We used a qPCR array to measure inflammatory gene expression in 12 RE specimens and 12 CD specimens, and then correlated the expression of genes that differed between the two groups with clinical parameters. Selection of the CD specimens from the UCLA Pediatric Surgery Program’s tissue bank was based on choosing patients whose ages at surgery were approximately the same as the much rarer RE cases that took approximately ten years to accrue. There were no other exclusion criteria. The array included genes encoding pro-inflammatory T helper 1 (Th1) and anti-inflammatory T helper 2 (Th2) cell cytokines, chemokines, Toll-like receptors and other factors involved in downstream signaling. The expression levels of only eight transcripts significantly differed between the RE and CD specimens. With the exception of HPRT mRNA, the transcripts were expressed at higher levels in the RE specimens. Reduced HPRT, a housekeeping gene on the array, was unexpected and may reflect disease-associated atrophy. Alternatively, a reduced level of HPRT in brain tissue may be significant since a decrease in this enzyme would be expected to affect purine metabolism, with possible effects on brain function . Analysis of HPRT protein levels in more RE samples will be necessary to establish the significance of this finding to the disease process.
In RE there is clear histopathologic evidence for the involvement of CD8+ T cells in the disease process [6–8, 23]. In agreement with these data, four of the seven genes whose mRNA levels were higher in RE compared with CD cases encode proteins involved in Th1-driven immune responses, namely IFN-γ, CCL5, CXCL9 and CXCL10. Activated CD8+ cytotoxic T cells (Tc1) and CD4+ Th1 cells produce IFN- γ [37, 38], thus infiltrating CD8+ T cells in neuropil and CD4+ T cells in Virchow-Robin spaces and leptomeninges (Figure 7) could be sources of IFN-γ transcripts in the RE brain specimens. Although the immunopathology in RE appears to be driven by Tc1 cells, CD4+ cells, in the perivascular space may play a role in sustaining Tc1 activity [39–41]. Natural killer (NK) cells, NKT cells, and γδ T cells produce IFN-γ [37, 42], which may also account for the IFN-γ transcripts in RE tissue. To date, we and others have found no evidence for significant numbers of NK cells in RE brain tissue . However, we have recently identified γδ T cells in brain infiltrating lymphocytes isolated from fresh RE brain tissue (unpublished data).
IFN-γ can induce major histocompatibility complex (MHC) class I molecules on the surface of neurons, rendering them vulnerable to attack by autoantigen-sensitized MHC class I-restricted Tc1 cells . Further, IFN-γ has been shown to induce bursting of hippocampal pyramidal neurons in vitro, providing a possible link between T cells and epileptogenesis.
It has been reported that IFN-γ induces the production of CXCL9 by microglia and CXCL10 by microglia and astrocytes , and promotes IL-1-induced synthesis of CCL5 by astrocytes . The positive correlation between the relative amounts of CXCL9, CXCL10, CCL5 and IFN-γ mRNA that we observed among the RE specimens is consistent with these reports. All three chemokines have been implicated in attracting Th1, Tc1, γδ T cells and NK cells to sites of inflammation . The presence of CXCR3, the receptor for CXCL9 and CXCL10 , and CCR5 (CCL5 receptor) on infiltrating T cells in brain sections from a single RE case has been documented . Other single patient studies have provided evidence for the expression of CCL5 and CXCL10 in RE brain tissue [50, 51].
Quantitative PCR allowed correlations to be made between the amount of inflammatory gene transcripts and clinical variables. Notably, much higher levels of IFN-γ and CXCL9 mRNAs were detected in specimens from patients that had undergone surgery within shorter times from disease onset compared to later times. This suggests that there is a pronounced Th1 immune response in the early phase of the disease that declines after 1 to 2 years. The observation of large numbers of CD4+ and CD8+ lymphocytes in sections from specimens in which high levels of IFN-γ mRNA were detected is consonant with an initial Th1 polarized response. In support of these data, it was previously reported that cerebrospinal fluid levels of IFN-γ were higher in the earlier stages of RE .
A role for the Fas/FasL-mediated cell death pathway in RE is indicated from the qPCR data. Based on the observation of granzyme B immunoreactivity in T cells in close apposition to neurons and astrocytes, it has been suggested that MHC class I-restricted killing occurs by the perforin lytic pathway [6, 9]. However MHC class I-restricted killing of neurons can also occur by Fas ligand-induced apoptosis . On the other hand, the detection of FasL transcripts in the present study could be explained by activation-induced cell death of activated T cells in the brain . In experimental autoimmune encephalitis, astrocytes expressing FasL have been implicated in T cell homeostasis .
In contrast to the chemokines associated with a Th1 response, CCL22, which binds CCR4 , is associated with a Th2 polarized response, which can facilitate B cell activation . The finding of higher levels of this chemokine in RE brain tissue is therefore consistent with the presence of circulating antibodies to neuronal proteins in some RE patients [10, 11, 14–16]. Unlike the Th1 cytokines, CCL22 expression did not strongly correlate with time from disease onset to surgery, supporting the notion that a B cell response may be a secondary consequence of tissue destruction mediated by Tc1 cells . CCL22 may also be involved in recruiting immunosuppressive T regulatory cells into the brain to modulate the Tc1 response .
A positive correlation was observed between the extent of tissue destruction as measured by MRI and the level of CCL23 mRNA. CCL23 can act as a chemoattractant for monocytes . A role for monocytes in RE pathogenesis is therefore possible as is the case in viral encephalitis . Since monocytes also produce vascular endothelial growth factor , this may explain the vascular changes that have been observed in resected RE but not CD brain tissue .