The balance of inhibitory and excitatory cytokines is differently regulated in vivo and in vitro among therapy resistant epilepsy patients

Epilepsy Research 59 (2004) 199–205

The balance of inhibitory and excitatory cytokines is differently regulated in vivo and in vitro among therapy resistant epilepsy patients Janne Hulkkonen a , Elina Koskikallio a , Sirpa Rainesalo a,b , Tapani Keränen b , Mikko Hurme a , Jukka Peltola b,∗ b

a Medical School, University of Tampere, Tampere, Finland Department of Neurology and Rehabilitation, Tampere University Hospital, Tampere, Finland

Received 4 February 2004; received in revised form 15 April 2004; accepted 23 April 2004 Available online 24 June 2004

Abstract Purpose: Excessive neuronal activity and seizures directly stimulate cytokine expression. In this study we investigated cytokine production in circulating blood and peripheral blood mononuclear cells (PBMC) in order to assess the cellular origin of these cytokines in patients with therapy resistant epilepsy. Methods: We compared the levels of plasma IL-1␤, IL-1Ra and IL-6 in 10 patients with therapy resistant localization-related epilepsy and in healthy volunteers. The spontaneous and exogenously stimulated production of these cytokines was studied in PBMC cultures using EIA. Moreover, cell-specific cytokine production was studied using flow cytometry. Results: Highly pro-inflammatory cytokine profile (high IL-6, low IL-1Ra and low IL-1Ra/IL-1␤ ratio) was observed in plasma from patients with epilepsy. Spontaneous and LPS stimulated cytokine release was similar in PBMC cultures of patients and control subjects. When cells were stimulated with OKT3 the cytokine response profiles in patients with epilepsy were almost opposite (anti-inflammatory) to the profile which was observed in circulating blood. Low IL-6 was observed in cell cultures of patients when stimulated with PDBu + A23187. Flow cytometric analysis revealed that the percentages of IL-1␤, IL-1Ra and IL-6 positive monocytes were similar in patients and control subjects. Conclusions: Patients with therapy resistant epilepsy display a pro-inflammatory profile of plasma cytokines without any evidence of increased production from PBMC. These results suggest that the most likely origin for these cytokines is the brain, where cytokines can exert neuromodulatory functions. © 2004 Elsevier B.V. All rights reserved. Keywords: Cytokines; Epilepsy; Interleukin-6; Interleukin-1; Interleukin-1 receptor antagonist

1. Introduction

∗ Corresponding author. Tel.: +358 3 247 4713; fax: +358 3 247 4351. E-mail address: [email protected] (J. Peltola).

There is a growing amount of experimental and clinical evidence suggesting that cytokines are involved in epilepsy as disease modifying molecules (Jankowsky and Patterson, 2001; Peltola et al., 2001a). Excessive neuronal activity (seizures) may directly

0920-1211/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2004.04.007

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increase cytokine expression (Allan and Rothwell, 2001). Seizures stimulate interleukin (IL)-1␣, IL-1␤ and IL-6 levels in the rat brain (Jankowsky and Patterson, 2001). We have previously reported increased concentrations of IL-6 and IL-1 receptor antagonist (RA) with unchanged IL-1␤ levels in cerebrospinal fluid (CSF) and plasma in patients after tonic-clonic seizures (Peltola et al., 1998, 2000a) without evidence of neuronal damage (Palmio et al., 2001). IL-1␤, IL-1␣ and IL-1RA gene pleomorphism has been associated with therapy resistant epilepsy (Kanemoto et al., 2000; Peltola et al., 2001b) and IL-1 levels are increased during epilepsy in the human brain (Sheng et al., 1994). In vitro production of cytokines from stimulated mononuclear cells from epileptic patients has been investigated in one study (Pacifici et al., 1995). The peripheral blood mononuclear cells showed greater production of IL-1␣, IL-1␤ and IL-6 in response to stimulation in patients than in control subjects (Pacifici et al., 1995). The soluble cytokines measured in peripheral blood may originate from the central nervous system, peripheral blood cells or endothelial cells (Peltola et al., 2000a). The purpose of this study is to investigate peripheral blood mononuclear cell production of cytokines by modern techniques in order to assess the cellular origin of these cytokines. We compared the levels of IL-1␤, IL-1Ra and IL-6 in circulating blood in patients with therapy resistant localization-related epilepsy and in healthy volunteers. The production of

these cytokines as well as cellular cytokine responses to various exogenous stimuli was studied using EIA and cell specific flow cytometric analysis of intracellular cytokines.

2. Patients and methods 2.1. Blood sampling Blood samples from ten patients with therapy resistant epilepsy, treated at the outpatient clinic of Tampere University Hospital, were obtained after a written informed consent. Clinical data on the patients are presented in Table 1. For all of the patients at least 48 h from previous seizure was elapsed before sampling. Blood samples for the determination of the reference range for plasma cytokines were obtained from healthy blood donors (n = 400, age range 18–60 years) of the Finnish Red Cross Blood Transfusion Centre, Tampere, Finland. For the in vitro studies the control blood samples were drawn from members of our laboratory staff. Citrate was used as anticoagulant in all samples. The study was approved by the Ethics Committee of Tampere University Hospital. 2.2. Cell separation The peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by centrifugation

Table 1 Clinical characteristics of the patients Patients (M/F)

Age

Duration of epilepsy

No. of seizures/month

Medication

Epileptic syndrome

M F M F F M F F M F

34 65 35 27 31 49 22 53 62 52

25 63 31 17 17 42 22 49 48 37

3 2 1 3 3 1 1 2 3 3

CBZ/VGB CBZ/VGB CBZ CBZ/LTG OXC/LTG CBZ/GBP CBZ/VGB CBZ/TPR CBZ/LTG/CLB CBZ/TGB

FLE TLE TLE-HS TLE TLE TLE FLE TLE-HS TLE TLE

CBZ: Carbamazepine, VGB: Vigabatrin, LTG: Lamotrigine, OXC: oxcarbazepine, GBP: Gabapentin, TPR: Topiramate, FLE: frontal lobe epilepsy, TLE: temporal lobe epilepsy, HS: hippocampal Sclerosis.

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over a Ficoll-Isopaque layer (Pharmacia, Upsala, Sweden). The cells were washed twice with complete medium consisting of RPMI 1640 (Gibco, Paisley, UK), 10 mM hepes (ICN Biomedical, Costa Mesa, CA, USA), 10% heat-inactivated fetal calf serum (Gibco), 2 mM l-glutamine and antibiotics. 2.3. Culture conditions and mitogens The PBMCs were tested for their capacity to release IL-1␤, IL-1Ra and IL-6, both spontaneously and after various stimuli. The cells were cultured in complete medium (above) at a concentration of 106 mL−1 in 96-well flat-bottomed plates (Falcon). Cells were alternatively stimulated with a pre-tested optimal dilution of plate-bound anti-CD3 antibody (OKT3; Pharmingen, San Diego, CA, USA) or phorbol dibutyrate (PDBu, 100 ng/mL; Sigma Chemicals Co., St. Louis, MO, USA) combined with calcium ionophore (A23187, 100 ng/mL; Calbiochem, La Jolla, CA, USA), or lipopolysaccaride (LPS, 1 ␮g/mL; Sigma). After 24 h in culture, cell culture supernatants were collected, centrifuged and stored at −70 ◦ C until cytokine determination. For a reference of cell stimulation protocols, see James (1991). 2.4. ELISAs IL-1␤, IL-1Ra and IL-6 concentrations were determined using commercially available enzyme linked immunosorbent assays (Pelikine Compact human IL-1␤ and human IL-6 ELISA kits, CLB, Amsterdam, Netherlands and Quantikine human IL-1Ra immunoassay, R&D systems, Mp, USA) following the manufacturer’s instructions. The optical density of individual wells was determined with a “Multiscan Biochromatic 348” spectrophotometer (Labsystems, Helsinki, Finland). The detection limits of the assays were 0.4 pg/mL for IL-1␤, 0.6 pg/mL for IL-6 and 46.9 pg/mL for IL-1Ra. The interassay variability of these assays is <10%. 2.5. Intracellular IL-1β, IL-1Ra and IL-6 detection Expression of intracellular IL-1␤, IL-1Ra and IL-6 was studied by flow cytometry using FacsCalibur

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flow cytometer (Becton Dickinson Immunocytometry systems, Palo Alto, CA, USA). Peripheral blood mononuclear cells (106 mL−1 ) were stimulated with LPS at concentration 1 ␮g/mL. After 4 h in culture, the cells were permeabilized and stained for intracellular cytokines with a commercially available cytostain kit (Cytofix/Cytoperm kit, Pharmingen, San Diego, CA, USA) following the manufacturer’s protocol. The protein transport inhibitor Brefeldin A (BFA, 10 ␮g/mL; ICN) had been added to the cell culture 4 h prior to staining. The PE-labeled antibodies for IL-1␤, IL-1Ra and IL-6 (FastImmune anti-Hu IL-1␤, IL-1Ra and IL-6), FITC/PE labelled CD45/14 (LeucoGate CD45/14) and PE-labeled intracellular isotype control (clone X40) were all purchased from Becton Dickinson Immunocytometry Systems (Mountain View, CA, USA). The FITC-labeled antibody for CD14 (clone Tuk4) and FITC-labeled cell surface isotype control (clone DAK-GO5) were purchased from Dako (Glostrup, Denmark). 105 cells were collected for flow cytometric data analyses. The proportions of cytokine positive monocytes were analysed by comparing the percentages of anti-cytokine labelled CD14+ cells against a control sample labeled with non-spesific antibodies. The cytokine production intensities were compared using mean fluorescence intensity (MFI) values of these same cells after subtracting the background MFI from the specific MFI. For a more through review of the method, see Prussin and Metcalfe (1995). 2.6. Statistical analyses Statistical analyses were performed using Statistica software (version Win 5.1D; StatSoft Inc., Tulsa, USA). Due to low number of patients non-parametric statistics based on Mann–Whitney U-test were used for the comparison of group medians. Correlations were calculated using the Spearman rank order correlation test. Differences were considered significant at significance level <0.05. In order to increase statistical power and improve control over confounding factors the number of control subjects substantially exceeded the number of patients in plasma analyses. As high effect size was expected in vitro the number of cases and controls was kept low in these analyses. By this approach the post-analysis power estimation for high effect size significant differences observed in

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Table 2 Plasma cytokine levels

3.2. In vitro cytokine production

Cytokine

Patients (n = 10)

Controls (n = 400)∗

P

IL-1␤ IL-1Ra IL-6

6.6 (1.3–9.9) 223 (131–266) 2.1 (1.22–2.49)

5.8 (2.2–13.6) 587 (372–852) 1.2 (0.7–2)

0.973 <0.001 0.057

Values are median (quartile range) given in pg/mL. ∗ n = 200 for IL-1Ra.

this study suggested a power from 75 to 100% at the probability level of 0.05. 3. Results 3.1. Cytokine plasma levels Plasma levels of IL-1␤, IL-1Ra and IL-6 are given in Table 2. The plasma IL-1Ra was significantly lower in epilepsy patients compared with controls. The patients had a trend towards high plasma IL-6. The IL-1Ra/IL-1␤ ratio in patients was highly pro-inflammatory (median ratio of patients 28.1, quartiles 10.4–109; median ratio of controls 114, quartiles 42.3–279, P = 0.020). A positive correlation was observed between plasma IL-1␤ and IL-6 in controls (R = 0.50, P < 0.0001). This trend was less distinct in epilepsy patients (R = 0.44, P = 0.21). A negative correlation between plasma IL-6 and IL-1Ra was observed in the patients (R = −0.64, P = 0.047), but not in the controls (R = 0.04, P = 0.54).

Spontaneous cell culture cytokine release was quite similar in patients and control subjects (Table 3). This also held true in LPS stimulated cell cultures. However, when cells were stimulated with OKT3 or PDBu + A23187, the cytokine profiles in patients with epilepsy were clearly different from those observed in the control group. After OKT3 stimulation the IL-1␤, IL-6 and IL-1Ra productions were markedly lower in patients than in control subjects. Low IL-6 response was observed also in cell cultures of patients after PDBU + A23187 challenge. Opposite to plasma, the median IL-1Ra/IL-1␤ ratio was higher in patients (129, quartiles 67.9–245) than in controls (31.2, quartiles 15–134, P = 0.013) in OKT3 stimulated cell cultures. As was the case with plasma IL-1␤ and IL-6, the production of these cytokines also correlated in vitro. Striking correlation was observed both in the spontaneous and OKT3 stimulated release of these factors in patients (R = 0.88 and 0.92, P < 0.001) as well as in control subjects (R = 0.87 and 0.95, P < 0.001). 3.3. Intracellular cytokine detection As the monocyte-macrophage lineage cells are the main cell types responsible for IL-1␤, IL-1Ra and IL-6 production in human, we analysed the cell-specific production of these cytokines from peripheral blood monocytes using flow cytometry. The monocyte

Table 3 In vitro cytokine responses for different stimuli among patients (n = 10) and controls (n = 10) Group IL-1␤ Patients Controls IL-1Ra Patients Controls IL-6 Patients Controls

No stimulus

OKT3

PDBu + A23187

LPS

0 (0–11.5) 5.3 (3.2–7.6) P = 0.435

3.5 (0–8.4) 53.1 (5.5–342) P = 0.069

549 (442–594) 617 (380–1420) P = 0.496

3990 (2670–10200) 4410 (2570–6050) P = 0.880

1075(472–1536) 1031 (504–1316) P = 0.705

660 (467–1345) 2900 (830–5600) P = 0.013∗

5580 (4480–7600) 5440 (4160–11080) P = 0.880

7180 (5360–13300) 7800 (6800–8480) P = 0.880

22.3 (0–49.6) 46.5 (11.4–61.6) P = 0.223

26.4 (0–45.9) 593 (17.7–1290) P = 0.068

719 (263–1420) 4310 (2720–4920) P = 0.016∗

21200 (17200–37000) 23800 (16000–26600) P = 0.762

Values are median (quartile range) given in pg/mL. ∗ Statistically significant values.

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Table 4 Cytokine responses for LPS stimulation in monocytes Parameter

Patients

Controls

P

IL-1␤ posive cells (%) IL-1Ra positive cells (%) IL-6 positive cells (%) IL-1␤ production MFI IL-1Ra production MFI IL-6 production MFI

78.3 (73.0–80.9) 32.8 (29.4–41.4) 74.3 (72.1–78.2) 489.4 (344–675) 60.9 (42.0–82.9) 1225.7 (694–1420)

78.0 (67.3–88.7) 30.0 (25.5–49.0) 76.3 (68.8–85.4) 379 (352–431) 53.9 (49.4–78.0) 1071.2 (903–1270)

0.545 0.940 0.597 0.257 0.940 0.762

Values are median (quartile range). MFI: mean fluorescence intensity.

derived cytokine production after LPS challenge is given in Table 4. The percentages of IL-1␤, IL-1Ra and IL-6 positive monocytes were similar in patients and in control subjects. Moreover, cell specific mean cytokine production intensities (MFIs) of were similar in these groups.

4. Discussion Our study shows that during the interictal state, patients with treatment resistant epilepsy have (1) decreased plasma levels of anti-inflammatory cytokine IL-1RA, (2) a trend towards elevated plasma levels of soluble pro-inflammatory cytokine IL-6 and (3) altered stimulus-dependent response of blood mononuclear cells (4) which is not derived from monocytes. These results suggest that other factors than increased cytokine responses in peripheral blood mononuclear cells are causing the net increase in the plasma levels of these cytokines observed postictally in patients with epilepsy (Peltola et al., 1998, 2000a). In addition to plasma cytokines we explored the effect of three different activation pathways on cell culture cytokine production in vitro. The pharmacological cell activation by phorbol dibutyrate and calcium ionophore A23187 leads to the direct induction of calcium-dependent intracellular signals (such as protein kinase C). This activation is independent of the cell type and cell surface antigens (James, 1991). In addition to this, we tested physiological activation pathways by inducing transmembrane signalling with LPS and OKT3. The LPS-induced activation is principally targeted at monocyte-macrophage lineage cells as LPS mediates its effect by binding with cell surface antigen CD14 on monocytes. In turn,

plate-bound OKT3 directly crosslinks T cell receptor and polyclonally activates T cells (James, 1991). Albeit we explored relatively low number of subjects (n = 10) in vitro in current information-intensive study, we could demonstrate clearly distinct cytokine profiles in patients and in control subjects. As was expected from the theoretical basis (see above), the magnitude of in vitro cytokine response was stimulus dependent. After OKT3 challenge the inducibility of the cytokine production was lower in therapy resistant epilepsy patients than in controls. Moreover, the IL-1RA/IL-1␤ ratio of patients in OKT3 stimulated cell cultures was opposite (i.e. anti-inflammatory) to the profile observed in their circulating blood. After the pharmacological stimulus with PDBU + A23187 the IL-6 production was markedly lower in patients than in controls, while no difference was observed in basal and LPS-stimulated cytokine responses. The lack of normal variation in the cytokine responses and the high effect size of difference after OKT3 stimulus indicate a biologically significant decrease in the T cell reactivity of epilepsy patients. Most of all, it can be further concluded from these cell culture data that other factors than altered PBMC production of cytokines are causing the net decrease in IL-1RA and IL-1RA/IL-1␤ ratios in the circulating blood of patients. The PBMC production of cytokines in epilepsy patients has been investigated only in one previous study (Pacifici et al., 1995). In contrast to our results, Pacifi et al. reported increased cytokine production in blood mononuclear cells. There are however, a number of important differences in the settings of their study and the present study. The patient population in the study by Pacifi et al. included only seizure-free patients whereas we investigated therapy resistant

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patients. Moreover, Pacifi et al. analyzed heparinized venous blood samples. Heparin may be, however, contaminated by endotoxin (i.e. LPS) which may non-specifically activate lymphocytes during the sampling and thus distort cytokine measurements (Riches et al., 1992). To exclude this possibility we consistently used citrate as an anticoagulant in the current study. As circulating and in vitro cytokine levels were differently regulated in patients with epilepsy it is evident that different mechanisms are responsible for maintaining the local and systemic balance of these neurostimulatory factors. These issues also raise the question whether other cell types, such as endothelial cells and central nervous system cells, may play a role in maintaining this systemic proinflammatory cytokine balance. It is known that increased IL-6 is one of the best markers for increased IL-1 activity (Dinarello, 1996). We also observed a strict correlation in the production of these two cytokines. This correlation is interesting, as a genetic association has been reported in a group of patients with therapy resistant localization-related epilepsy (Kanemoto et al., 2000; Peltola et al., 2001b). In patients with temporal lobe epilepsy and hippocampal sclerosis an overrepresentation of the homozygotes for Il-1␤-511 allele 2 was observed; allele 2 is associated with high IL-1␤ production (Hulkkonen et al., 2000). We observed that haplotypes with a propensity to increased inflammatory cytokine responses (increased production of IL-1␤ and decreased production of IL-1RA) were associated with therapy resistant epilepsy (Peltola et al., 2001b). In the present study, we observed a trend towards elevated plasma levels of whereas the concentrations of IL-1RA were decreased in a patient population comparable to that in which an association with relevant cytokine pleomorphism was observed. The possible consequences for constant pro-inflammatory stimulation of peripheral tissues include stimulation of the acute phase reaction and autoantibody production. In patients with recent single tonic-clonic seizures plasma IL-6 levels correlated with laboratory indicators of inflammation (Peltola et al., 2002). These immunological aspects are also interesting in patients with therapy resistant localisation-related epilepsy in whom increased production of anti-nuclear and antiphospholipid antibodies was found (Peltola et al., 2000b).

To what extent the changes in the peripheral circulation of soluble cytokines reflect the central nervous system compartment remains conjectural in this study. In our previous studies CSF concentrations of cytokines have been substantially higher than those in plasma suggesting the possibility of increased production in the brain (Peltola et al., 1998, 2000a). Cytokines may have neuromodulatory roles in the brain. Experimental studies suggest that IL-1␤ prolongs the duration of kainic acid induced seizures and promotes neuronal damage (Vezzani et al., 1999), whereas its effects are blocked by IL-1 receptor antagonist (Vezzani et al., 2000). IL-6 has both neuroprotective and neurotoxic effects in the brain, and its role in seizures is not yet well documented. Our study gives additional evidence for activation of the cytokine network in patients with therapy resistant epilepsy. Since this activation may promote important neuromodulatory functions in the brain and also cause immunological activation further clinical and experimental studies to clarify its role in epilepsy are needed. References Allan, S.M., Rothwell, N.J., 2001. Cytokines and acute neurodegeneration. Nat. Rev. Neurosci. 2, 734–744. Dinarello, C.A., 1996. Biologic basis for interleukin-1 in disease. Blood 87, 2095–2147. Hulkkonen, J., Laippala, P., Hurme, M., 2000. A rare allele combination of the interleukin-1 gene complex is associated with high interleukin-1 beta plasma levels in healthy individuals. Eur. Cytokine Netw. 11, 251–255. James, S.P., 1991. Measurement of basic immunologic characteristic of human mononuclear cells. In: Coligan, J., Kruisbeek, A., Marquelies, D. (Eds.), Current Protocols in Immunology, vol. 1. Wiley Interscience, pp. Section 7.10.12. Jankowsky, J.L., Patterson, P.H., 2001. The role of cytokines and growth factors in seizures and their sequelae. Prog. Neurobiol. 63, 125–149. Kanemoto, K., Kawasaki, J., Miyamoto, T., Obayashi, H., Nishimura, M., 2000. Interleukin (IL)1beta, IL-1alpha, and IL-1 receptor antagonist gene polymorphisms in patients with temporal lobe epilepsy. Ann. Neurol. 47, 571–574. Pacifici, R., Paris, L., Di Carlo, S., Bacosi, A., Pichini, S., Zuccaro, P., 1995. Cytokine production in blood mononuclear cells from epileptic patients. Epilepsia 36, 384–387. Palmio, J., Peltola, J., Vuorinen, P., Laine, S., Suhonen, J., Keranen, T., 2001. Normal CSF neuron-specific enolase and S-100 protein levels in patients with recent non-complicated tonic-clonic seizures. J. Neurol. Sci. 183, 27–31. Peltola, J., Eriksson, K., Keranen, T., 2001a. Cytokines and seizures. Arch. Neurol. 58, 1168–1169.

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