بسم الله الرحمن الرحيم

Estimation of Serum Interlukin-12 and soluble Vascular Adhesion Molecule-1 in Multiple Sclerosis patients By Ibtesam Fahmy, Omaima Elkholy, Hanan Amer, Amira Hassouna,(2) and Reda Saad

Introduction

Introduction Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system (CNS) in which gradual destruction of the myelin occurs in patches throughout the brain or the spinal cord or both. MS is a dynamic disease, with almost constant lesion formation and a progressive clinical course leading to physical disability. The disease can present in different clinical forms, such as primary progressive, relapsing remitting, relapsing progressive, and secondary progressive phenotypes.

Introduction The cause of MS is elusive, although autoimmune mechanisms, possibly triggered by environmental factors in genetically susceptible individuals, are thought to be important. MS is postulated to be a T cell-mediated autoimmune disease. The progressive nature of the disease is due to the memory of the immune system. After the initial attack, the myelin sheath is recognized by the immune system as an invader. To begin a new attack only the activation of T cells have to occur.

Introduction Interleukin-12 (IL-12) is an immuno-regulatory cytokine, which is produced by antigen presenting cells as dentritic cells and macrophages in response to antigens and mitogens. It has long been considered essential in T-cell-mediated autoimmune diseases, including multiple sclerosis (MS) and its experimental animal model (EAE).‎ Moreover, it is a potent inducer of interferon-gamma (IFN-γ), and other Th1 cytokines that may play an important role in MS pathogenesis. The main function of IL-12 is to signal with an antigen into Th1 cells or inflammatory T cells. Th1 cells further stimulate the production of complement proteins by B cells that lead to the production of a membrane attack complex that lyses the cell.

Introduction After the initial attack of MS, the myelin sheath is recognized by the immune system as an invader. To begin a new attack only the activation of T cells have to occur. So increased levels of IL-12 have a high probability of beginning the relapsing phase of the disease. The mode of action of IL-12 in MS may be due to up regulation of IL-18 receptor expression. It was reported that IL-18 has an important role in cellular adhesion, being the final common pathway used by IL-1 and TNF-α. IL-1 and TNF-α. synergize to stimulate IFN-γ release; that may have an important role in the BBB disruption in MS.

Introduction Increased levels of IL-12 has been found to correspond to more damage observed by magnetic response imaging of the brain of MS patients. Levels of IL-12 positive monocytes were also found to correspond to new lesions produced in the brain seen by MRI. Endothelial activation is a key feature of multiple sclerosis (MS) pathogenesis. Attention has been directed towards the role of the vascular endothelium in actively supporting trans-vascular migration. Activated cerebral vascular endothelial cells express The endothelial micro-particles (EMPs) which facilitate leukocyte adhesion to endothelium and migration into inflammatory lesions.

Introduction The endothelial micro-particles (EMPs) include P- and E- selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Soluble adhesion molecules; ICAM-1, VCAM-1 and platelet-endothelial cell adhesion molecule-1 (PECAM-1), have been reported as promising biomarkers of BBB damage in multiple sclerosis (MS). These EMPs also bind to and activate monocytes; so, EMP-monocyte conjugate were found to be a marker of inflammation in multiple sclerosis (MS).

Aim of Work

The present study is designed to: 1- Estimate the serum levels of IL-12 (as an immuno-regulatory cytokine) and s-VCAM-1 (as a marker of endothelial dysfunction) in different clinical types of (MS). 2- Find out possible correlations between serum levels of IL-12 and s-VCAM-1 and disease activity, clinical grade of disability and MRI findings of cerebral atrophy.

Patients and Methods

Subjects Fifty three Egyptian female patients, diagnosed as clinically definite MS according to the modified Poser criteria, were included in this study. Patients were divided into three groups: Group I: included 20 patients suffering from relapsing remitting MS in remission. Group II: included 16 patients suffering from relapsing remitting MS in relapse. Group III: included 17 patients suffering form secondary progressive MS.

Subjects Patients were previously subjected to the therapeutic protocols accepted in the neurology department of Cairo university hospital. Pulse therapy in the form of methylprednisolone 1 gm by intravenous infusion for 5-7 days for acute exacerbation in relapsing remitting patients followed by oral prednisolone in a tapering schedule. In secondary progressive patients, the pulse therapy was followed during exacerbations and immunosuppressive drugs were taken as maintenance to slow the progression of the disease. Fifteen age matched healthy control female subjects were included as a control group.

Patients were submitted to the following: Methods Patients were submitted to the following: Thorough clinical evaluation. Clinical grading of the disability using the Expanded Disability Status Scale (EDSS). Conventional MRI of the brain (T1 and T2 weighted axial, sagittal, coronal and fluid attenuated inversion recovery images). Routine Laboratory investigations. Assessment of the serum levels of Interlukin-12 and sVCAM-1 using Enzym Linked Immuno-Sorbed Assay (ELISA) technique.

Results

Results Demographic and clinical results: Age: The age of patients ranged from 27 to 45 years with a mean of 34.49 ± 5.5 years. The age of the controls ranged from 25 to 45 years with a mean of 34 ± 5.3 years. (p > 0.05). Duration of illness: The duration of illness ranged from 1 to7 years with the mean of 3.95 ± 1.8 years in group I, and from 2 to 10 years with a mean of 3.68 ± 2.0 years in group II , and from 1 to 9 years with a mean of 4.47 ± 2.8 years in group III.

Table (1): Age range, mean and SD of the 3 groups of patients with definite MS. Max Min 6.4 35.2 45 27 Group 1 4.9 33.3 43 28 Group 2 33.8 41 Group 3 NS > 0.05 P value

3.85 ± 1.56 a 7 1 8.05 ± 2.63 b 12 4 6.25 ± 2.40 b Patients Mean ± SD Number of relapses: The mean number of relapses was significantly higher in the RRMS in relapse and SPMS patients compared to RRMS in remission patients (P=0.004 and 0.01, respectively). However, no significant difference was detected between RRMS in relapse and SPMS patients, (P> 0.05). Table (2): The range, mean and SD of the number of relapses of MS in the patient groups. Number of relapses Patients Mean ± SD Max Min 3.85 ± 1.56 a 7 1 Group 1 8.05 ± 2.63 b 12 4 Group 2 6.25 ± 2.40 b Group 3

Expanded Disability Status Scale (EDSS): Expanded Disability Status Scale (EDSS): The mean score was significantly higher in the secondary progressive (SP) patients compared to patients with RRMS in remission (P=0.0001) and in relapse (P=0.0001). However, no significant difference was detected between group I (RRMS in remission) and group II (RRMS in relapse), (P>0.05). Table(3): Range, mean and SD of EDSS score in the patient groups. EDSS Score Patients Mean ± SD Max Min 3.3 ± 1.0 a 6 1.5 Group 1 3.82 ± 1.03 a 2 Group 2 5.50 ± 1.20 b 8 3.5 Group 3

MRI results: MRI of the brain revealed cerebral atrophy in 17 patients (32%); 4 from RRMS in remission group, 3 from RRMS in relapse group and 10 patients from SPMS group. Cerebral atrophy was significantly encountered in SPMS group as compared to RRMS in remission group and RRMS in relapse group, (p= 0.01). The mean value of the lesion load was significantly higher in the RRMS in relapse as compared to RRMS in remission and SPMS (P= 0.0001, 0.01 respectively). SPMS also showed significantly higher lesion load as compared to RRMS in remission (P=0.01).

Table (4): Range, mean and SD of the lesion load in MRI in the patients groups. Mean ± SD Max Min 8.5 ± 3.05 a 16 4 Group 1 15.2 ± 3.94 b 21 7 Group 2 11.93 ± 4.24 c 20 Group 3

Laboratory results: Table (5): Range, mean and SD of IL-12 in control subjects and the patients groups. IL-12 (pg/mL) Mean ± SD Max Min 1.5 ± 0.51 2.6 0.9 Control group 3.04 ± 0.95 4.5 1.6 Group 1 6.53 ± 1.0 8.3 4.3 Group 2 11.7 ± 3.11 16.3 5.2 Group 3

Table (6): Range, mean and SD of sVCAM-1 in control subjects and the patients groups. sVCAM-1 (ng/mL) Mean ± SD Max Min 508.33 ± 40.5 570 430 Control group 580.5 ± 93.0 750 450 Group 1 710.62 ± 83.2 890 590 Group 2 888.23 ± 95.34 1050 700 Group 3

The mean levels of serum IL-12, and sVCAM-1 in all patients groups were significantly higher as compared to the control group. Moreover, the mean levels of these parameters were significantly higher in the SPMS patients compared to that of RRMS in remission and RRMS in relapse patients, (P=0.0001 for each). A statistically significant difference was also detected in the mean serum levels of IL-12 and sVCAM-1 in group II (RRMS in relapse) versus group I (RRMS in remission) (P= 0.001).

Table (7): Comparison between mean serum levels of IL-12 and sVCAM-1 in the control group and in different patient subgroups. Group 3 Group 2 Group 1 Controls Variable 11.7± 3.11 d 6.53± 1.0 c 3.04± 0.95 b 1.50 ± 0.51 a IL-12 888.23± 95.34 d 710.62 ± 83.2 c 580.5± 93.0 b 508.33± 40.5 a sVCAM-1

According to MRI, patients were subdivided into patients with cerebral atrophy (17 patients) and patients without cerebral atrophy (36 patients). Significantly higher levels of serum IL-12, sVCAM-1 and mean EDSS score in patients with cerebral atrophy versus those without atrophy (P= 0.004, 0.01 and 0.0003, respectively). However, no statistically significant difference was detected between patients with and without MRI signs of cerebral atrophy in the total number of relapses or lesion load, (P>0.05).

Patients without atrophy Table (8): Comparison between patients with and without MRI signs of cerebral atrophy as regards number of relapses, mean EDSS score mean lesion load and mean serum levels of IL-12, sVCAM-1. P value Patients without atrophy Patients with atrophy Variable NS 5.72 ± 2.8 6.35 ± 2.82 No of Relapses 0.0003* 3.63 ± 1.12 5.17 ± 1.46 Mean EDSS Score 11.58 ± 4.56 12.0 ± 4.9 Lesion Load 0.004* 5.73 ± 3.38* 9.28 ± 4.56 IL-12 0.01* 677.5 ± 115.1* 805.2 ± 205 sVCAM-1

Correlations: No significant correlation was detected between the mean serum levels of IL-12, sVCAM-1 with either age, duration of illness, total number of relapses or lesion load (p < 0.05). No significant positive correlation was detected between mean serum levels of IL-12 and sVCAM-1 and EDSS score (r= 0.57; P=0.0001 and 0.001 respectively).

Fig (1): Correlation between IL-12 (pg/mL) and EDSS score in MS patients.

A significant positive correlation was detected between mean serum levels of IL-12 and sVCAM-1, (r=0.73; P=0.0001). Fig (2):  Correlation between serum IL-12 (pg/mL) and  sVCAM-1 (ng/mL) in MS patients.

Correlations: Non-parametric correlation detected the presence of a highly significant positive correlation between MRI signs of cerebral atrophy with EDSS score (r=0.51, P=0.001*). However, no significant correlation could be detected between MRI signs of cerebral atrophy with either IL- 12 or sVCAM-1 serum levels (p>0.05).

Conclusions

Conclusions The mean serum level of IL-12 was significantly elevated in all MS groups compared to the control group. Significantly higher levels of IL-12 were detected in SPMS group versus RRMS groups (whether in relapse or in remission). The level of IL-12 was also significantly higher in RRMS in relapse versus RRMS in remission. the progressive phase of MS may be secondary to neuronal degenerative changes triggered by inflammation. This may explain the higher levels of IL-12 in SPMS versus RRMS in remission and in relapse detected in our study.

Conclusions The mean serum level of sVCAM-1 was elevated in MS patients compared to controls. Significantly higher levels were detected in SPMS versus RRMS groups. The levels of sVCAM-1 were also significantly higher in RRMS in relapse versus RRMS in remission. Up regulated sVCAM-1 in blood and CSF indicate sustained potential for inflammation in the CNS throughout the clinical spectrum of MS. The functional alteration of hematoencephalic barrier endothelium manifest in constant pathologic expression of sVCAM-1 molecule is one of the patho-chemical mechanisms promoting progression of MS. This might explain the significantly higher levels of sVCAM-1 in SPMS as compared to RRMS in our patients.

Conclusions Significant positive correlation was detected between EDSS scores and serum levels of IL-12 and sVCAM-1 suggesting their utility as reliable markers of disability in MS patients. No significant correlation was detected between the mean serum levels of IL-12 and sVCAM-1 with the total number of relapses or lesion load. Patients with MRI signs of cerebral atrophy have significantly higher serum levels of IL-12 and sVCAM-1 as compared to patients without cerebral atrophy. This finding suggests that inflammation does not decrease in the stage of brain atrophy and that brain atrophy may occur early in the course of MS.

Thus Immunomodulatory therapy targeting IL-12 and sVCAM-1 might be beneficial to delay disease progression and/or reduces lesion activity in patients with MS.