Clinical and laboratory study of pro-inflammatory and anti-inflammatory cytokines in women with multiple sclerosis

29

Folia Medica 2011; 53(2): 29-35Copyright © 2011 Medical University Plovdiv

doi: 10.2478/v10153-010-0034-x

Correspondence and reprint request to: A. Trenova, Department of Neurology, Medical University, Plovdiv;E-mail: [email protected] Vassil Aprilov St, 4002 Plovdiv, Bulgaria; Received 21 April 2010; Accepted for publication 14 April 2011

clInIcal and labORaTORy STudy OF PRO-InFlaMMaTORy and anTI-

InFlaMMaTORy cyTOkIneS In WOMen WITh MulTIPle ScleROSIS

Anastasiya G. Trenova, Mariya G. Manova, Ivanka I. Kostadinova1, Mariana A. Murdjeva2, Dimka R. Hristova, Tonka V. Vasileva, Zahari I. Zahariev Department of Neurology, 1Department of Pharmacology and Clinical Pharmacology, 2Department of Microbiology and Immunology, Medical University, Plovdiv, Bulgaria

AbstRACtMultiple sclerosis (Ms) is an autoimmune demyelinating disorder of the central nervous system characterised with a complex system of interactions between proinflammatory and anti-inflammatory cytokines in its course.AIm: The aim of the present study was to investigate the serum levels of cytokines TnF-α, IFn-γ, Il-4 and Il-10 in female patients with MS and healthy individuals, the changes occurring in the relapse and remission phases of the disease and their correlation with the severity of the neurological deficit. PAtIents And methods: Thirty-five women with relapsing-remitting MS were examined. The patients’ age ranged between 18 and 50 years and MS was verified clinically and by magnetic resonance imaging according to the Mcdonald criteria. Thirteen of the patients were treated with interferon-β-1b. The serum concentrations of TnF-α, IFn-γ, Il-4 and Il-10 were determined twice – in relapse and in remission – using an enzyme-linked immu-nosorbent assay (elISa). The control group consisted of 35 age-matched healthy females. results: the comparison of cytokine serum concentrations during the two phases of the disease showed significant elevation of the TnF-α serum levels in the relapse phase and of Il-4 – in the remission phase. The comparison between the patients and the healthy control subjects demonstrated statistically significant lower concentrations of TnF-α in remission patients and higher concentrations of Il-10 in relapse patients. The patients with interferon-β-1b treatment showed different profile of cytokine secretion from the patients without interferon-β-1b treatment. Interferon-β-1b-treated patients showed significantly lower serum levels of TnF-α and IFn-γ during the relapse phase and higher TnF-α and Il-10 serum levels during the remission phase compared with the untreated patients.conclusIons: Serum levels of TnF-α and Il-4 objectively reflect the immune response dur-ing relapse and remission of the disease. The severity of neurological deficit as estimated with the expanded disability status scale (edss) does not depend on the serum levels of TnF-α, Il-10 and IFn-γ in the two phases of MS.

key words: multiple sclerosis, cytokines, interferon-β-1b

IntRoduCtIon

Multiple sclerosis is an immunological organ-specific disease with persisting immunological dysbalance in the patient serum and cerebrospinal fluid. Ex-perimental and clinical studies have revealed dif-ferent profiles of cytokine secretion during relapse and remission of MS. It is thought that it is the Тh1-dependent secretion of TNF-α, IFN-γ, IL-12 that triggers the autoimmune reaction, while the Th2 synthesis of IL-4, IL-6, and IL-10 has anti-inflammatory effects.1,2 There is a large body of evidence showing that some mediators of inflam-

mation, which include cytokines, can protect the neurons from degeneration at some phases of the disease.3,4

The studies of cytokine profiles in MS patients and the correlations with the clinical picture and progress of the disease have yielded contradictory results. Finding what roles play each of the immune components of the MS pathological process is of crucial importance if we want to determine the “targets” for the drugs and the intensity of their therapeutic effect determined very accurately.

The AIm of the present study was to investigate

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A. Trenova et al

Folia Medica 2011; 53(2): 29-35© 2011 Medical University Plovdiv

the serum levels of cytokines TNF-α, IFN-γ, IL-4 and IL-10 in female patients with MS and healthy individuals, the changes which occur during the relapse and remission phases of the disease and their correlation with the severity of the neuro-logical deficit.

PaTIenTS and MeThOdS

patients

We conducted an open, prospective and randomized study at the Department of Neurology, Medical University, Plovdiv between 2003 and 2007. The study included 35 female patients with MS and a control group of 35 healthy women aged between 18 and 50 years.

Inclusion criteria1. Gender – females;2. Age – between 18 and 50 years;

3. Relapsing-remitting MS diagnosed according to the McDonald criteria (2000) and verified by magnetic resonance tomography.

Exclusion criteria1. Endocrinological, renal, hepatic and immu-

nological disorders;2. Acute and chronic infections;3. Therapy with immunosuppressive drugs; 4. Therapy with corticosteroids three months

before the first examination.The serum levels of TNF-α, IFN-γ, IL-4 and

IL-10 were measured twice:• during the relapse phase defined as the appear-ance of new symptoms or deterioration of already existing ones, lasting more than 24 hours and at least a 30-day period of improvement or stabiliza-tion of the neurological status after the previous relapse. • during the remission phase (at least 2 months after the previous exacerbation).

methoDs

Clinical tests• McDonald criteria (2000) for diagnosis of MS; • Expanded Disability Status Scale (EDSS) to determine the severity of the neurological deficit.

Laboratory testsSerum TNF-α, IFN-γ, IL-4 and IL-10 levels of the MS patients and the control subjects were measured twice; venous blood for examination was taken using ordinary procedures. The obtained serum was stored at -70°С until the analysis was car-ried out. The laboratory analyses of MS patients

were performed during relapse and remission. For the control group the cytokine serum levels were estimated as the mean of two measurements. The TNF-α, IFN-γ, IL-4 and IL-10 serum levels were determined by ELISA using kits of BIOSOURCE Europe S.A. The absorption, which is proportional to the cytokine concentration, was measured colori-metrically using TECAN ELISA reader at 450 and 630 nm. Тhe concentration of each of the cytokines was calculated with the help of a standard curve.

statistiCs

Statistical analysis were performed using SPSS 13.0; the level of significance was accepted at p < 0.05. We used the Independent Samples t-test, the paired-samples t-test, the Wilcoxon signed-rank test and correlation analysis.

The study was approved by the Scientific Ethics Commission of the Medical University, Plovdiv, Proceedings №3/28.03.2005.

Results

The clinical characteristics of the study contingent are presented in Table 1.

Thirteen of the study patients (37.14%) were treated with interferon-β-1b – group А, unlike the rest 22 patients (62.86%) included in group B.

The mean level of neurological deficit as es-timated with the EDSS was significantly lower in remission compared with the one in relapse p < 0.001 (t = 9.62).

The results of the investigated immune indicators (serum cytokines) are presented in Fig. 1.

The changes of the cytokine serum levels were analyzed using the Independent Samples t-test. The comparison between the results of the patients and the healthy controls showed statistically significant lower concentrations of TNF-α in the patients during remission (p < 0.05, t = 1.99) and higher concentrations of IL-10 during relapse (p < 0.01, t = 3.18). We noticed the following tendencies: higher mean values of IFN-γ and IL-10 in the patients in remission compared with the controls. During the two phases of clinical manifestations the changes of the cytokine levels were analyzed using the Wilcoxon signed-rank test. The following statisti-cally significant correlations were found: significant elevation of IL-4 levels (p < 0.05) during remission compared to the levels during relapse and of TNF-α levels (p < 0.01) during exacerbation compared to the levels during remission (Fig. 1).

Tables 2 and 3 present the results from the investigation of the cytokine levels in group A



Clinical and Laboratory Study of Pro-Inflammatory and Anti-Inflammatory Cytokines in Women with Multiple Sclerosis

31Folia Medica 2011; 53(2): 29-35© 2011 Medical University Plovdiv

(interferon-β-1b-treated patients) and group B (interferon-β-1b non-treated patients) analyzed using the paired-samples t-test.

The IFN-γ levels were significantly elevated in group A during remission compared with the relapse levels (p < 0.05, t = 2.19). The changes in TNF-α, IL-4 and IL-10 levels during the two periods of clinical manifestations could not reach statistical significance.

In group B statistically significant elevation of TNF-α (p < 0.05, t = 2.56) and of IL-10 (p < 0.01, t = 3.28) as well as significant decrease of IL-4 (p < 0.05, t = 2.31) were found during exacerba-tion compared to the remission levels.

The concentrations of the investigated cytokines in group A and group B during the different phases of disease are compared in Figs 2 and 3.

The group A patients had significantly lower concentrations of TNF-α (p < 0.05, t = 4.20) and

Table 1. Clinical characteristics of studied contingent

* p < 0.001.

groupsn

Agemean ± se

(years)

duration of the diseasemean ± se

(years)

edssmean ± se

relapse remission

Patients 35 34.80 ± 1.53 5.66 ± 0.91 3.63 ± 0.25 2.26 ± 0.22*

Controls 35 30.45 ± 1.01 - - -

Figure 1. Serum cytokine concentrations of studied contingent.

IFN-γ (p < 0.01, t = 6.56) during relapse and el-evated concentrations of TNF-α (p < 0.05, t = 4.42) and IL-10 (p < 0.05, t = 4.07) during remission in comparison with group B patients.

The correlation analysis revealed moderate posi-tive correlation between the degree of disability as estimated with the EDSS in remission and the concentration of IL-4 during the same period (p < 0.05, r = 0.38).

dIsCussIon

The immune response to the myelin antigens in MS is still not enterely clear. TNF-α is given as one of the major pro-inflammatory cytokines in demyelinating disorders; it is considered to possess myelinotoxic effects.8 The present study revealed statistically significantly lower levels of TNF-α in remission and elevated levels of IL-10 in relapse in the MS patients compared with healthy individuals.

25.5

5 ±

2.99

4.44

± 0

.51

6.89

± 0

.91

2.22

± 0

.35

16.1

3 ±

2.20

5.43

± 0

.68

4.94

± 0

.83

3.49

± 0

.52

22.1

4 ±

2.07

4.68

± 0

.36

3.76

± 0

.38

3.07

± 0

.28

0

5

10

15

20

25

30

35

TnF-α IFn-γ Il-10 Il-4

Con

cent

ratio

n p

g/m

l

in relapse

in remission

controls



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A. Trenova et al


The significant decrease of TNF-α levels in remis-sion which become lower than those of controls is probably an expression of abating autoimmune demyelination during clinical improvement. It is supposed that the elevated IL-10 serum levels during the relapse phase result from the activation of the regulatory mechanisms of the body which suppress autoimmune reactions. On the other hand several experimental studies reported exacerbation of the disease clinical manifestations after administration of IL-10 in doses higher than the physiological ones.2 The significant elevation of IL-10 in patients during remission, which was found in the present study and compared to the healthy controls sup-ported this point of view. The revealed tendency of increased secretion of mediators with the opposite effect (IFN-γ and IL-10) in remission patients com-pared with the controls showed persisting regulatory dysbalance in the profile of the cytokines from the Th1/Th2 system. This hypothesis was supported by the significant elevation of the IL-10 serum levels

during relapse in the interferon-β-1b non-treated patients. Similar data were reported by K. Hohnoki et al., M. C. Rodriges-Sanz, et al. and de Andres S, et al.5-7

The significant elevation of IL-4 plasma levels in all patients in remission compared with that in exacerbation is probably an indication of a shift from a Th1 to a Th2 immune response. The results of our study are consistent with the conclusion F Weber makes that a shift of the immune reaction from type Th1 to type Th2 should result in sup-pression of the inflammatory process in the central nervous system.2

The hypothesis of pro-inflammatory pathogenic effect of TNF-α and IFN-γ is based on clini-cal evidence: statistically significant elevation of TNF-α during relapse and increased frequency of disease exacerbations after intravenous treatment with IFN-γ.8,9 The significant elevation of TNF-α serum levels during exacerbation we found in this study in comparison with the levels in remission

Table 2. Serum cytokine concentrations in interferon-β-1b-treated MS patients

Cytokines Msphase n Mean ± se

pg/ml sd t p

TNF-αrelapse 13 21.72 ± 3.18 11.48

1.53 > 0.05remission 13 19.63 ± 3.68 13.25

IFN-γrelapse 13 2.86 ± 0.81 2.92

2.19 < 0.05remission 13 4.35 ± 0.89 3.21

IL-10relapse 13 6.70 ± 1.85 6.68

0.13 > 0.05remission 13 7.03 ± 1.69 6.12

IL-4relapse 13 2.36 ± 0.60 2.17

0.72 > 0.05remission 13 3.14 ± 0.86 3.11

Table 3. Serum cytokine concentrations in interferon-β-1b-non-treated MS patients

Cytokines Msphase n Mean ± se

pg/ml sd t p

TNF-αrelapse 22 27.81 ± 4.34 20.40

2.56 < 0.05remission 22 14.06 ± 2.72 12.74

IFN-γrelapse 22 5.37 ± 0.59 2.74

0.75 > 0.05remission 22 6.07 ± 0.94 4.39

IL-10relapse 22 7.00 ± 0.98 4.59

3.28 < 0.01remission 22 3.70 ± 0.78 3.69

IL-4relapse 22 2.13 ± 0.43 2.03

2.31 < 0.05remission 22 3.70 ± 0.66 3.10





Figure 2. Cytokine serum concentrations in relapse patients.

Figure 3. Cytokine serum concentrations in remission patients.

confirms the role of this cytokine as a factor trig-gering the immune inflammatory process. There are some difficulties in the clinical interpretation of the changes we found in the IFN-γ concentrations dur-ing the two phases of the disease. We established significant differences in all patients individually in the changes of IFN-γ and a tendency towards elevation of its plasma levels during remission compared with the relapse phase. Y. Miyazaki et al. reported that interferon-β-1b exerted a signifi-cant suppressive effect on the IFN-γ synthesis by the peripheral myelin-reactive CD4+ CD28+ T cell subpopulations, but had an insignificant effect on the production of IFN-γ by the CD4+ CD28- T

lymphocyte branches.10 We suggest that the re-sults of the present study reflect such an aspect of dysbalance of the cytokine-regulatory system. During remission interferon-β-1b-treated patients demonstrated statistically significantly elevated IL-10 plasma levels compared with this indica-tor in non-treated patients for the same clinical period. We accept that these results are associ-ated with the increased synthesis of IL-10 by the auto-reactive Т lymphocytes under the influence of interferon-β-1b which was experimentally proved by Zang YC, et al. and S Chabot and VW Yong in their studies.11,12

We found no causal relationship between the

p < 0.05

p < 0.01

0

5

10

15

20

25

30

pg/m

l

TnF-α IFn-γ Il-4 Il-10Cytikines

group A

group b

p < 0.05

p < 0.05

02468

101214161820

pg/m

l

TnF-α IFn-γ Il-4 Il-10Cytokines

group A

group b



34

A. Trenova et al


severity of disability (EDSS) and the level of TNF-α, IL-10 and IFN-γ during the two phases of clinical manifestations. That is why we suppose that the regulatory cytokine dysbalance and EDSS reflect the degree of immunological inflammation with different specificity. Literature data show direct correlation between the severity of neurological deficit and the loss of axons, but the axonal le-sions are of different mechanisms: immunological-inflammatory, degenerative (Wallerian degeneration) and ischemic.4

ConClusIons

The serum levels of TNF-α and IL-4 objectively reflect the activity of the immune response during relapse and remission of MS.

The severity of neurological deficit as estimated by EDSS does not depend on the serum levels of TNF-α, IFN-γ and IL-10 during the two periods of MS.

ReFeRenceS

1. Hartung H-P, Bar-Or A, Zonkas I. What do we know about the mechanism of action of disease-modi-fying treatment in MS. J Neurol 2004;251 (Suppl 5): 12–19.

2. Weber F. Effects of autoreactive T cells and cytokines in the pathogenesis of multiple sclerosis. Int MSJ 2001;9(1):29–35.

3. Lubetzki C, Zale B. Myelin repair in multiple sclero-sis. In: Antel S, Birnbaum G, Hartung H-P, Vincent A, eds. Clinical Neuroimmunology. New York: Oxford University Press; 2005:399–402.

4. Wilkins A, Scolding N. Protecting axons in multiple sclerosis. Multiple Sclerosis 2008;14:1013–25.

5. Hohnoki K, Inoue A, Koh CS. Elevated serum lev-

els of IFN gamma, IL 4 and TNF alpha/unelevated serum levels of IL 10 in patients with demyelinating diseases during the acute stage. J Neuroimmunol 1998;87:27–32.

6. Rodriguez-Sainz MC, Sanchez-Ramon S, de An-dres C, et al. Th1/Th2 cytokine balance and nitric oxide in cerebrospinal fluid and serum from pa-tients with multiple sclerosis. Eur Cytokine Netw 2002;13(1):110–4.

7. de Andres C, Rodriguez-Sainz MC, Muñoz-Fernan-dez MA, et al. Short-term sequential analysis of sex hormones and helper T cells type 1 (Th1) and helper T cells type 2 (Th2) cytokines during and after multiple sclerosis relapse. Eur Cytokine Netw 2004;15(3):197–202.

8. Evans R, Smith K. Multiple Sclerosis: effects of immune mediators on neurophysiological function. In: Antel S, Birnbaum G, Hartung H-P, Vincent A, eds. Clinical Neuroimmunology. New York: Oxford University Press; 2005:209–16.

9. Mahad D, Trapp B, Ransohoft M. Tissue pathol-ogy of multiple sclerosis. In: Antel S, Birnbaum G, Hartung H-P, Vincent A, eds. Clinical Neuroim-munology. New York: Oxford University Press; 2005:173–83.

10. Miyazaki Y, Iwabuchi K, Kikuchi S, et al. Expan-sion of CD4+CD28- T cells producing high levels of interferon-γ in peripheral blood of patients with mul-tiple sclerosis. Mult Scler 2008;14(8):1044–55.

11. Zang YC, Yang D, Hong J, et al. Immunoregulation and blocking antibodies induced by interferon beta treatment in MS. Neurol 2000;55(3):397–404.

12. Chabot S, Yong VW. Interferon beta 1b increases interleukin 10 in a model of T cell-microglia inte-raction: relevance to MS. Neurol 2000;55(10): 1497–505.





клИНИкО-лАбОРАТОРНОЕ ИССлЕДО-вАНИЕ пРОвОСпАлИТЕльНых И АНТИ-вОСпАлИТЕльНых цИТОкИНОв у ЖЕНщИН С мНОЖЕСТвЕННым СклЕ-РОзОм

А. Тренова, М. Манова, И. Костадинова, М. Мурджева, Д. Христова, Т.Василева, З. Захариев

РЕзюмЕВВедение: Множественный склероз (МС) представ-ляет собой аутоиммунное, демиелинизирующее за-болевание центральной нервной системы в ходе которого развивается сложная сеть взаимодействий между проинфляматорными и антиинфляматорными цитокинами.

Цель: Исследовать сывороточные уровни TNF-α, IFN-γ, IL-4 и IL-10 у женщин с МС и у здоровых лиц, а также и изменения, наступающие во время приступа и во время ремиссии заболевания и их связь с тяжестью неврологического дефицита.

матеРиал и методы: Обследовано 35 женщин в возрасте от 18 до 50 лет с приступно-ремитент-ным, клиническим и резонансно подтвержденным МС по критериям Мс Donald. 13 женщин лечены интерфероном β-1b. Сывороточные концентрации TNF-α, IFN-γ, IL-4 и IL-10 определены двукратно –

во время приступа и во время ремиссии с помощью энзимосвязанного иммуносорбентного теста (ELISA). Контрольная группа включает 35 здоровых женщин того же возрастного интервала.

Результаты: При сравнении сывороточных концен-траций цитокинов во время обеих фаз заболевания устанавливаются сигнификантно более высокие уровни TNF-α во время приступа и IL-4 – во время ремиссии. При сопоставлении результатов пациенток и здоровых женщин регистрированы статистически значимо более ниская концентрация TNF-α у больных во время ремиссии и более высокая концентрация IL-10 во время приступа. У пациенток с и без интерферона β-1b наблюдается различный профиль секреции цитокинов. Пациентки, леченные препара-том интерферон β-1b, имеют сигнификантно более ниские TNF-α и IFN-γ во время приступа и более высокие TNF-α и IL-10 в состоянии ремиссии по сравнению с нелеченными.

ВыВоды: Сывороточные уровни TNF-α и IL-4 объ-ективно отражают активность иммунной реакции во время двух периодов клинических проявлений МС (приступ и ремиссия). Тяжесть неврологического дефицита, оцененная с помощью EDSS, не зависит от сывороточных уровней TNF-α, IL-10 и IFN-γ во время двух периодов клинических проявлений.



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Clinical and laboratory study of pro-inflammatory and anti-inflammatory cytokines in women with multiple sclerosis