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January2008 Vol.45 Issue:      1 Table of Contents
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Serum IgA, IL-2, IL-6 and TNF- α Concentrations in Patients with Pharmacoresistant Epilepsy

Ehab S Mohammed1, Mohammed F ELShater1, Hazem A. Fayed1, Ehab A El-Seidy1, Salwa A Essa2
Departments of Neuropsychiatry1, Clinical Pathology2, Tanta University

ABSTRACT

 

Purpose: The concept that the immune system plays a role in the epileptogenesis was first proposed more than 20 years ago. Since then, several laboratory and clinical studies have reported on the existence of a variety of immunological abnormalities in epileptic patients, on the observation of favourable responses of refractory epilepsy syndromes to immunomodulatory treatment, and on the association of epilepsy with certain well-known immune-mediated disorders. In this study we try to verify if there is a link between pharmacoresistant epilepsy and immune system. Methods: We compared the serum levels of IgA, IL-2, IL-6, and TNF-α, in 45 patients with pharmacoresponsive epilepsy, in 30 patients with pharmacoresistant epilepsy and in 15 healthy reference subjects. Results: Low serum levels of IgA were found in epileptic patients than reference subjects and in pharmacoresistance epilepsy group than pharmacoresponsive epileptic group .There were significant high serum levels of IL-2, IL-6 and TNF-α in epileptic patients than reference subjects and in pharmacoresistance group than pharmacoresponsive epileptic group. There was significant association between the serum levels of the IL-2, IL-6 and TNF-α and frequency of seizures and not the duration of the epilepsy or its type. Conclusion: These results give additional evidence for activation of the cytokine network and the magnitude of these changes is related to severity of seizures. Since this activation may promote important neuromodulatory functions and may serve as a link between excessive neuronal activity and various immunological changes that can lead to refractoriness of seizures.

(Egypt J. Neurol. Psychiat. Neurosurg., 2008, 45(1): 185-192)

 




INTRODUCTION

 

Despite of the introduction of many novel antiepileptic drugs (AEDs) over recent years, approximately one-third of people with epilepsy will never achieve remission1. In the literature, there is no universally accepted definition of pharmacoresistant epilepsy, some define it as a failure of two to three AEDs, or lack of control within a defined time period after diagnosis (e.g. 1-2 years)2.

The biological basis of pharmacoresistant epilepsy has not been well established. There is good evidence that pharmacoresistant seizures may be self perpetuating in some individuals, potentially causing irreversible neuroplastic changes including denteretic sprouting, synaptic reorganization, glial proliferation and neuronal cell death3. Pharmacological refractoriness may be genetically determined as a consequence of ion channel mutations, or over expression of multidrug resistance gene,syndromic classification, causative neuropathology and reactive autoimmunity4.

Based on the bidirectional interaction between the central nervous system and immune system, it is attractive to speculate that immune mechanisms may involved in the pathogenesis of at least some forms of epilepsy or that epileptic seizures may affect the immune system 5. Many studies have reported on aberrant levels of one or more Ig subclasses in epileptic patients, but the data are inconsistent and sometimes conflicting6,7. Also, there is a growing amount of experimental and clinical evidence suggesting that cytokines are involved in epilepsy as a disease modifying molecules8. In this study we try to verify a link between pharmacoresistant epilepsy and immune system.

 

PATIENTS AND METHODS

 

Study subjects:

The present study was conducted at outpatient clinic of Neuropsychiatry and Clinical Pathology Departments, Tanta University Hospital. The target group consisted of 41 male subjects (54.6%) and 34 female subjects (45.3%). The mean±SD of age of the patients was 33.56±9.13 years. The patients were divided into 2 groups. Group A consisted of 45 patients with pharmacoresponsive epilepsy (seizures free at least in the last 6 months) and their mean±SD of age was 29.40±6.47 years. Group B consisted of 30 patients with pharmacoresistant epilepsy (more than one seizure /month) and their mean±SD of age was 39.80±9.05 years. A structured questionnaire was used to record the demographic data, type (the type of epilepsy was determined according to ILAE, 1989), duration of epilepsy, AEDs in use, frequency of seizures. According to ILAE, 1989, Fourty-one patients (54.6%) had partial epilepsy (with and without secondarily generalized seizures), thirty-four patients (45.3Κ%) had primary generalized epilepsy. The most commonly used antiepileptic drugs (as monotherapy or polytherapy) were carbamazipine, valproate, phenytoin, oxcycarb-zepine and lamotrigine. A reference group consisted of 15 healthy volunteer subjects of matched age and sex with the patients group. The mean age of the reference group was 34.07±5.71 years.

 

Sampling:

        Three ml of venous blood were withdrawn under complete aseptic precautions from each subject in a plain tube which was incubated at 37 c for 30 minutes and then centrifuged at 4000 rpm for 10 minutes, then the serum was collected and stored at -70 c till the time of assay of IL-2,IL-6 , TNF-α and IgA.

All cases and control groups were subjected to the following investigations:

1-      Quatitative measurement of serum cytokines IL-2, IL-6 and TNF α - using ELISA technique by commercially available Kit, the Quantikines immunoassay supplied by R&D systems (Minneapolis, USA). The assay employs the quantitative sandwich enzyme immunoassay technique .The test was done according to the manufacturer's instructions.

2-      Quantitative estimation of serum IgA level. The assay was done using sandwich enzyme immunoassay (EIA) technique by a kit supplied by R&D systems (Minneapolis, USA). The manufacturer's procedure was followed.

 

Statistical analysis:

       Data are presented as mean±SD. Analyses were performed with SSPS statistical package version 12 (SSPS, USA).

 

RESULTS

 

Immunoglobulin IgA:

Low serum IgA was significantly more in epileptic patients than reference subjects (Table 1). Serum IgA concentrations were lower in group B than group A (Table 2 and Fig 1). There was no significant association between low serum IgA and neither the duration of the epilepsy nor its type. There was no significant association between frequency of seizures and low serum IgA (Table 3).

 

Serum cytokines:

There were significant high serum levels of IL-2, IL-6 and TNF-α in epileptic patients than reference subjects (Table 1).

There were significantly high levels of IL-2, IL-6 and TNF- α in group B than group A (Table 2 and Figs. 2, 3, 4).

There was significant association between the serum levels of the IL-2, IL-6 and TNF-α and frequency of seizures (Table 3) and not the duration of the epilepsy or its type.


Table 1. Patients versus reference subjects as regard serum IgA, IL-2, IL-6, TNF- α.

 

 

Patients

No=75

Reference group

No=15

t

P

Mean

SD

Mean

SD

IgA (g/l)

1.55

0.86

2.34

1.16

2.4

0.024

IL-2(pg/ml)

31.40

9.56

23.91

6.52

3.72

0.009

IL-6(pg/ml)

30.84

8.19

24.42

1.83

6.07

0.001

TNF- α (ng/ml)

31.2

12.7

21.64

7.91

3.81

0.002

 

 

 

 
Table 2. Group A versus Group B as regard serum IgA, IL-2, IL-6 and TNF-α.

 

 

Group A

No=45

Group B

No=30

t

P

 

Mean

SD

Mea

SD

IgA (g/l)

1.79

0.86

1.18

0.73

3.27

0.001

IL-2(pg/ml)

28.07

9.13

36.39

7.98

4.17

0.001

IL-6(pg/ml)

26.79

4.60

36.91

8.69

5.85

0.001

TNF- α (ng/ml)

25.40

8.74

39.9

12.8

5.43

0.001

 

  

Fig. (1): The serum level of IgA among different groups.

 

Fig. (2): Serum IL-2 among different groups.

 


Fig. (3): Serum levels of IL-6 among different groups

 

Fig. (4): Serum TNF among different groups

 

Table 3. Correlations between frequency of seizures and serum levels of IgA, IL-2, IL-6, and TNF- α.

 

Frequency of seizures

 

 

 

P

r

 

 

0.5

0.064

Serum IgA

 

0.001

0.394

Serum  IL-2

 

0.01

0.291

Serum  IL-6

 

0.001

0.501

Serum TNF- α


DISCUSSION

 

In the present study, low serum IgA concentrations were lower in patients with epilepsy than reference subjects and among pharmacoresistant patients than pharmaco-responsive patients. These findings are going with previous studies that showed, low serum IgA concentrations in patients with epilepsy than in the reference subjects9,10. This is may be explained by the immunological effects of antiepileptic drugs, especially phenytion which had permanent immunological effects in some patients11. Many reports have been published concerning the effects of carbamazepine and valproic acid on humoral and cellular immunity12,13. On the other hand, it has been reported that a subgroup of epilepsy patients may have low serum IgA levels due to the immunological effects of the epilepsy per se14. Low serum IgA concentrations have been reported to be associated with autoimmune disorders15, but no such association found in this study. We found no IgA deficiency in this study while other previously published studies showed such deficiency5,10. The clinical significance of low serum IgA concentration is controversial. It has been reported that low serum IgA is associated with increase risk of upper respiratory tract infections, possibly through changes in nasal secretion and changes in secretory IgA formation9.

Evidence on the involvement of pro-inflammatory cytokines in seizure-associated processes has been obtained in various experimental models of seizure induction16. In the present study, epileptic patients demonstrate high levels of serum cytokines IL-2, IL-6 and TNF-α than reference subjects and these changes were more in patients with pharmacoresistant epilepsy .Theses changes had significant association with severity of epilepsy (frequency of seizures) and not its duration or the types of epilepsy. These results confirm the previous findings in which there were increase in the levels of IL-1, IL-6 and TNF- α in both serum and CSF in epileptic patients8, 17.

The potential significance of IL-2, IL-6, TNF-α in epilepsy is unknown 18. It was demonstrated that high doses of exogenous IL-2 can promote seizure generation through, decrease latency to epileptiform electrocortical discharges and increase the duration of seizure activity, while the role of endogenous IL-2 is much less clear 19. Experimental studies suggest that IL-6 may act as a protective factor against seizure induced neuronal damage20. IL-6 has been also reported to inhibit spread of excitation in the rat cerebral cortex21. On the other hand, when chronically overproduced, IL-6 has been reported to result in seizures and accompanying histological changes such as neuronal loss and gliosis22. These changes, commonly referred as temporal or mesial sclerosis, are frequently seen in temporal lobe structures of patients with chronic refractory epilepsy. Therefore, it can be hypothesized that IL-6 response after acute seizures may have beneficial effects. However, when chronically produced due to recurrent seizures, IL-6 may contribute to formation of structural changes in the neuronal tissue eventually leading to refractory seizures

In conclusion, these results give additional evidence for activation of the cytokine network in patients with pharmacoresistant epilepsy and the magnitude of these changes is related to severity of seizures. Since this activation may promote important neuromodulatory functions and immunological changes that can lead to refractoriness of seizures. So, early and tight control of seizures is important to prevent such immunological and neuromodulatory changes.

 

REFERANCES

 

1.      Kwan P, Brodie MJ. Early identification of refractory epilepsy.N Engl J Med 2000;342:3149.

2.      Kwan P, Brodie MJ. Refractory epilepsy: a progressive,intractable but preventable condition? Seizure 2002;11:77–84.

3.      Brodie MJ.Diagnosing and predicting refractory epilepsy.Acta Neurol Scand 2005;112(suppl.181):36-39.

4.      Kwan P, Brodie MJ. Potential role of drug transperters in the pathogenesis of medically interactable epilepsy. Epilepsia 2005; 46:224-35.

5.      Aarli JA. Epilepsy and the immune system. Arch Neurol 2000; 57:1689-1692.

6.      Haraldsson A, van Engelen BG, Renier WO, Bakkeren JA, Weemaes CM. Light chain ratios and concentrations of serum immunoglobulins in children with epilepsy. Epilepsy Res 1992; 13:255-260.

7.      van Engelen BG, Renier WO, Weemaes CM, Gabreels FJ, Meinardi H. Immunoglobulin treatment in epilepsy, a review of the literature. Epilepsy Res 1994; 19:181-190.

8.      Joanna LJ, Paul HP. The role of cytokines and growth factors in seizures and their sequelae.Progress in Neurobiology 2001;63:125-149.

9.      Jouni R, Katja L, Anssi A, Jukka P, Anna-Maija H, Jani R, Jouko I. SerumIgA, IgG and IgM concentrations in patients with epilepsy and matched controls: a cohort-based cross-sectional study. Epilepsy &behavior 2005;6: 191-195.

10.    Billiau AD, Wouters HC, Lagae GL. Epilepsy and immune system: is there a link?. Euro J Pad Neurol 2005;9:29-42.

 

11.    Ponti F, Lecchini S, Cosentino M, Castelletti CM, Malesci A, Frigo GM. Immunological adverse effects of anticonvulsants. Drug Saf 1993;3:235-250.

12.    Callenbach  PMC  , Jol-Van Der Zijd CM , Geerts AT, Arts WFM, Van Donselaar CA,Peters ACB, Stroink H, Brouwer OF, Van Tol MJD.Immunoglobulins in children with epilepsy: the Dutch Study of Epilepsy in Childhood.Clin Exp Immunol 2003;132:144-151.

13.    Basaran N, Hincal F, Kansu E, Ciger A. Humoral and cellular immune parameters in untreated and phenyoin or carbamazepine treated epileptic patients. Int J immunopharmacol 1994; 16: 1071-7.

14.    Aarli JA. Immunological aspects of epilepsy. Brain Dev 1993; 15:41-50.

15.    Palace J, Lang B. Epilepsy: an autoimmune disease?. J Neurol Neurosurg Psychiatry 2000;69:711-714.

16.    Jankowsky JL, Patterson PH. The role of cytokines and growth factors in seizures and their sequelae. Prog Neurobiol 2001; 63:125-149.

17.    Lehtimaki KA, Keranen T, Huhtala H, Hurme M, Ollikainen J,Honkaniemi J, Palmio J, Peltola J. Regulation of IL-6 system in cerebrospinal fluid and serum compartments by seizures: the effect of seizure type and duration. J Neuroimmunol 2004;152:121-125.

18.    Peltola J, Laaksonen J, Haapala AM, Hurme M, Rainesalo S, Keranen,T. Indicators of inflammation after recent tonic-clonic epileptic seizures correlate with plasma interleukin-6 levels. Seizure 2002;11: 44–46.

19.    De Sarro G, Rotiroti D,Audino MG, Gratteri S, Nistico G. Effects of IL-2 on various models of experimental epilepsy in DBA/2 mice.Neuroimmunomodulation 1994;1:361-369.

20.    Penkowa M, Molinero A, Carrasco J, Hidalgo J. Interleukin-6 deficiency reduces the brain inflammatory response and increases oxidative stress and neurodegeneration after kainic acid-induced seizures. Neuroscience. 2001;102: 805– 818.

21.    D’Arcangelo G, Tancredi V, Onofri F, D’Antuono M, Giovedi S, Benfenati F. Interleukin-6 inhibits neurotransmitter release and the spread of excitation in the rat cerebral cortex. Eur J Neurosci 2000;12,1241–1252.

22.    Campbell IL, Abraham CR, Masliah E, Kemper P, Inglis JD, Oldstone MB, Mucke L. Neurologic disease induced in transgenic mice by cerebral overexpression of interleukin 6. Proc Natl Acad Sci USA.1993; 90:10061–10065.

 

 

الملخــص العربـــى

 

قياس ارتباط مستوي بعض دلالات نشاط الجهاز المناعي بممانعة بعض مرضي الصرع للعلاج الدوائي

 

فكرة وهدف البحث:

ارتبط مفهوم العلاقة بين الجهاز المناعي والأسباب المؤدية لحدوث النوبات الصرعية منذ حوالي عشرين عاما. ولقد أجريت العديد من الدراسات التي أثبتت حدوث العديد من التغيرات المناعية في مرضي الصرع كما أثبتت بعض الدراسات أن العلاجات التي تنظم عمل الجهاز المناعي قد تفيد في بعض حالات الصرع الذين لا يستجيبون للعلاجات الدوائية المعتادة وقد ربطت بعض الدراسات بين حدوث بعض أمراض جهاز المناعة ومرض الصرع ذاته ولهذا فقد حاولت الدراسة بحث ما اذا كانت هناك علاقة بين ممانعة بعض حالات مرضي الصرع للعلاج وحدوث أي تغيرات في عمل الجهاز المناعي.

 

طريقة البحث:

تمت مقارنة مستوي بعض دلالات جهاز المناعة بالدم مثل ((IgA , IL-2,IL-6,TNF- α عند مرضي الصرع الممانعين للعلاجات الدوائية (ثلاثون مريضا) مع خمس وأربعين مريضا يستجيبون بشكل تام للعلاج الدوائي. وقد تم أخذ عينة ضابطة تتكون من خمس عشرة شخصا سليما.

 

النتائج:

كان مستوي gA  أقل في مرض الصرع عنه في أشخاص العينة الضابطة كما كان المستوي أقل في المرضي الممانعين للعلاج عنه في تلك العينة كما كان مستوي  IL-2,IL-6 ,TNF- α أعلي بنسبة إحصائية ملحوظة في مرضي الصرع عنه في العينة الضابطة وكذلك كانت هذه النسبة أعلي في المرضي الممانعين للعلاجات عنه في المرضي المستجيبين للعلاج. كان هناك علاقة ذو دلالة إحصائية بين مستوي  IL-2,IL-6 ,TNF- α وعدد النوبات الصرعية.

 

الاستنتاجات :

هذه الدراسة تقدم دليلا جديدا علي أن  نشاط الجهاز المناعي يرتبط ارتباطا مباشرا بتطور مرض الصرع وأن التغيرات المناعية ترتبط بشكل أساسي بشدة هذا المرض (عدد النوبات وشدتها). وعلي هذا فكلما كان علاج مرضي الصرع محكما منذ البداية, كانت فرصة حدوث تغيرات في الجهاز المناعي أقل وفرصة ممانعة المرض للعلاجات الدوائية أقل.

 



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