Online ISSN : 1687-8329

    




Quick Search 
 
Author  
Year    
Title  
Vol:  

 
 
April2014 Vol.51 Issue:      2 Table of Contents
Full Text
PDF


Evaluation of Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) as a Response Marker for Interferon beta in Multiple Sclerosis

Maha Nada1, Salma H. Khaleel1, Iman M. Bayoumy1, Rania Ahmed Hassan2

Departments of Neurology1, Medical Microbiology & Immunology2, Ain Shams University; Egypt



ABSTRACT

Background: Multiple sclerosis is the most common disease of the CNS that causes long and severe disability in young adults. Some patients with relapsing-remitting MS (RRMS) are unresponsive to IFN-β therapy. The Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL) level was proposed as a response marker for IFN beta treatment. Objective: To recognize patients who will benefit most from treatment by assessing the functional relevance of TRAIL, to the clinical treatment response. Methods: This study was done on 24 patients from the Neurology department, Ain Shams University Hospitals. Patients had relapsing-remitting multiple sclerosis, for longer than 1 year. Patients were selected with no immunomodulatory treatment, and no exacerbations 4 weeks before start of study. Patients were examined clinically. MRI examinations were done before, 6 months and at the end of treatment. Estimation of TRAIL levels on monocytes and lymphocytes surfaces was done before treatment. Patients were given 44µg interferon beta 1a by subcutaneous injection three times per week for 1 year. Responders were defined as patients with no relapses and non-responders with one or more relapses during treatment (one year). Results: Responders showed higher values in the number of lymphocytes and monocytes having TRAIL and in the amount of TRAIL on them. Clinically worse patients showed higher number of monocytes having TRAIL. Conclusion: All the markers of TRAIL were increased in the responder group .The monocyte level was increased in clinically worse patients, which suggests that monocytes can be a better marker than lymphocytes. [Egypt J Neurol Psychiat Neurosurg.  2014; 51(2): 201-206]

Key Words: TRAIL, Multiple Sclerosis, Interferon beta.

Correspondence to Iman Bayoumy, Neurology Department, Ain Shams University, Egypt. Tel.: +201002595522. Email: imanbayomy@yahoo.com






INTRODUCTION

 

Multiple sclerosis (MS) affects about 1 million individuals worldwide and is the most common disease of the central nervous system (CNS) that causes long and severe disability in young adults.  Although its cause remains unknown, strong evidence supports the notion that underlying its pathogenesis is an inflammatory process against self molecules within the white matter of the brain and spinal cord that is mediated by T cells.1 Since T cells reactive to myelin are present in patients with multiple sclerosis and in healthy individuals2, the main immune abnormality in multiple sclerosis probably involves failed regulatory mechanisms that lead to enhanced T-cell activation and less stringent activation requirements.3

The tumor necrosis factor (TNF) family genes are strong candidates for involvement in MS risk because they play important roles in the interaction between the CNS and the immune system in both defense and apoptosis of neurons and glial cells in neuroinflammatory diseases.4

TNF Related Apoptosis Inducing Ligand (TRAIL) is a type II transmembrane protein, belonging to the TNF/nerve growth factor superfamily, capable of inducing apoptosis in susceptible cells through interaction with its receptors TRAILR-1 and TRAILR-2. Two other cell-bound receptors; TRAILR-3 and TRAILR-4, and a soluble receptor; osteoprotegerin, do not contain functional death domains and act as decoy receptors for TRAIL. It is thought that the balance between death and decoy receptors underlies the sensitivity to TRAIL-induced apoptosis in different cell types.5 TRAIL plays immunosuppressive, immunoregulatory and immune-effector functions, and it is involved in the pathogenesis of MS as well as in other autoimmune diseases, although its precise role is not completely understood.5

It may inhibit activation of autoreactive T cells that initiate autoimmune responses in vitro and in vivo or may induce apoptosis of inflammatory cells. Recently, it has been reported that activation of T cells with IL-2 resulted in TRAIL-mediated death of antigen-specific memory CD8+ T cells and that human T helper (Th) 1 cell clones are sensitive to TRAIL-induced apoptosis, whereas Th2 cell lines are not. Whether this is also true in vivo remains to be clarified.4

CNS inflammation in MS is associated with elevated expression of TRAIL, both within the CNS and autoreactive immune cells.6 TRAIL has been implicated in MS pathogenesis and in the mechanism of action of interferon-beta (IFN-β), a disease modifying therapy that has been used to treat MS for over twenty years.6 Recombinant IFN-β therapy is typically employed for the treatment of relapsing-remitting MS (RRMS). Although the precise mechanism responsible for the beneficial effects of IFN-β in the treatment of MS remains unclear, the abilities of this cytokine to inhibit T-cell activation and proliferation as well as to facilitate the apoptotic elimination of autoreactive T cells are thought to be therapeutically relevant.6 IFN-β increases circulating levels of soluble TRAIL (sTRAIL) and the expression of membrane-bound TRAIL (mbTRAIL) in immune cells derived from the peripheral blood of MS patients. Therefore, TRAIL may have a rule in the mechanism of action of IFN-β, by stimulating the apoptosis of autoreactive immune cells. IFN-β is not curative, however it reduces disease progression, hence decreasing relapses frequency and severity. However, some patients are unresponsive to IFN-β therapy and continue to experience relapses and disease progression while treated with this therapeutic agent.  TRAIL level has been proposed as a response marker for IFN beta treatment.6

Aim of work: to recognize patients who will benefit most from IFN-β1a treatment by assessing the functional relevance of tumor necrosis factor (TNF) -Related Apoptosis Inducing Ligand (TRAIL), to the clinical treatment response.

 

PATIENTS AND METHODS

 

This study took place in the Neurology department, Ain Shams University Hospitals in the period from January 2012 to June 2013.

This study was done on 24 patients (20 females and 4 males) with age ranging from 25 to 55 years. They were recruited from inpatient and outpatient clinic, Ain Shams University Hospitals.

All patients had to fulfill the inclusion criteria of having clinically definite or radiologically supported multiple sclerosis.7 Relapsing-remitting course for longer than 1 year. They were with no immune-modulatory treatment before the study and with no exacerbations 4 weeks before the start of the study. Patients with any other neurological or systemic disease were excluded from the study.

All patients were examined clinically and were chosen to fulfill the inclusion and exclusion criteria. The expanded disability status scale (EDSS)8 was used in the evaluation of patients before and at the end of treatment. MRI examinations were done before the onset of the study,6 months and at the end of treatment(1 year). Estimation of TRAIL levels on monocytes and lymphocytes surfaces was done for all patients before interferon beta 1a treatment.

Whole blood was collected in evacuated tubes containing EDTA as the anti-coagulant.

Cells have been washed three times in an isotonic phosphate buffer (supplemented with 0.5% BSA) followed by centrifugation at 500xg for 5 minutes to remove contaminating serum components. Fifty μL of packed cells were transferred to a 5 mL tube for staining with the monoclonal antibody.

The Fc-blocked (25 μL) cells treated with 1 μg of human IgG/105 cells for 15 minutes at room temperature; were transferred to a 5ml tube. Then 10 μL of PE-conjugated anti-TRAIL reagent were added and incubated for 30 minutes at 2°-8° C.  Following washing of the cells twice with 4 mL of PBS buffer, to remove unreacted anti-TRAIL reagent; the RBCs were lysed using lysing solution supplied by Sigma (Chemical Co., St. Louis Mo). Cells were re-suspended in 200-400 μL of PBS buffer for final flow cytometric analysis. As a control for this analysis, cells in a separate tube were treated with PE-labeled mouse IgG1 antibody.

Estimation of TRAIL levels on monocytes and lymphocytes’ surfaces was done by flow cytometry using R&D Systems, Inc.  Monoclonal  Anti-human TRAIL/TNFSF10-Phycoerythrin (Catalog Number: FAB687P) containing Phycoerythrin (PE)-conjugated mouse monoclonal anti-human TRAIL/TNFSF10: Supplied as 50μg of antibody in 1mL saline containing up to 0.5% BSA and 0.1% sodium azide. Cytometric analysis was done using flow cytometry device (Coulter Epics XL TM USA, 1999), (Miami, Florida, System II, TM software).  Cell surface expression of TRAIL was determined at 488 nm wave length laser excitation and monitoring emitted fluorescence with a detector optimized to collect peak emissions at 565-605nm. Lymphocytes and monocytes phenotyping was done by gating according to forward scatter (size) and side scatter (granularity) strategy. All patients were given 44µg interferon beta 1a by subcutaneous injection three times per week9 for one year. Drug responders were defined as patients who had no further relapses and no deterioration in the EDSS during interferon-beta treatment and follow up MRI showing no enhanced or newly developed lesions in T2 or flair. Patients who continued to have one or more relapses were defined as nonresponders. A clinical relapse was defined as substantial worsening of pre-existing symptoms or appearance of new neurological deficits in the absence of fever and lasting for longer than 24 hours confirmed by MRI showing newly developed lesions corresponding to the clinical presentation.

Statistical Analysis

Kolmogorov–Smirnov’s test was used to evaluate normal distribution of continuous data. All results are presented as mean and SD values or as median according to the distribution of data. Categorical results are presented as numbers of cases and percentages. Continuous variables were compared using Student t test or the Mann–Whitney U-test, depending on the distribution of raw data. Categorical variables were compared using fisher exact test. Spearman correlation coefficient (rho) was used for assessment of correlation between variables. A significance level of p <0.05 was used in all tests. All statistical procedures were carried out using SPSS version 15 for Windows (SPSS Inc, Chicago, IL, USA).

 

RESULTS

 

The age of patients ranged from 25 to 55years with a mean age of 39.5±12.2.years. Regarding the sex distribution, this study included 4 males (16.7%) and 20 females (83.3%). There was no significant difference between responders and non-responders as regard Patients’ sex (p=1.00). There was a significant difference between responders and non-responders regarding number of lymphocytes and monocytes having TRAIL and amount of TRAIL on lymphocytes. There was a highly significant difference between responders and non responders as regard amount of TRAIL on monocytes, with responders showing higher values. However   no significant difference between responders and non responders was found regarding EDSS (Table 1). There was no significant difference between females and males as regard, number of monocytes having TRAIL, amount of TRAIL on lymphocytes and monocytes, with exception of number of lymphocytes having TRAIL. Males had higher value 26.1±2.5 whereas females had 21.7±9 (p=0.013). There was a significant negative correlation between number of lymphocytes having TRAIL and Number of attacks/one year, otherwise no significant correlation was found between number of monocytes having TRAIL, amount of TRAIL on lymphocytes and monocytes and number of attacks / one year (Table 2). There were no significant differences between cases with EDSS below or equal to 5 and cases above 5 as regard sex and response to treatment. There was no significant difference between cases with EDSS below or equal to 5 and cases above 5 as regard number of lymphocytes having TRAIL, amount of  TRAIL on lymphocytes and monocytes, with exception of number of monocytes having TRAIL where cases with EDSS>5 had higher value (Table 3).


 

Table 1. Comparison between responders and non responders as regard patients’ TRAIL results.

 

Items of TRAIL studies

Responders

Non responders

P-value

Mean

±SD

Median

Mean

±SD

Median

Number of lymphocytes having TRAIL a

27.59

10.17

23

18.70

4.23

19

0.026  *

Number of monocytes having TRAIL a

65.82

6.57

66

55.48

14.91

58

0.028 *

Amount of TRAIL on lymphocytes b

1.79

0.57

2

1.35

.18

1

0.042**

Amount of TRAIL on monocytes a

3.47

1.42

3

1.71

.33

2

0.001 *

a Mann-Whitney U, b student t-test, TRAIL TNF Related Apoptosis Inducing Ligand,

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

 

Table 2. Correlation between Number of attacks / one year and TRAIL results.

 

Items of TRAIL studies

Number of attacks / 1 year

Number  of lymphocytes having TRAIL

rho

-0.836

P

0.001

Sig

p<0.01*

Number  of monocytes having TRAIL

rho

0.398

P

0.200

Sig

NS

Amount of TRAIL on lymphocytes

 

rho

-0.314

P

0.320

Sig

NS

Amount of TRAIL on monocytes

 

rho

-0.440

P

0.153

Sig

NS

TRAIL TNF Related Apoptosis Inducing Ligand, Sig. Significance, NS Non-significant, HS Highly significant

Table 3. Comparison between cases with EDSS below or equal to 5 and cases above 5 as regard TRAIL results.

 

Items of TRAIL studies

EDSS=<5

EDSS>5

P-value

Mean

±SD

Median

Mean

±SD

Median

Number of lymphocytes having TRAIL

19.31

3.97

19.95

28.60

11.51

25.80

0.058

Number  of monocytes having TRAIL

55.59

13.3

58.65

68.18

7.61

66.55

0.004*

Amount of TRAIL on lymphocytes

1.37

0.18

1.34

1.85

0.63

1.73

0.070

Amount of TRAIL on monocytes

2.65

1.52

1.77

2.03

0.35

2.04

0.806

EDSS Expanded disability status scale, TRAIL TNF Related Apoptosis Inducing Ligand

*Significant at p<0.01

 

 


DISCUSSION

 

MS is a complex and chronic demyelinating autoimmune disorder that presents through an interaction of environmental and genetic factors.10-14 The onset of MS occurs at an individual’s most productive years (20–40 years)15,16 and affects considerably more women than men.17-19 These results go with our study that showed that 83.3% (20) patients were females and only 16.7% (4) were males. Interferons (IFNs) are proteins that belong to the cytokine network and are involved in the regulation of immune response against microbial and viral antigens.20 IFN-β suppresses the proliferation of myelin-basic protein-specific T cells, reduces the production of proinflammatory cytokines (as IFN-γ), and induces anti-inflammatory cytokines, such as interleukin (IL)-1021-23, therefore, maintaining the cytokine balance that protects neurons from demyelination. This is achieved through suppressing the proliferation of T cells that are required for advancing the autoimmune process and inhibiting them from crossing the blood–brain barrier (BBB).22 In our study, we used a dose of 44ug subcutaneous three times a week as this dose proved to be more effective. Its efficacy was demonstrated in the 2-year PRISMS trial, as SC IFNβ-1a 44 µg three times weekly (ttw) significantly reduced relapse rates, with ≈30% relative risk reduction compared with placebo. SC IFNβ-1a was also associated with significantly delayed progression of disability, and lower disease activity according to MRI, relative to placebo. CNS inflammation in MS is associated with elevated expression of TRAIL, both within the CNS and autoreactive immune cells.24,25

In this study, we compared levels of TRAIL on lymphocytes, T cells, B cells, and monocytes isolated from the peripheral blood of MS patients treated with IFN-β. We classified the patients into two groups .The first group was the responders who did not have any attacks during the period of treatment (one year). The second group was the non-responders that had one or more attacks during the same period. There was a significant difference between responders and non-responders as regard number of lymphocytes having TRAIL, number of monocytes having TRAIL and amount of TRAIL on lymphocytes .There was a highly significant difference between responder and non-responder as regard amount of TRAIL on monocytes, with responders showing higher values. These results are matched with the results of Wandinger KP and colleagues 200326 that found that the overall TRAIL exression pattern differed significantly between the responders and the non-responders.  They also found that in patients with multiple sclerosis, expression of TRAIL in peripheral blood mononuclear cells increased significantly both in the immediate short term, and in the long term after treatment with interferon beta. Responders also had significantly higher baseline concentrations of soluble TRAIL than non-responders (p =0.004), indicating underlying differences in posttranscriptional regulation of TRAIL expression. There was an indirect significant correlation between number of lymphocytes having TRAIL and Number of attacks / one year (table 2). This means that the more the patient has attacks, the more the decrease in the number of lymphocytes that have TRAIL.  This finding was not discussed in other studies and needs further assessment in future studies. There was significant difference between cases with EDSS below or equal to 5 and cases above 5 as regard , number of monocytes having TRAIL where cases with EDSS>5 had higher value (table 3). This means that the worse the condition of the patient, the higher the number of monocytes that have TRAIL. These findings go with those of Hebb and colleagues 20116 that found that monocytes had a significant increase in the level of TRAIL in relapsing remitting MS in comparison to level of TRAIL on T and B lymphocytes.

 

Conclusion

We found that TRAIL is increased in responders before commence of treatment; all the markers were increased in the responder group. The monocyte level was increased in patients with worse clinical condition, which suggests that monocytes can be a better marker than lymphocytes. We recommend future study of TRAIL during and after treatment with interferon.

 

[Disclosure: Authors report no conflict of interest]

REFERENCES

 

1.        Martin R, McFarland HF, McFarlin DE. Immunological aspects of demyelinating diseases. Annu Rev Imumnol. 1992; 10:153-87.

2.        Pette M, Fujita K, Kitze B, Whitaker JN, Albert E, Kappos L,  et al. Myelin basic protein-specific T lymphocyte lines from MS patients and healthy individuals. Neurology. 1990; 40:1770-6.

3.        Markovic-Plese S, Cortese I, Wandinger KP, McFarland HF, Martin R. CD4+CD28- costimulation-independent T cells in multiple sclerosis. J Clin Invest. 2001; 108:1185-94.

4.        Falschlehner C, Schaefer U, Walczak H. Following TRAIL’s path in the immune system. Immunology. 2009; 127:145-54.

5.        Todaro M, Zeuner A, Stassi G. Role of apoptosis in autoimmunity. J Clin Immunol. 2004; 24(1):1-11.

6.        Hebb AL, Moore CS, Bhan V, Robertson GS. Effects of IFN-B on TRAIL andDecoy Receptor Expression in Different Immune Cell Populations from MS Patients with Distinct Disease Subtypes. Autoimmune Diseases. 2011; 485752. doi:10.4061/2011/485752.

7.        Poser CM, Paty DW, Scheinberg L, McDonald WI, Davis FA, Ebers GC, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol .1983; 13:227-31.

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

9.        Sanford M, Lyseng-Williamson KA. Subcutaneous Recombinant Interferon-β-1a (Rebif®). Drugs. 201171(14):1865-91.

10.     Polman CH, O'Connor PW, Havrdova E, Hutchinson M, Kappos L, Miller DH, et al. A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med. 2006; 354(9):899-910.

11.     Williams R, Rigby AS, Airey M, Robinson M, Ford H. Multiple sclerosis: its epidemiological, genetic, and health care impact. J Epidemiol Community Health. 1995; 49(6):563-9.

12.     Compston A, Coles A. Multiple sclerosis. Lancet. 2002; 359(9313):1221-31.

13.     Compston A, Coles A. Multiple sclerosis. Lancet. 2008; 372(9648): 1502-17.

14.     Keegan BM, Noseworthy JH. Multiple sclerosis. Ann Rev Medic. 2002; 53:285-302.

15.     Koch-Henriksen N. The Danish Multiple Sclerosis Registry: a 50-year follow-up. Mult Scler. 1999; 5(4):293-6.

16.     Mayr WT, Pittock SJ, McClelland RL, Jorgensen NW, Noseworthy JH, Rodriguez M. Incidence and prevalence of multiple sclerosis in Olmsted County, Minnesota, 1985–2000. Neurology. 2003; 61(10): 1373-7.

17.     Hawkins SA, McDonnell GV. Benign multiple sclerosis? Clinical course, long term follow up, and assessment of prognostic factors. J Neurol Neurosurg Psychiatry. 1999; 67:148-52.

18.     Whitacre CC, Reingold SC, O’Looney PA. A gender gap in autoimmunity: Task Force on Gender, Multiple Sclerosis and Autoimmunity. Science. 1999; 283(5406):1277-8.

19.     Orton SM, Herrera BM, Yee IM, Valdar W, Ramagopalan SV, Sadovnick AD, et al. Sex ratio of multiple sclerosis in Canada: a longitudinal study. Lancet Neurol. 2006; 5(11):932-6.

20.     Weinstock-Guttman B, Ransohoff RM, Kinkel RP, Rudick RA. The interferons: biological effects, mechanisms of action, and use in multiple sclerosis. Ann Neurol. 1995; 37(1):7-15.

21.     Weber F, Janovskaja J, Polak T, Poser S, Rieckmann P. Effect of interferon beta on human myelin basic protein-specific T-cell lines: comparison of IFNbeta-1a and IFNbeta-1b. Neurology. 1999; 52(5):1069-71.

22.     Yong VW, Chabot S, Stuve O, Williams G. Interferon beta in the treat­ment of multiple sclerosis: mechanisms of action. Neurology. 1998; 51(3): 682-9.

23.     Kieseier BC. The mechanism of action of interferon-beta in relapsing multiple sclerosis. CNS Drugs. 2011; 25(6):491-502.

24.     Aktas O, Smorodchenko A, Brocke S, Infante-Duarte C, Schulze Topphoff U, Vogt J,   et al. Neuronal damage in autoimmune neuroinflammation mediated by the death ligand TRAIL Neuron. 2005; 46(3):421-32.

25.     Aktas O, Waiczies S, Zipp F. Neurodegeneration in autoimmune demyelination: recent mechanistic insights reveal novel therapeutic targets. J Neuroimmunol. 2007; 184:17-26.

26.     Wandinger KP, Lünemann JD, Wengert O, Bellmann-Strobl J, Aktas O, Weber A, et al. TNF-related apoptosis inducing ligand (TRAIL) as a potential response marker for interferon-beta treatment in multiple sclerosis. Lancet. 2003; 361(9374):2036-43.


 

 

 

 

 

 

 

الملخص العربى

 

تقييم الجزئ المحفز لموت الخلايا المبرمج المرتبط بعامل نخر الورم كعلامة تجاوب

فى علاج  مرضى التصلب المتناثر بعقار الانترفيرون – بيتا

 

التصلب المتناثر من أكثر أمراض الجهاز العصبى المركزى التى تحدث إعاقة شديدة وطويلة المدى فى الشباب.يستعمل عقار الانترفيرون –بيتا عادة لعلاج التصلب المتناثر ذات الانتكاسات المتعددة.بعض المرضى لا يستجيبون للعلاج بعقار الانترفيرون –بيتا.تم اقتراح تقييم نسبة  الجزئ المحفز لموت الخلايا المبرمج المرتبط بعامل نخر الورم كعلامة تجاوب للعلاج بعقار الانترفيرون – بيتا.

لهدف من البحث هو التعرف على المرضى الذين سوف يستفيدون أكثر من العلاج بتقييم الأهمية الوظيفية للجزئ المحفز لموت الخلايا المبرمج المرتبط بعامل نخر الورم و علاقته بالتحسن السريرى على العلاج.تم عمل البحث على 24 مريض من قسم الأعصاب بمستشفيات جامعة عين شمس.يعانى المرضى من التصلب المتناثر ذات الانتكاسات المتعددة لأكثر من عامين.لم يخضع المرضى لأى علاج مناعى لمدة ستة اشهر ولا يعانوا من انتكاسات لمدة أربعة أسابيع قبل بدء البحث.تم فحص المرضى سريريا.تم تقييم نسبة الجزئ المحفز لموت الخلايا المبرمج المرتبط بعامل نخر الورم علي سطح  الحيدات والخلايا الليمفاوية  قبل بدء العلاج.

تم علاج المرضى بعقار الانترفيرون –بيتا 1أ 44µ جم عن طريق الحقن تحت الجلد ثلاث مرات أسبوعيا لمدة ستة اشهر.تم متابعة المرضى لمدة ستة اشهر.المرضى المستجيبين للعلاج هم الذين لم يتعرضوا لأى انتكاسات أم الغير مستجيبين فهم الذين تعرضوا لانتكاسة أو أكثر.

أظهرت النتائج ارتفاعات ذات دلالة إحصائية في نسبة الخلايا الليمفاوية والحيدات الحاملة للجزئ المحفز لموت الخلايا المبرمج المرتبط بعامل نخر الورم وكميتة على هذه الخلايا فى المرضى المستجيبين للعلاج. كما كان عدد الحيدات الحاملة لهذا الجزئ أعلى فى المرضى الأسوأ سريريا.كل مؤشرات الجزئ المحفز لموت الخلايا المبرمج المرتبط بعامل نخر الورم كانت مرتفعة فى المجموعة المستجيبة للعلاج. عدد الحيدات الحاملة لهذا الجزئ أعلى فى المرضى الأسوأ سريريا مما يشير الى ان الحيدات من الممكن ان تكون مؤشر أفضل من


2008 � Copyright The Egyptian Journal of Neurology,
Psychiatry and Neurosurgery. All rights reserved.

Powered By DOT IT