INTRODUCTION

 

Multiple sclerosis is considered a chronic demyelinating neuro-inflammatory disease of the central nervous system. Inflammation composed of mononuclear cells, breakdown of the blood brain barrier, focal plaques of demyelination and axonal damage characterize acute MS lesions and can be effectively targeted by anti-inflammatory therapies¹.

The genetics of multiple sclerosis are thought to comprise a variety of genes, influencing both disease susceptibility and disease characteristics. Among these, the contribution of genes encoding cytokines is currently under elaborate investigation, as cytokines are important mediators in immune and inflammatory conditions, such as multiple sclerosis.²

Interleukin-l is a pro-inflammatory cytokine that has numerous biological effects, including activation of many inflammatory processes (through activation of T cells, for example), induction of expression of acute-phase proteins, an important function in neuroimmune responses and direct effects on the brain itself. There is now extensive evidence to support the direct involvement of interljsukin-1 in the neuronal injury that occurs in multiple sclerosis.³

Differences in either absolute cytokine production or in the ratio of pro-inflammatory and anti-inflammatory cytokines may influence multiple sclerosis course; for example, by maintaining inflammatory activity².

IL-lb (member of interleukin 1 family) is a pleiotropic pro-inflammatory cytokine whose production is tightly controlled at several levels/In multiple sclerosis, IL-lb is mainly expressed by microglial cells and infiltrating monocyte/macrophages throughout the white matter and in acute lesions^4.

The aim of this work was to study the relation between IL-1b as well as IL-1b gene polymorphism and multiple sclerosis.

 

SUBJECTS AND METHODS

 

Subjects

The participants were recruited from neurology department, Cairo University Hospital during the period from December 2011 to December 2011.

Twenty seven patients (group I) (14 females and 13 males) with relapsing remitting multiple sclerosis (in acute attack and 3 months after the attack) diagnosed by revised McDonald’s criteria of multiple sclerosis 2010⁵. Patients’ age ranged from 23 to 32 years with a mean age of 27.3±2.3 years.

No participants had a history of myocardial infarction, trauma and surgery at the time of sampling.

Twenty-five healthy subjects (group II) (15 females and 10 males) were enrolled as control subjects, their age range from 24 to 53 ears (mean age of 29.9±7.5 years).

Excluded from the study were patients were with infections prior or complicating the attack. In addition, patients with liver, kidney and autoimmune diseases were excluded.

 

Methods

Patients were evaluated by clinical examination and diagnosed clinically by revised McDonald’s criteria of multiple sclerosis⁵.

Assessment of disease severity and level of impairment during and in between attack using expanded disability status scale (EDSS)⁶, which is a standardized neurologic severity scale developed to quantify M.S patients deficits in clinical trials. A score up to 4.5 (have no impairment to their daily activity). A score up to five or more defined by severe disability enough to impair full daily activity.

Assessment of functional out come using multiple sclerosis progression index (PI) in order to calculate progression of the disease.⁷

 

Laboratory, investigations were done and they included routine laboratory profile, C.S.F examination and specific laboratory, investigations including:

a)        Quantities measurement of human IL-B in serum by enzyme-linked immunosorbant assay (ELISA), normal values of serum IL-B are 0.00 pg/m to 3.72 pg/m.

b)        Genetic profile: DNA gene polymorphisms (one single base pair) at position 511 promater region of IL-1b gene were assessed by PCR technique magnetic resonance imaging was performed on a 1.5 Tesla Phillips Intera® scanner at the magnetic resonance unit (Radio diagnosis department, Kasr Al-Ainy hospital).

c)        The control group underwent the same routine and specific laboratory tests (IL-1b) and genetic testing.

 

Statistical Methods

               Quantitative data were summarized as means and standard deviations. Categorical data were summarized percentages. Qualitative data were compared by Chi-square of fisher exact tests according to the expected frequencies. Person's correlation coefficient, which is a test to measure the strength of the linear relationship between two variables, was used. A 5% probability level (P < 0.05) was considered statistically significant.

 

RESULTS

 

The demographic and clinical characteristics of the subjects are summarized in Table (1).

 

Specific laboratory results:

a)        Serum level of interleukin B levels (IL-1b): elevated serum IL-1b was found in 8 patients (29.6%) during the attack and 5 patients (18.5%) in between the attack, while normal serum level of IL-1b was found in 19 patients (70.3%) during the attack and 22 patients (81.4%).

b)        IL1-b genotyping: Human IL-1b-115 polymorphism genotype results revealed that (TT allele) was found in nine patients (33.33%) and 10 persons of control group (40%), while (CC allele) was found in 11 patients (40.7%), and in 5 persons (20%) in control group. The third allele (CT) was found in seven patients (25.9%) in patients, and in 10 persons (40%) of control group. In patients having the homozygous allele TT, the mean IL-1b level during and in between the attack was 5.3±4.1, 3.8±3.3 respectively. The mean level of IL-1b in patients having CC allele during attack was 1.7±0.6 and in between attack were 30.0±1.9. Where as the mean level of CT allele (during and in between the attack) were 2.6±0.3, 2.8±1.5 respectively (Table 4).

 

In between the attack, where as all subjects in control group had normal serum IL-1b levels. There was a highly significant difference between multiple sclerosis patients (during and in between attack) and control subjects regarding the mean level of IL-1b (P-value < 0.01). (Tables 2 and 3).

Female patients had high significant IL-1b serum level during the attacks and significantly in between the attacks when compared to male patients (Table 5 and Figure 1).

 

Correlation study between IL-1b serum level and age of onset and the age of patients:

1.        There was negative correlation between the serum level of IL-1b and the age of onset, but these data did not reach a statistical significance (Table 6).

2.        In addition, positive correlation was present between IL-1b and age of MS patients. The correlation did not reach statistical significant values (Table 7).

 

Table 1. Clinical and radiological characteristics of M.S patients.

 

 

 

Table 2. MS patients with normal and abnormal serum level of IL-b during and in between attack.

 

	During attack	In between attack	P-value
Normal	19 (70.3%)	22 (81.4%)	0.191
Abnormal	8 (29.6%)	5 (18.54)

MS Multiple sclerosis

 

 

Table 3. Serum level of IL-1b in patients (during and in between attack) and healthy control group.

 

IL-b	Patients in attack	Patients in between attack	Control	P-value
Minimum	0.9	1	0.5	0.001*
Maximum	18.8	10.5	3.3
Mean ± SD	8.0±5.4	5.0±3.42	1.6± 0.8

* Significant at p<0.01

 

 

Table 4. Comparison between genotypes and serum level in patients (during and in between attack).

 

	During attack	In between attack
TT	5.3± 4.1	3.8±3.3
CC	1.7±0.6	3.0±1.9
CT	2.6±0.3	2.8±1.5

 

 

Table 5. Mean±SD of serum level of IL-1b in males and females (during and in between attack)

 

	Male	Female	p-value
During attack	2.1±0.7	7.2±2.1	0.001**
In Between Attack	1.9±1.7	3.9±3.1	0.046*

* Significant at p<0.05 ** Significant at p<0.01

 

 

 

Figure 1. Mean±SD of serum level of IL1 beta in males and females (group I) (during and in between the attacks).

 

 

Table 6. Correlation serum level of IL-1b during and in between attack and age of onset of M.S

 

	r-value	P-value
Serum level during attack	-0.2	0.328
Serum level in between attack	-0.1	0.646

 

 

Table 7. Correlation between serum level (during and in between serum level during and in between attack) and age of MS patients

 

Serum level	r-value	P-value
During attack	-0.03	0.892
In between attack	-0.16	0.445

 

 

DISCUSSION

 

Many genetic association studies evaluating the relationship between cytokine gene polymorphisms and MS risk, have already been published. Among many pro inflammatory cytokines considered as being essential for MS pathogenesis, IL1 cluster gene was the most widely investigated, including different ethnic populations.^8,9

Our study was conducted on 27 patients with relapsing remitting multiple sclerosis and 25 normal volunteers, aimed at finding an association between IL-lb serum level, also its gene polymorphism T 51 1C and multiple sclerosis.

Results of our study revealed that serum IL-lb in multiple sclerosis patients during and in between the attacks was high, it reaches a highly significant and significant difference respectively when compared with its serum level in healthy controls with a notable difference in its mean level during and in between the attacks.

This was in accordance with several preceding studies conducted by others.^10-12

Rossi et al.¹³ stated that brain focal inflammation in MS perturbs the cytokine milieu within the circulating CSF, and causes reduced GABAA-mediated inhibition in neurons. Reduced GABAA-mediated neuronal inhibition in the grey matter was postulated to occur in the acute phases of MS and they demonstrated IL-1 p action at GAB A synapses. In MS, the frequency of inflammatory episodes in the early stages of the disease correlates with late neurodegeneration and progressive clinical course. Accordingly, inflammatory cytokines released during acute MS attacks have the property of both increasing glutamate mediated synaptic transmission, and reducing GABA mediated synaptic signaling, resulting in unbalanced synaptic hyper ex citation and possibly excitotoxic neurodegeneration.

In our study, we found a highly statistically significant differences in serum level of IL-lb in female patients than males in group 1 (p-value = 0.001**), also female patients had significant higher serum level of IL-lb three months after the relapse (p-value =0.046*), while no statistical significant gender difference was found in its serum level in controls.

This was in agreement to O'Mahony et al.¹⁴, who found that there was gender differences, in IL-lb values with female predominance, while Hans et al.² found that IL-lb production showed no significant differences regarding the gender, as female hormones such as estrogen may influence the immune response in women^15,16 and it acts as enhancer of the humoral immunity, while androgens and progesterone (and glucocorticoids) act as natural immunosuppressors¹⁶.

Although the earlier the age of onset of multiple sclerosis, the higher the serum level of IL-1b, no statistically significant correlations was found between IL-lb serum level during and in between the attacks and age of onset of multiple sclerosis (p-value=0.328, 0.646) respectively.

In addition, it was found that the younger the multiple sclerosis patient, the higher the IL-lb serum level during and in between the attacks, but no statistically significant correlations was found (P-value=0.892, 0.445) respectively.

Hans et al.² found that IL-lb production was lower in MS compared with controls. No significant differences were observed with regard to age or gender. Although other studies have shown that inter- individual cytokine production is highly variable, intra-individual production is remarkably stable over time and appears to be characteristic of an individual.

No significant correlations were detected between IL-lb serum level and demographic and clinical characteristics (gender, age, disease duration) of MS as stated by Irena et al.¹².

Specific cytokines and chemokines, are signal molecules produced by innate immune cells, have been reported to substantially alter with age, especially proinflammatory cytokines such as interleukin (IL)-6, IL-1b, tumor necrosis factor (TNF)α, and TGFB.¹⁴

Small sample size in our study and most patients with the age range occurs in the second decade, which may limit studying the relation between interleukin-1b and age or age of onset of multiple sclerosis.

Our study revealed that, there were no statistically significant differences in the distribution of gene polymorphism (IL-lb-511 CT) and its different alleles between patients and controls (p-value = 0.252).

In our study, the number of patients in the different subtype groups were too small to allow for more detailed statistical analysis, which is also a limitation of our study.

A number of studies have suggested that gene polymorphisms in the IL-1b gene family influence susceptibility to MS¹⁷ and others found no evidence for a role of these cytokine genes on the risk of developing MS¹⁸.

The genes encoding for interleukin 1 family molecular are located close to one another on chromosome 2q, within which there are several common polymorphisms. IL-1 alpha has two variants at position-889 and 845, both are C to T substitution and are in linkage disequilibrium (LD). Two single nucleotide polymorphisms (SNPs) in the IL-1b gene have been described- at position -511in the promoter region and at position 3953, in the fifth exon.¹⁹

Supporting our results, Dagmara et al.¹ also Tieneke et al.²⁰, stated that no evidence of the role of the IL-1 genes in MS.

In patients having the homozygous allele TT, the mean IL-lb level was higher during the attack (5.3±4.1pg/dl) and also in between the attacks (3.8±3.3pg/dl), but it does not reach a statistical significant values. Patients having the homozygous allele CC, had lower mean level IL-lb during the attack (1.7±0.6 pg/dl), whereas the mean level of IL-lb in patients having CT allele was 2.6 ±0.3 pg/dl during the attack. There was no statistical difference between serum IL-l beta during and in between the attacks in patients having different alleles.

C to single base polymorphism in the promotor of IL-lb gene (C-511àT) have been reported to affect the levels of IL-l.²¹

Involving combined gene polymorphisms, may alter the results of IL-lb serum level. Hans et al.², investigated whether IL-lb and TL-lra production differed depending on carrier ship of this gene combination. Twenty MS patients and 20 controls were selected based upon carrier ship of the specific combination.

Carriers for the specific combination, produced more IL-lra, especially in MS patients, although not significantly. IL-lra production was significantly higher in individuals homozygous for IL-1RN allele. In patients, Il-lra production was higher and IL-­lb production was lower, compared with controls.

Hence, the findings concerning cytokine genes and MS are conflicting. The role of the IL-1 gene family in MS remains controversial. Part of the controversy was caused by a relatively low number of patients further, the immunology of MS is complex, as suggested by studies in which no clear shift of cytokine profile in pathology²² or after intervention²³.

The diversity of the results of previously mentioned studies including ours; may be also explained by differences in patients' selection criteria or ethnic factors.

The network effects of cytokines with each other and with other inflammatory factors may underlie the complexity. In addition, there is a disease locus in the chromosome region where the IL-1 gene family is located, it is unknown where this would be. In this region, several polymorphisms have been associated with effect, and complex patterns of the functional variants exist.²⁰

In conclusion, elevated IL-1b level in multiple sclerosis patient is considered as a biomarkers, however, its genetic polymorphism did not influence its level. Both serum level of interleukin 1beta and its gene polymorphism T-511C are not associated with any of the demographical characteristics of multiple sclerosis (Age, gender, age of onset of the disease).

Further larger studies are needed on a more stratified Ms groups to find out the exact role of IL-1b in MS patients

 

[Disclosure: Authors report no conflict of interest]

 

REFERENCES

 

1.      Dagmara M-G1, Grazyna G, Anna M, Andrzej C, Anna C. Association of ILIA, IL1b, ILRN, 1L6, 1L10 and TNF-a polymorphisms with risk and clinical course of multiple sclerosis in a Polish population. J Neuroimmunol. 2011; 236: 87-92.

2.      Hans M S, Jaco van As, J Bart A C, Christien DD, and Bernard M. J U. Interleukin, IL-1 gene polymorphisms: relevance of disease severity associated alleles with IL-1 beta and IL-1ra production in multiple sclerosis. Mediators Inflamm.2003;12(2):89-94.

3.      Allan SM, Tyrrell PJ , Rothwell NJ. Interleukin-1 and neuronal injury. Nat Rev Immunol. 2005; 5(8): 629-40.

4.      Dinarello CA. Historical insights into Cytokines. Eur J Immunol. 2007 ; 37, suppl 1: S34-45.

5.      Polman CH, Reingold SC, Edan G, Filippi M, Hartung HP, Kappos L, et al. Diagnostic criteria for multiple sclerosis:2005 revisions to the "McDonald Criteria". Ann Neurol .2005; 58(6): 840-6.

6.      Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale, (EDSS). Neurology. 1983; 33(11): 1444-52.

7.      Akcali A, Pehlivan S, Pchlivan M, Sever T, Akgul P, Neyal M. TNP-a promoter polymorphisms in multiple sclerosis: no association with -308 and - 238 alleles, but the -857 alleles is associated with the disease in Turkish patients. Int J Immunogenet.2010; 37 (2): 91-5.

8.      Aggelakis K, Zacharaki F, Dardiotis E, Xiromerisiou G, Tsimourtou V, Ralli S, et al. Interleukin-lb and interleukin-1 receptor antagonist gene polymorphisms in Greek multiple sclerosis (MS) patients with bout-onset MS. Neurol Sci. 2010; 31 (3): 253-7.

9.      Borzani T, Tola MR, Caniatti L, Collins A, De Santis G, Luiselli D, et al. The interleukin-1 cluster gene region is associated with multiple sclerosis in an Italian Caucasian population. Eur J Neurol.2010; 17(7): 930-8.

10.    Heesen C, Sieverding F, Buhmann C, Gbadamosi J. IL-lra serum levels in disease stages of MS—a marker for progression. Acta Neural Scand. 2000; 101: 95-7

11.    Cindy M, De Jong C, Rene` Lutter R, Marten Verberk M, et al. Differential cytokine expression in skin after single and repeated irritation by sodium lauryl sulphate. Exp Dermatol 2007; 16 (12):1032-40.

12.    Dujmovic I, Mangano K, Pekmezovic T, Quattrocchi C, Mesaros S, Stojsavljevic N, et al. The analysis of IL-1 beta and Us naturally occurring inhibitors in multiple sclerosis: The elevation of IL-1 receptor antagonist and IL-1 receptor type 11 after steroid therapy. J Neuroimmunology. 2009; 207 (1-2): 101-6

13.    Rossi S, Studer V, Motta C, De Chiara V, Barbieri F, Bernardi G, et al. Inflammation inhibits GABA transmission in multiple sclerosis. Mult scler. 2012; 18 (11): 1633-5.

14.    Oprica M, Eriksson C,  Schultzberg M. Inflammatory mechanisms associated with brain damage induced by kainic acid with special reference to the interleukin-l system. J Cell Mol. 2003; 7( 2): 127-40.

15.    Beagley WK, Christine M Gockcl. Regulation of innate and adaptive immunity by the female sex hormone oestradiol and progesterone. FEMS immunology and medical microbiology. 2003; 38 (4): 13-22.

16.    Cutolo M, Sulli A, Capellino S, Villaggio B, Montagna P, Seriolo B, et al. Sex hormones influence on the immune system: basic and clinical aspects in autoimmunity. Lupus. 2004; 13(9) :635-8.

17.    Kantarci OH, Schaefer-Klein JL, Hebrink DD, Achenbach SJ, Atkinson EJ, McMurray CT, et al. A population based study of IL-4 polymorphisms in multiple sclerosis.J Neuroimmunol. 2003; 137(1-2): 134-9.

18.    Feakes R, Sawcer S, Broadley S, Coraddu F, Roxburg R, Gray J, et al. Interleukin-1 receptor antagonist (IL-1ra) in multiple sclerosis. J Neuroimmunol. 2000; 105 (1): 96-101.

19.    Huang J, Xie ZK, Lu RB, Xie ZF. Association of interleukin-1 gene polymorphisms with multiple sclerosis: a meta-analysis. Inflammation Research. 2013; 62(1): 97-106.

20.    Jiangtao LI, Dongchen Z, Shudong X, Yunpeng S, Jianhua Z. Jiaqi P, et al. Association of interleukin-1 gene cluster polymorphism and ischemic stroke in Chinese population. Biomarker laboratory.2006; 54 (4) 366-9.

21.    Hooper-van Veen T, Schrijver HM, Zwiers A, Crusius JB, Knol DL, Kalkers NF, et al. The interleukin-1 gene family in multiple sclerosis susceptibility and disease course. Mult Scler. 2003; 9 (6): 535-9.

22.    Baranzini SE, Elfstrom C, Chang SY, Butunoi C, Murray R, Higuchi R, et al. Transcriptional analysis of multiple sclerosis brain lesions reveals a complex pattern of cytokine expression. J Immunol. 2000; 165 (11): 6576-82.

23.    Wandinger KP, Sturzcbecher CS, Bielekova B, Detore G, Rosenwald A, Staudt LM, et al. Complex immunomodulatory effects of interferon-beta in multiple sclerosis include the upregulation of T helper 1-associated marker genes. Ann Neurol. 2001; 50(3): 349-57.