Human epilepsy disorders exhibit peripheral cytokine changes that may originate at sites of neural dysfunction and contribute to seizure propagation (reviewed in [
12]). Sinha et al. [
15] analyzed serum IL-1β, IL-2, IL-4, IL-6, IFN-γ, or TNF-α detectability in 100 postictal patients and 100 healthy controls. None of the age- and sex-matched control subjects had detectable levels of any of these cytokines, whereas among epilepsy patients, the overall incidence was 74 %. Of the nine epilepsy patients who had a lumbar puncture, CSF levels of these cytokines were detectable in subject(s) with no serum levels. Significantly, none of the subjects who were serum-positive for a cytokine were CSF-negative for that cytokine. Serum IL-6, IL-1β, TNF-α, and MIP-1α levels were elevated in AED-resistant epilepsy patients and, in patients undergoing resection of the epileptogenic region, IL-1β, TNF-α, and MIP-1α levels decreased after 8 weeks postoperatively [
11]. These results implied that the epileptogenic brain tissue removed was either a direct or indirect source of these mediators. Until now, there has been a paucity of data on human brain tissue inflammation-related mediator levels [
10].
In the current study, however, none of these mediators appeared to be elevated proximal to the site of epileptogenesis. In fact, IL-6, IL-1β, and MIP-1α levels were higher in the temporal cortex than in the hippocampus, and TNF-α showed no regional or epilepsy-related differences, though levels in entorhinal cortex may have been lower than either the hippocampus or temporal cortex (see Additional file
2: Figure S2S). If both studies are valid, and the patient samples represent the greater population, then it must be concluded that the resected tissue had been an indirect stimulus for production of these mediators in the blood.
Proinflammatory mediator levels were hypothesized to be higher in the hippocampus at the site of epileptogenesis. This was true for eotaxin, IFN-γ, IL-2, IL-4, IL-12 p70, IL-17A, TNF-α, and ICAM-1. Interestingly, Sinha et al. [
15] found that blood levels of most of these were more detectable in epilepsy patients than in age-matched controls. Eotaxin (CCL11) recruits eosinophils by activating their CCR3 receptors. CCL11 transport across the blood-brain barrier (BBB) resulted in region-specific alterations of eotaxin brain levels [
36] and age-related increases in humans have been implicated in cognitive decline in a novel mouse model [
37]. IFN-γ, an important activator of macrophages and inducer of major histocompatibility class II immune/inflammatory activities, is associated with a number of autoinflammatory and autoimmune diseases. Its brain levels were virtually undetectable except in 10/49 hippocampus specimens exclusively from epileptic cases. IL-17A, also highest in the hippocampus, has synergistic effects with IFN-γ, IL-1, and TNF-α, acting as a proinflammatory mediator not unlike IFN-γ [
38]. TNF-α levels were among the lowest measured in this study (Additional file
2: Table S3B). Some variability in the convulsive effect of TNF-α has been noted previously and its relatively reduced levels in the entorhinal cortex may attest to this variability. The proconvulsive effect may be concentration-dependent, as with its role in
Shigella dysenteriae-related seizures at low concentrations and an anticonvulsive role at high concentrations [
39]. Lower picomolar concentrations may preferentially affect the p55 receptor pathway, increasing synaptic activity [
40], and promoting epileptogenicity in the longer term. ICAM-1 signaling is proinflammatory via recruitment of macrophages and leukocytes across the BBB [
22,
41]. In a mouse model of mTLE, ICAM-1 levels were locally induced in the hippocampus [
22].
Neurosurgical resection of the epileptic focus significantly reduced elevated blood levels of IL-1β, TNF-α, and MIP-1α in mTLE patients [
11]. TNF-α levels were highest in the hippocampus, consistent with the implications of prior findings. However, brain levels of IL-1β and MIP-1α were lower in the hippocampus compared to the temporal cortex. Moreover, none of these mediators showed any epilepsy-associated increases, as might have been predicted from that study [
11].
MIP-1α, IL-β, and IL-8 levels were highest in the temporal cortex. MIP-1α belongs to the C–C chemokine family (CCL3) and is involved in the recruitment and activation of macrophages, monocytes, and neutrophils. IL-8 (CXCL8) functions in inflammatory cell chemotaxis and phagocytosis, as well as angiogenesis. IL-8 has also been postulated, through NF-kB- and TNF-α-related mechanisms, to contribute to local inflammatory mechanisms in response to oxidative insults [
42]. IL-β and IL-α showed graded levels with cortical levels exceeding those in the hippocampus. IL-1β is involved in a plethora of inflammatory activities, including the induction of many other proinflammatory mediators; the induction of cyclooxygenase-2 (PTGS2) by this cytokine in the CNS is just one response to inflammation. IL-1β secreted by hypoxic astrocytes upregulated MCP-1 and ICAM-1 levels that are thought to play a crucial role in leukocyte recruitment [
43]. IL-α also mediates numerous inflammation-related activities, including TNF induction. Both are acute-phase cytokines that operate in the picomolar-femtomolar range. MIP-1β, which acts as a chemoattractant for natural killer cells, monocytes, and other immune cells, was also greater in the temporal cortex than in the entorhinal cortex.
Intriguingly, CRP levels were lower in the temporal cortex than in the hippocampus or entorhinal cortex. As a well-documented inflammatory biomarker, its levels follow those of proinflammatory cytokines; this may reflect an increased inflammatory load proximal to the site(s) of epileptogenesis. Paradoxically, IL-10, an anti-inflammatory cytokine, was found at higher levels in the hippocampus than in the entorhinal cortex but not significantly different from temporal cortical levels.
In the entorhinal cortex, IL-α and TNF-β levels were highest, compared to the hippocampus and temporal cortex. TNF-β (aka lymphotoxin alpha) is a highly inducible, cell surface molecule that mediates a variety of inflammatory responses, as well as apoptotic cell death.
Findings of neuroanatomic mediator level differences suggest brain-specific regulation; however, these might be confounded by blood levels, with brain tissue levels reflecting only differences in vascularization. If true, it could be expected that more vascularized brain tissue would regularly demonstrate higher mediator levels, but this was not the case. Moreover, a review of the recent literature on blood and CSF inflammatory mediator levels indicated, at least for many mediators, that individual CSF levels were greater than blood levels [
35,
44,
45]. In comparing brain tissue levels in this study with consensus blood levels from multiple studies (Additional file
2: Table S4J, K), median brain levels exceeded median blood levels for IP-10 (~threefold), MCP-1 (~fourfold), MIP-1β (~threefold), IL-8 (~fivefold), IL-17A (~fourfold), VEGF (~threefold), CRP (>tenfold), and ICAM-1 (~sixfold). Thus, the neuroanatomic differences observed were likely due to brain-related regulatory mechanisms rather than contamination from the blood or CSF.
Differences in inflammation-related mediators between epileptic and nonepileptic subjects were further hypothesized to suggest some significance for AED-resistant epileptogenicity. Caution must be taken when comparing these epilepsy cases with nonepilepsy cases as the nonepileptic subjects were (i) emergent neurosurgical patients and not healthy controls, (ii) comprised only four cases, and (iii) had an age distribution (64.5 ± 6.3) different from that of the epileptic group (38.5 ± 1.5)(nonetheless, there was no instance where an epilepsy-related difference could be fully explained by the age difference). Moreover, no data were available regarding circulating AED levels (or other medications) at the time of surgery for these cases, so confounding influences on mediator levels due to drug effects cannot be ruled out.
Eotaxin levels were lower in epilepsy specimens overall and particularly in the entorhinal cortex. Interestingly, phase 2 patients appeared to have higher eotaxin levels than cases without prior electrode placement. Higher levels in the hippocampus would suggest eotaxin is involved in both acute (nonepileptic) and chronic (epileptic) neuroinflammation. Although IP-10 levels were also reduced in the entorhinal cortex of epilepsy cases, this finding is suspect due to several high nonoutlier values in the nonepileptic group.
Proinflammatory cytokines IL-1β and IL-6 were higher in the nonepileptic cases. Both are acutely upregulated, so it is likely the emergent conditions warranting neurosurgical intervention contributed to the elevations. It is interesting to note that, for both mediators, the primary region of increase was in the hippocampus, suggesting that local control of these cytokines dominated, perhaps in the early phase of neuroinflammation.
Epileptic brain exhibited higher levels of IL-12 p70 overall; most of the nonepileptic specimens had no measurable levels. IL-12 p70 was also elevated in the cerebral cortex of pediatric epilepsy patients, compared to nonepileptic controls [
46]. The ratio of bioactive IL-12 p70 (p40:p35 heterodimer) to its p40 subunit appeared elevated both in the hippocampus and temporal cortex, indicating a more active IL-12 in these regions relative to the entorhinal cortex. IL-12/23 p40 levels were much greater than IL-12 p70 levels (Additional file
2: Table S3B), indicating that the manifestation of IL-12 bioactivity would be more dependent upon p35 subunit availability and/or the rate of heterodimer formation. In the periphery, IL-12 p70 stimulates T cell proliferation and differentiation, as well as natural killer cell activation. It also promotes induction of IFN-γ and TNF-α in T cells, through which it may block the formation of new blood vessels. Elevated brain IL-12 p70 in epileptics may be associated with transient leukocyte changes observed postictally, manifested by increased lymphocytes, neutrophils, NK cells, and NK-like T cells, with decreased T cells and CD4
+/CD8
+ ratios [
19]. These peripheral changes were all resolved by 24 h postictally. Elevated IL-12 p70 may also relate to alterations in specific blood cytokines and leukocyte numbers in adult epileptics that were differentially ameliorated by various AEDs [
13].
Vascular mediators CRP, ICAM-1, and VCAM-1 all showed epilepsy-related differences. Epileptic CRP levels were about half those of nonepileptics in the hippocampus and temporal cortex and 63 % in the entorhinal cortex. This was likely due to the emergent medical condition(s) of the nonepileptic cases. Unexpectedly, epileptic ICAM-1 levels in the entorhinal cortex were about half those of nonepileptic cases. Interestingly, blood levels of ICAM-5, a related gene located predominantly on neural cells, were ~fivefold lower in a group of epileptic patients than in age-matched controls [
47]. ICAMs are endothelial-, leukocyte-, and tissue-associated proteins important in cell-cell adhesion and leukocyte extravasation [
41,
48]. In this study, ICAM-1 levels were highest in the hippocampus, particularly among the epilepsy cases. Conversely, VCAM-1 levels were much lower in the epileptic cases across all three brain regions (11 % in the hippocampus, 23 % in the entorhinal cortex, 24 % in the temporal cortex). Moreover, the regional VCAM-1 differences noted in the nonepileptic cases were not apparent in the larger group of epileptic cases. While nonepileptic cases comprised a very small, nonage-matched sample, this might have implications for epilepsy-related changes in hippocampal VCAM-1. Not much is known about the contribution of human VCAM-1 to neuroinflammation, though its levels increased acutely in the blood and CSF after brain injuries [
49,
50]. Again, the emergent nature of the nonepileptic cases might explain these differences.
Though the epilepsy-related differences will require further investigation to corroborate, several other notable findings were derived from these results. Distinct regional levels of inflammation-related mediators likely have significance in neuroinflammatory physiology. For example, correlations of mediator levels within and between individual brain regions reveal the influences of intrinsic and extrinsic controls on neuroinflammatory regulation. These are summarized below.
Of the inflammation-related mediators with significant correlation(s), 70 % were with other mediators within a single brain region. Intraregional correlations support the notion of local control of multiple mediators, whereas correlations between different brain regions support more external mechanisms. Half of the observed correlations within a single brain region were in the temporal cortex, suggesting greater local regulatory mechanisms, compared to those of the hippocampus and entorhinal cortex.
Correlations of different mediators in different regions suggested more external control mechanisms (e.g., by diffusible factors or distributed neural cell inputs). Over 90 % of these types of correlations were between the hippocampus and the cortical brain regions (temporal cortex, 57 %; entorhinal cortex, 36 %). Only 7 % of interregional correlations of different mediators were between the entorhinal and temporal cortices. Although it cannot be known from these data in which direction controls may be exerted, the preponderance of hippocampal involvement along with their neuroanatomical relationships suggest a focus of hippocampal control of inflammation-related mediator levels between these regions.
Only IL-1α, IP-10, MCP-1, IL-2, VEGF, ICAM-1, and VCAM-1 levels were correlated between different brain regions. Of these, except for VCAM-1, tissue level differences were observed. IL-1α was greatest in the entorhinal cortex and diminished from the temporal cortex to the hippocampus; IP-10 was also lowest in the hippocampus, whereas MCP-1 was greater in the hippocampus and temporal cortex than in the entorhinal cortex; and IL-2, VEGF, and ICAM-1 were higher in the hippocampus than in both cortical regions. Thus, individual mediators showing tissue level differences and interregional correlations suggest the existence of common, external regulatory mechanisms with local gain controls.
Functional implications
Systemic inflammatory cells, as well as activated intrinsic neuroinflammatory cells, likely contribute to neurochemical and neurophysiologic dysfunction in the affected brain regions. Inflammatory mediators produced by both types of cells can uniquely affect the brain, with little or no manifestation in the periphery [
23]. It has been proposed that glial cells establish a cytokine/chemokine network in the ischemic brain—“activated microglia produce… various types of cytokines… which activate astrocytes to synthesize chemokines…. Chemokines in turn activate and/or recruit microglial cells in the injured site” [
43]. Cytokines IL-1 and TNF-α, as well as chemokines MCP-1, RANTES, and IL-8 were implicated in these networks [
43].
Interestingly, the majority (110/141) of mediator-to-mediator associations observed in this study were accounted for by only eight mediators, namely, eotaxin (22), ICAM-1 (17), MCP-1 (16), IL-4 (14), IL-6 (14), IL-8 (11), MIP-1α (9), and VCAM-1 (8). This observation implies a hierarchical dynamic in which mediators with multiple correlations may regulate, directly or indirectly, the levels of other mediators. These could be one basis of neuroinflammatory functional networks.