INTRODUCTION

 

There are two principally different and partly opposite ways in which apoptosis could affect an auto immune attacks on a target organ.  First, the induction of apoptosis in B-cells of the langerhans islets^1,2 or in oligodendrocytes ⁽³⁾ may contribute to disease in diabetes or MS.  Secondly, activation of T cells is normally followed by "activation–induced cell death" (AICD), which is a physiologic mechanism to limit an immune response.  Thus failure in this process, i.e. decreased apoptosis, may lead to inappropriate persistence of activated T cells, thereby contributing to autoimmunity⁴.

                Apoptosis is the effect of complex chain of intracellular events leading to the activation of a number of pathways⁵. These pathways are triggered by a number of cell-surface receptors, the most important of which belong to the tumor necrosis factor (TNF) receptor family, of these Fas (CD95) together with its ligand Fas-L (CD95L)⁶.

                Fas mediate apoptosis of T cells. T cells apoptosis contributes to resolution of the central nervous system inflammation and clinical recovery form attacks of experimental autoimmune encephalomyelitis [EAE], an

animal model of multiple sclerosis (MS)⁷. Apoptosis of auto reactive T cells in the CNS is likely to be important in preventing the development of MS⁸.

NF-кB represents a family of dimeric transcription factor that play a central role in inflammatory responses by regulation of gene expression and inhibition of apoptosis⁹.

             Bcl-2 family proteins include the death antagonists Bcl-2 and Bcl-X(L), and death agonists Bax and Bad the commitment of T lymphocytes to die is partly regulated by the Bcl-2 family proteins and altered expression of these families in T lymphocytes is involved in promoting cellular  resistance to apoptosis in patients with MS¹⁰.

This work aims at clarifying the role of pro- and anti-apoptotic mediators in pathogenesis of MS.

 

PATIENTS AND METHODS

 

This study included 24 patients with multiple sclerosis selected from neurology clinics, Tanta University hospital and 16 healthy individuals as control. Patients were divided into 3 groups: Group I: Included 8 patients with remitting-relapsing multiple sclerosis in remission. Group II: Included 9 patients with remitting-relapsing multiple sclerosis in relapse. Group III: Included 7 patients with secondary progressive multiple sclerosis.

Inclusion criteria:

All patients had clinically definite MS patients according to McDonald criteria¹¹. Relapsing, remitting, and secondary progressive courses based on criteria of Lublin and Reingold¹².

 

Exclusion criteria:

1.      Patients aged less than 15 years or more than 55 years.

2.      Patients administrating any medications that suppress the immune system as corticosteroids and immune- suppressant drugs within two months from inclusion in the study.

3.      Patients suffering from any neurological or medical problems including e.g. diabetes, liver, kidney or other autoimmune diseases.

 

Patients and controls were subjected to the following:

I.       Morphological assessment of apoptosis by Giemsa¹³, acridine orange¹⁴ and propidium iodide stain¹⁵ at time 0, 24, 48 and 72 hours.

II.     Morphological assessment of apoptosis by determination of DNA fragmentation¹⁶ using agarose gel electrophoresis.

III.   Immunological studies of apoptotic and antiapoptotic factors.

Determination of Fas (CD 95)¹⁷, Fas ligand¹⁸. Bcl-2¹⁹ and Nuclear factor Kappa B (NF-kB)²⁰ in serum sample by ELISA (kits were supplied from Sumimoto Co, Ltd, Japan, Oncogene Science, Cambridge UK and Vector laboratories inc., Burlingame, CA respectively.

IV.    Gene expression study of apoptotic factors (Fas and Fas ligand) and antiapoptotic factors (Bcl₂ and NF-_κB).^21,22.

 

Statistical analysis:

Statistical analysis was performed using SPSS v.13 for Windows. Data were expressed as Mean±SD. Comparison of variables among more than 2 groups was performed using analysis of variance (ANOVA). A p-value of <0.05 was considered significant.

 

RESULTS

               

The percentage of apoptotic PBMCs was significantly lower in patients versus controls, with no difference between patients groups (Table 1 and Figs. 1, 2, 3).

                Apoptotic factors [Fas and Fas ligand] showed highly significant decrease in patients versus control with no significant correlation between patient's groups (Table 2 and Figs. 4,5).  DNA fragmentation showed significant  increase in control versus patients (Table 3; Figure 6). Antiapoptotic factors [Bcl-2 and NF-_kB] showed significant increase in patients than control (Table 4 and Fig. 7).

 

Table 1. Percentage of apoptotic peripheral blood mononuclear cells in the patients with multiple sclerosis and controls

 

RRMS relapsing remitting multiple sclerosis, SPMS secondary progressive multiple sclerosis

*Statistically significant at p<0.01

 

Table 2. Apoptotic factors (Fas CD₉₅ and Fas ligand) by ELISA in mg/ml in patients with multiple sclerosis and controls

RRMS relapsing remitting multiple sclerosis, SPMS secondary progressive multiple sclerosis

*statistically significant at p<0.01

 

Table 3. Assessment of apoptosis of the studied by DNA electrophoresis patients with multiple sclerosis and controls

 

RRMS relapsing remitting multiple sclerosis, SPMS secondary progressive multiple sclerosis

 

Table 4. Antiapoptotic factors (Bcl-2 and NF-kB) determined by ELISA in mg/mL and RT-PCR in patients with multiple sclerosis and controls

RRMS relapsing remitting multiple sclerosis, SD standard deviation, SPMS secondary progressive multiple sclerosis

*Statistically significant at p<0.01

 

Figure 1. Apoptosis of PBMCs using Giemsa stain after 72h.	Figure 2. Apoptosis of PBMCs using acridin orange after 72h.
Figure 3. Apoptosis of PBMCs using propidium iodide stain after 72 h.	Figure 4. Gel electrophoresis of PCR products stained  by ethidium bromide in estimation of Fas-LmRNA (lane 2,3,4) and Fas mRNA (lane 5,6,7) by RT-PCR
Figure 5. Agarose gel electrophoresis for DNA fragmentation	Figure 6. Gel electrophoresis of PCR products stained by ethedium bromide in estimation of NF-kB mRNA by RT-PCR
Figure 7. Gel electrophoresis by PCR products stained by ethedium bromide in estimation of Bcl-2mRNA by RT-PCR.

 

 

DISUSSION

 

The pathogenesis of MS is under strong genetic control involving several genes each of modest effect. It has been hypothesized that either decreased apoptosis of auto reactive T cells in the CNS, or increased apoptosis of oligodendrocytes may play an important role²³.

                Abnormal expression of many apoptosis-related molecules, including TNF, Fas, and their corresponding receptors, has been observed in MS lesion^24,25. The data vary, however, regarding the extent of apoptosis associated with this pattern of expression. Whereas some authors have observed little evidence for apoptosis of oligodendrocytes²⁶, others report intense DNA fragmentation as in apoptosis both in lymphocytes and oligodendrocytes²⁷.

                Our study revealed highly significant decrease in the percentage of apoptotic PBMCs in patients versus control subjects  as demonstrated morphologically by Giemsa stain, Acridin orange stain,  propidium  iodide stain and by DNA fragmentation.

         In MS, spontaneous apoptosis of unfractionated peripheral mononuclear cells was significantly reduced, and activated intrathecaly, and peripheral T cells were found to be predominantly resistant to independent apoptosis. These results indicate that (in clinically active MS) the reduced susceptibility of cells to apoptosis is partly due to impairment of Fas- independent apoptotic pathway²⁸. An alternative explanation is that increased expression levels reflect a constitutional over activity of apoptosis-inducing molecules. This, however, would be contrary to the hypothesis that MS may be influenced by a genetically determined failure of activation-induced apoptosis of auto reactive T cells^29. This view is supported by experimental autoimmune encephalitis data which show that CNS apoptosis is more prevalent in effector (lymphoid) cells than in target cells (oligodendrocytes)³⁰.

                Our study revealed highly significant decrease in the expression of apoptotic factors (Fas & Fas L) in patients versus control and highly significant increase in the expression of anti-apoptotic factors (Bcl-2 & NF-κB) in patients compared to control.

                Ichikawa³⁰ found that the expression rate of Fas antigen on T cells in peripheral blood (PB) in MS patients was higher than that of healthy control and the expression rate in MS patients was higher in CSF than in peripheral blood, the results suggested that there is acceleration or impairment of apoptosis on activated T cells in MS. 

         Zipp³¹, Dianzani et al.³² and Gomes et al.³³ are in agreement with our results about Fas and suggested that development of autoimmune lymphoproliferative patterns may involve several alterations hitting the Fas system, but might also involve alterations in other systems contributing to the switching–off or proliferation of lymphocytes. Moreover Lopatins Kaya et al.³⁵ found that FasL mRNA was increased prior to exacerbation in relapsing remitting (RR) MS and decreased during clinical activity which may be due to the migration of inflammatory cells to the central nervous system.

            Sharief et al.^35,36 suggested that altered expression of Bcl-2 family proteins in T lymphocytes is involved in promoting cellular resistance to apoptosis in MS. However, the relationship between these alterations in Bcl-2 proteins expression and clinical disease activity has not yet been evaluated. They observed a significant reduction in the expression ratios of the pro- to anti- apoptosis Bcl-2 members in peripheral lymphocytes from patients with active MS when compared to corresponding ratios in patients with stable MS or other controls. This imbalance in the expression ratios of pro- and anti- apoptosis proteins was functionally active in reducing cellular susceptibility to apoptosis and my allow for continuing cellular proliferation and tissue destruction within the central nervous system in active  MS. It also correlated with clinical features of disease activity. This finding indicate that dysregulated expression of Bcl-2 family proteins in peripheral lymphocytes is a feature of clinically active MS. Heesen³⁷ detected the same results and also found that the cellular expression of Bcl-2, Bcl-X(L),Bax or Bad in MS patients was independent of the expression of other apoptotic regulatory molecules, such as Fas receptor protein.

                Seidi³⁸ suggested that cellular over expression of apoptosis–inhibitory proteins in patients with relapsing MS may promote apoptotic resistance of potentially pathogenic, auto reactive B lymphocytes and consequently, may allow for continuing autoimmune tissue destruction.

                Bonetti and JoAnn^39,40 detected increased expression of NF-κB in multiple sclerosis with minimization of initial innate  cytokine and chemokine  responses and prevention of further  propagation of the inflammatory response after  using NF-κB antagonist.  Moreover, activation of NF-κB may exert anti-apoptotic effects and contribute to the absence of an apoptotic response by oligodendrocytes in MS.

The MS plaque microenvironment is able to activate the NF-κB pathway in oligodendrocytes and microglia, this in concert with other protective mechanisms such as Bcl-2 and ciliary neurotrophic factor, activation of NF-κB may exert anti –apoptotic effects and contribute to the absence of an apoptotic response by oligodendrocytes in MS.

           Many recent studies have utilized NF-κB inhibitors in attempts to control various inflammatory diseases, including multiple sclerosis⁴².

                Conclusion: decreased expression of Fas and FasL- and increased expression of Bcl-2 and NF-κB may serve as indicators of multiple sclerosis susceptibility and further studies are required for evaluation of apoptosis in MS.

 

REFERENCES

 

1.          Lynch DH, Ramsdell F, Alderson MR. Fas and Fasl in the homeostatic regulation of immune response. Immunol Today. 1995; 16: 569-74.

2.          Chervonsky AV, Wang Y, Wong FS, Visintin I, Flavell RA, Janeway CA Jr, et al. The role of Fas in autoimmune diabetes.  Cell. 1997; 89: 17-24.

3.          Akassoglou K, Bauer J, Kassiotis G, Pasparakis M, Lassmann H, Kollias G, et al. Oligodendrocyte apoptosis and primary demyelination induced by local TNF/p55 TNF receptor signaling.  Am J Pathol. 1998; 153:801-13.

4.          Sneller MC, Wang J, Dale JK, Strober W, Middelton LA, Choi Y, et al. Clinical, immunologic, and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis.  Blood. 1997; 89: 1341-8.

5.          Hale AJ, Smith CA, Sutherland LC, Stoneman VE, Longthorne V, Culhane AC, et al. Apoptosis; molecular regulation of cell death. Eur J Biochem. 1996; 236: 1-26.

6.          Alderson MR, Tough TW, Davis-Smith T, Braddy S, Falk B, Schooley KA, et al. Fas-ligand mediates activation induced cell death.  In human T lymphocytes. J Exp Med; 1995.181:71-7.

7.          Schmied M, Breitschopf H, Gold R, Zischler H, Rothe G, Wekerle H, et al. Apoptosis in experimental autoimmune encephalomyelitis: Evidence for programmed cell death as a mechanism of inflammation in the brain.  Am J Pathol. 1993; 143:446-52.

8.          Pender MP, Rist MJ. Apoptosis of inflammatory cells in immune control of the nervous system.  Glia. 2001; 36: 137-44.

9.          Karin M, Lin A. NF-κB at the crossroads of life and death.  Nat Immunol. 2002; 3: 221-7.

10.       Sharief MK, Mathew SH, Noori MA. expression of ratios of the Bcl-2 family proteins and disease activity in MS. Mult Scler. 2003; 192: 64-7

11.       McDonald W, Compston A, Edan gG, Goodkin D, Martung H, Lublin F, McFarland M, Wolheim McDonald, W. I. The diagnosis of multiple sclerosis. Brain. 1989; 299: 635-7

12.       Lublin FD, Reingold SC. Defining the clinical course of multiple sclerosis: results of an international survey. National Multiple Sclerosis Society (USA) Advisory Committee on Clinical Trials of New agents in Multiple Sclerosis.  Neurology. 1996; 46: 907-11.

13.       McGahon AJ, Martin SJ, Bissonnette RP, Mahboubi A, Shi Y, Mogil RJ, et al. The end of the (cell) line: methods for the study of apoptosis in vitro.  Methods cell Biol. 1995; 46:153-185.

14.       Osawa Y, Banno Y, Nagaki M, Brenner DA, Naiki T, Nozawa Y, et al. TNF-alpha-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidyl inositol 3-kinase/Akt pathway in human hepatocytes. J Immunol. 2001; 167 (1):173-80.

15.       Matute-Bello G, Liles WC, Radella F 2nd, Steinberg KP, Ruzinski JT, Jonas M, et al. Neutrophil apoptosis in the acute respiratory distress  syndrome. Am J Respir Crit Care Med. 1997; 156: 1969-77.

16.       Shou I, Tashiro K, Kurusu A, Kaneko S, Hayashi T, Fukui M, et al. Serum levels of soluble Fas and disease activity in patients with IgA neuropathy. Nephron. 1991; 81:387-92.

17.       Hohlbaum AM, Saff RR, Marshak-Rothstein A. Fas-ligand--iron fist or Achilles' heel?  Clin Immunol. 2002; 103: 1-6.

18.       Salomons GS, Smets LA, Verwijs-Janssen M, Hart AA, Haarman EG, Kaspers GJ, et al. Bcl-2 Family members in childhood, in vitro and in vivo drug response and long term clinical outcome. Leukemia. 1999; 13:1574-80.

19.       Pahl HL. Activators and target genes of Rel/NF-κB transcription factors.  Oncogene. 1999; 6853-66.

20.       Bargou RC, Daniel PT, Mapara MY, Bommert K, Wagener C, Kallinich B, et al. Expression of Bcl-2 gene family in normal and malignant breast tissue. Int J Cancer. 1995; 60:854-59.

21.       Villunger A, Egle A, Marschitz I, Kos M, Böck G, Ludwig H, et al. Constitutive expression of Fas (Apo-1/CD95) a potential mechanism of tumor suppression of immune surveillance.  Blood 1997, 90: 12-20.

22.       Huang QR, Teutsch SM, Buhler MM, Bennetts BH, Heard RN, Manolios N, et al. Evaluation of the Apo-Fas promoter  MVaI polymorphis in MS. Mult Scler. 2000; 6(1): 14-8.

23.       D'Souza SD, Bonetti B, Balasingam V, Cashman NR, Barker PA, Troutt AB, et al. Multiple sclerosis: Fas signaling in oligodendrocyte cell death.  J Exp Med. 1996; 184:2361-70.

24.       Raine CS, Bonetti B, Cannella B. Multiple sclerosis: expression of molecules of the tumor necrosis factor ligand and receptor families in relationship to the demyelinated plaque.  Rev Neurol. 2003; 154: 577-85.

25.       Bonetti B., Raine CS. Multiple sclerosis: oligodendrocytes display cell death related molecules in situ but do not undergo apoptosis. Ann Neurol. 1997; 42: 74-84.

26.       Dowling P, Husar W, Menonna J, Donnenfeld H, Cook S, Sidhu M. Cell death and birth in multiple sclerosis brain J Neurol Sci. 1997; 149: 1-11.

27.       Sharief MK. Impaired Fas-independent apoptosis of T lymphocytes in patients with MS. J Neuroimmunol. 2000; 109 (20): 236-43

28.       Pender MP. Genetically determined failure of activation-induced apoptosis of autoreactive T cells as a cause of multiple sclerosis. Lancet. 1998; 351: 978-81.

29.       Bonetti B, Pohl J, Gao YL, Rain CS. Cell death during autoimmune demyelination: effector but not target cells are eliminated by apoptosis. J Immunol.  1997; 159 (11): 5733-41.

30.       Ichikawa H, Ota K, Iwata M. Increased Fas antigen on T cells in MS. J Neuroimmunol. 1996;   71(1-2): 125-9.

31.       Zipp F, Weller M, Calabresi PA, Frank JA, Bash CN, Dichgans J, et al. Increased serum levels of soluble CD95 (APO-1/Fas) in relapsing-remitting multiple sclerosis.  Ann Neurol. 1998; 43:116-20.

32.       Dianzani U, Choiocchetti A, Ramenghi U.  Role of inherited defects decreasing Fas function in autoimmunity. Life Sci. 2003; 72 (25): 2803-24.

33.       Gomes AC, Morris M, Stawiarz L, Jönsson G, Putheti P, Bronge L, et al. Decreased levels of CD95 and Capase-8 mRNA in multiple sclerosis patients with gadolinium-enhancing  lesions on MRI. Neurosci Lett. 2003; 4: 352 (2): 101-4.

34.       Lopatinskaya L, van Boxel-Dezaire AH, Barkhof F, Polman CH, Lucas CJ, Nagelkerken L. The development of clinical activity in relapsing-remitting MS is associated with a decrease of FasL mRNA and an increase of Fas mRNA in peripheral blood. J Neuroimmunol. 2003; 138 (1-2): 123-31.

35.       Sharief MK, Douglas M, Norri M, Semra YK. The expression of pro- and anti-apoptosis Bcl-2 family proteins in lymphocytes from patients with multiple sclerosis. J Neuroimmunol. 2002; 125 (1-2): 155-62.

36.       Sharief MK, Matthews H, Noori MA. Expression ratios of Bcl-2 family proteins and disease activity in MS.  J Neuroimmunol. 2003; 134 (1-2): 158-65.

37.       Heesen C., Georghiu S, Gbadamosi J, Schoser BG.  CD95-mediated apoptosis and DNA fragmentation in MS.  Acta Neurol Scand. 2000: 102(5): 333-6.

38.       Seidi OA, Sharief MK. The expression of apoptosis-regulatory proteins in B lymphocytes from patients with multiple sclerosis. J Neuroimmunol. 2002: 130 (1-2): 202-10.

39.       Bonetti B, Stegagno C, Cannella B, Rizzuto N, Moretto G, Raine CS.  Activation of NF-κB and C. Jun transcription factors in MS lesions.  Am J Pathol. 1999; 155:1433-8.

40.       Palma JP, Kwon D, Clipstone NA, Kim BS. Infection with Theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via NF-kappaB activation: potential role for the initiation of demyelinating disease. J Virol. 2003; 77 (11): 5322-6331.

41.       Muijsers RB, Goa KL. Balsalazide: a review of its therapeutic use in mild-to-moderate ulcerative colitis.  Drugs. 2002; 62: 1689-705.

42.       Wiendle H, Hohlfeld R. Therapeutic approaches in multiple sclerosis: lessons from failed and interrupted treatment trials.  BioDrugs. 2002; 16: 183-200.