, which then become powerful amplifiers of inflammation in the brain.
The NPSLE patients included in this study were heterogeneous with regard to their clinical manifestations (), so it is, at present, unclear whether CSF IFG activity is more commonly associated with one or more subtypes. Since there are 19 different specific NPSLE subtypes (23
), large multicenter studies comprising several hundred NPSLE patients will be needed to address this important question in the future. Similarly, knowledge regarding association with disease subtypes is also important since our studies have implications for therapy of NPSLE, including targeting of IFN-α and consideration of IVIG administration. IVIG has successfully been used to treat anecdotal cases of NPSLE (42
) as well as other neuroinflammatory and neurodegenerative disorders (43
The striking difference in the IFG activity in CSF compared with serum normalized for IgG concentrations was shown to be due to inhibitor(s) present in normal serum. Inhibitor(s) were absent or present in low concentrations in NPSLE CSF since dilution of CSF generally led to reduction in IFG activity, and addition of normal CSF to bioassays failed to attenuate IFG responses. In view of the 600-fold lower IgG concentration in CSF and the known inhibitory effect of IgG in IVIG in attenuating inflammation (33
), we examined the role of IgG as a possible inhibitory factor. Serum with spontaneous deficiencies of Ig (patients with CVID or XLA) or normal serum depleted of IgG had a significantly lower inhibitory activity for IFN-α compared with normal serum. Furthermore, addition of IgG to PBMC cultures also attenuated IFN-α stimulation by either NPSLE+
CSF or serum. Taken together, these findings indicate that IgG is one functional inhibitor of IFG activity in serum, consistent with the results of Bave et al. (45
). Interestingly, the lack of IgG in serum does not fully abrogate inhibition, suggesting that other inhibitors are present. Note that IgG is unlikely to inhibit IFN-α production simply by competition for FcγR since the only FcγR detected on pDCs is FcγRII (45
), a low-affinity receptor that should not bind monomeric IgG in preference to immune complexes. Furthermore, cell depletion experiments have revealed that the inhibitory effect is mediated, in part, by cells other than pDCs (D. M. Santer and K. B. Elkon, unpublished observations). Finally, since high concentrations of IgG are necessary for inhibition of IFN-α, we propose that a biochemically distinct subfraction of IgG, possibly arising through differential glycosylation (46
), may be responsible for the inhibitory effect observed in these studies.