Overexpression of the apoptosis inhibitor FLIP in T cells correlates with disease activity in multiple sclerosis

The cellular caspase-inhibitory protein FLIP has been recently identified as a potent regulator of T lymphocyte susceptibility to Fas-mediated programmed cell death (apoptosis). Since impairment of apoptosis may be involved in multiple sclerosis (MS), we investigated the dynamics of cellular FLIP in unstimulated and activated T lymphocytes from MS patients, inflammatory and non-inflammatory neurological disorders, and healthy subjects. Cellular expression of the long and short forms of FLIP protein was similar in unstimulated T cells from MS patients and controls, but was significantly higher in activated T cells from patients with clinically active MS. This high FLIP expression in active MS correlated with cellular resistance to Fas-mediated apoptosis. In contrast, cellular expression of the anti-apoptotic protein Bcl-2 did not differ between active and stable disease, and was relatively similar between the MS group and controls. These findings suggest that cellular overexpression of the anti-apoptotic protein FLIP is a feature of clinically active multiple sclerosis.     

Heightened intrathecal release of soluble CD137 in patients with multiple sclerosis

Human CD137 (ILA/4-1BB), a member of the tumour necrosis factor (TNF) receptor family, regulates the activation and proliferation of immune cells, and may induce apoptosis (programmed cell death) of activated lymphocytes. A soluble form of CD137 (sCD137) released by activated lymphocytes may interfere with the activities of the membrane-bound CD137. This study reports the detection of significantly high intrathecal and systemic levels of sCD137 in patients with clinically active multiple sclerosis (MS) when compared with corresponding levels from patients with clinically stable MS or those with inflammatory and non-inflammatory neurological disorders, or from healthy individuals. Intrathecal concentrations of sCD137 in patients with active MS correlate with the intrathecal release of the soluble death receptor protein Fas, but not with the release of interleukin-2, TNF or the synthesis of immunoglobulins G and M. Results presented here suggest that heightened release of sCD137 is a feature of clinically active MS.     

Expression ratios of the Bcl-2 family proteins and disease activity in multiple sclerosis

There is emerging evidence that failure of apoptosis (programmed cell death) of potentially pathogenic T lymphocytes may be involved in the pathogenesis of multiple sclerosis (MS). The commitment of T lymphocytes to die is partly regulated by the Bcl-2 family proteins, which act as a checkpoint upstream of mitochondrial dysfunction. These proteins include the death antagonists Bcl-2 and Bcl-X(L), and death agonists Bax and Bad. Recent studies suggest that altered expression of Bcl-2 family proteins in T lymphocytes is involved in promoting cellular resistance to apoptosis in patients with MS. However, the relationship between these alterations in Bcl-2 proteins expression and clinical disease activity has not yet been evaluated. In this study, we analyzed the expression ratios of pro- to anti-apoptosis Bcl-2 family proteins in patients with clinically active MS and compared results to corresponding ratios in patients with stable MS and relevant control groups. We observed a significant reduction in the expression ratios of 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 in active MS. It also correlated with clinical features of disease activity, such as the number of gadolinium-enhancing MRI lesions and clinical relapses. Our findings indicate that dysregulated expression of Bcl-2 family proteins in peripheral lymphocytes is a feature of clinically active multiple sclerosis.     

Disease activity in multiple sclerosis correlates with T lymphocyte expression of the inhibitor of apoptosis proteins.

The pathogenesis of multiple sclerosis (MS) is thought to involve failure of programmed cell death (apoptosis) to eliminate potentially pathogenic, autoreactive T lymphocytes. This failure may be caused by multiple abnormalities of the cell death machinery. The inhibitors of apoptosis (IAP) proteins are central regulators of cell death that inhibit apoptosis induced by a variety of stimuli. In this study, we investigated the dynamics of cellular IAP-1, IAP-2, and X-linked IAP, in resting and mitogen stimulated T lymphocytes from MS patients and relevant controls. The expression of IAP proteins was significantly higher in mitogen stimulated T lymphocytes from patients with clinically active MS when compared to corresponding expressions from patients with stable MS or from other controls. Heightened expression of IAP proteins in patients with active MS correlated with clinical features of disease activity, and with T lymphocyte resistance to apoptosis. In contrast, cellular expression of the anti-apoptosis protein Bcl-2 did not differ between active and stable MS, and was relatively similar between MS patients and controls. These findings suggest that overexpression of IAP proteins in stimulated T lymphocytes is a feature of clinically active multiple sclerosis.     

Elevated Bcl-X(L) levels correlate with T cell survival in multiple sclerosis

T cell resistance towards apoptotic elimination by activation-induced cell death (AICD) might be a crucial pathogenic feature of multiple sclerosis (MS). Since the Bcl-2 family is critically involved in the regulation of apoptosis, we investigated the protein expression of Bcl-2, Bcl-X(L), and Bax in peripheral blood mononuclear cells (PBMC) of 23 MS patients and 29 control subjects. An in vitro model of AICD, which exemplifies the elimination of antigen-reactive T cells in vivo, was used as an indication of T cell susceptibility or resistance towards apoptosis. Increased expression of the survival factor Bcl-X(L), which directly correlated with a resistance towards AICD, was observed in peripheral immune cells of MS patients. In contrast to Bcl-X(L), no differences were found in the protein expression of Bcl-2 and Bax between patients and controls. Our data indicate that the anti-apoptotic factor Bcl-X(L), responsible for T cell resistance towards apoptosis, might be an important factor in the MS pathogenesis and a potential target for therapeutic intervention.     

Impaired apoptosis in mitogen-stimulated lymphocytes of patients with multiple sclerosis

We investigated the sensitivity to cell death of peripheral blood mononuclear cells (PBMCs) from patients with multiple sclerosis (MS). PBMCs from MS patients, following PHA stimulation, were less sensitive to cell death than those from healthy donors (mean +/- s.e.m., 22.5 +/- 1.9 in MS patients vs 36.5 +/- 2.8 in healthy controls; p = 0.0003). However, when Fas-agonist antibody was added, the increase in respect to apoptosis induced by mitogen alone was even higher in MS patients than in controls. In addition, PHA-activated PBMCs from MS patients showed higher surface expression of Fas than controls, while Bcl-2 expression was decreased. This finding raised the question of whether an impaired generation of apoptotic signals may be contributing to the immune component of MS.     

The expression of apoptosis-regulatory proteins in B lymphocytes from patients with multiple sclerosis

The pathogenesis of multiple sclerosis (MS) is thought to involve T- and B-lymphocyte-mediated autoimmunity. However, the mechanisms that regulate lymphocyte activity in MS are poorly understood. In normal circumstances, programmed cell death (apoptosis) contributes to the maintenance of lymphocytes homeostasis and the deletion of autoreactive cells. Cellular commitment to apoptosis is partly regulated by the cell death receptor Fas, and the anti-apoptosis proteins Bcl-2 and FLIP. Although there is emerging evidence that dysregulations of apoptotic pathways play a role in T-cell autoimmunity in MS, the expression of apoptosis-regulatory proteins in B cells from MS patients is largely unknown. In this study, we analyzed the expression profiles of Fas, Bcl-2, and FLIP proteins in peripheral B lymphocytes from patients with relapsing-remitting and progressive MS, and from appropriate controls. We observed a significant up-regulation of Bcl-2 and FLIP proteins in B cells from relapsing-remitting MS when compared to corresponding expression in progressive MS, or in noninflammatory neurologic controls and healthy individuals. This cellular overexpression of Bcl-2 and FLIP proteins was not affected by treatment with interferon-beta, but was also observed in B cells from patients with systemic inflammatory diseases. Our findings suggest that cellular overexpression of the apoptosis-inhibitory proteins in patients with relapsing MS may promote apoptotic resistance of potentially pathogenic, autoreactive B lymphocytes and consequently, may allow for continuing autoimmune tissue destruction.     

Catecholamine production and tyrosine hydroxylase expression in peripheral blood mononuclear cells from multiple sclerosis patients: effect of cell stimulation and possible relevance for activation-induced apoptosis

Sympathoadrenergic mechanisms may play a role in multiple sclerosis (MS). We examined catecholamine (CA) levels and production and tyrosine hydroxylase (TH) expression in peripheral blood mononuclear cells (PBMCs) from MS patients, and the correlation between CA production and apoptosis in PBMCs. PBMCs from MS patients had increased norepinephrine (NE) levels. However, phytohaemagglutinin (PHA)-stimulated PBMCs from MS patients with active disease synthesized less dopamine (DA) than cells from both healthy controls and patients with inactive disease. PBMCs from patients with inactive disease showed lower expression of TH. Pharmacological inhibition of TH in cultured PBMCs stimulated with PHA reduced the percentage of apoptotic cells. Since a failure of activation-induced apoptosis in immune cells may be involved in MS, it is suggested that altered CA production by PBMCs may be implicated in such dysregulation.     

Interferon-β therapy downregulates the anti-apoptosis protein FLIP in T cells from patients with multiple sclerosis

Interferon-β reduces clinical exacerbations in multiple sclerosis (MS) through several immunomodulatory mechanisms that may involve augmentation of programmed cell death (apoptosis) of T lymphocytes. The anti-apoptosis protein FLIP (Fas-associated death domain-like interleukin-1β-converting enzyme inhibitory protein) has been recently identified as a potent regulator of T lymphocyte susceptibility to apoptosis. In a prospective study, we evaluated the expression of FLIP and other apoptosis regulatory proteins in ex vivo activated T lymphocytes from MS patients, before and serially after treatment with interferon-β. We also investigated the long-term effects of interferon-β on T cell apoptosis in a cross-sectional study of MS patients receiving chronic drug therapy. Treatment with interferon-β reduced the expression of FLIP isoforms in activated T lymphocytes. This reduced expression correlated with augmented T cell susceptibility to apoptosis and with clinical response to treatment. In contrast, interferon-β therapy did not alter cellular expression of the anti-apoptotic protein Bcl-2. This downregulatory effect of interferon-β on cellular FLIP expression was maintained following long-term therapy. Our findings suggest that interferon-β therapy exerts a regulatory effect on peripheral T lymphocytes through a pro-apoptosis mechanism that involves the downregulation of cellular FLIP expression.     

Upregulation of the apoptosis regulators cFLIP,CD95 and CD95 ligand in peripheral blood mononuclear cells in relapsing-remitting multiple sclerosis

Multiple sclerosis (MS) is a chronic disease involving an inflammatory reaction within the white matter of the CNS, mediated by T cells, B cells and macrophages. The pathogenesis of MS may involve impaired activation-induced cell death of activated myelin-specific mature T cells. We investigated the mRNA expression of the apoptosis mediators cellular FLICE-inhibitory protein (cFLIP), caspase-8, CD95 and CD95L in peripheral blood mononuclear cells (PB MNCs) from MS patients using real-time PCR. The overall increased expression of the four key players in the CD95 pathway in relapsing-remitting MS suggests their involvement in the inflammatory process in this disease.     

Heightened expression of survivin in activated T lymphocytes from patients with multiple sclerosis

The perpetuation of the inflammatory process in multiple sclerosis (MS) may arise from the failure to eliminate potentially pathogenic autoreactive lymphocytes by programmed cell death (apoptosis). Such impairment may be caused by multiple abnormalities of apoptosis regulatory proteins. In this study, we investigated the expression of survivin, a recently described cell cycle-regulated antiapoptosis protein, in lymphocytes from patients with active relapsing-remitting MS and appropriate controls. Survivin reactivity was detected in intrathecal lymphocytes from some MS patients, but not in resting peripheral lymphocytes. However, mitogen stimulation of resting lymphocytes induced survivin expression, which was significantly higher in stimulated intrathecal and peripheral T lymphocytes from MS patients when compared to controls. In contrast, cellular expression of the antiapoptosis protein Bcl-2 was relatively similar between MS patients and the control groups. Moreover, heightened survivin expression in MS patients correlated with T lymphocyte resistance to apoptosis, and was independent of cellular expression of the death receptor Fas. These findings suggest that upregulation of the antiapoptotic protein survivin in mitogen-stimulated T lymphocytes is a feature of multiple sclerosis.     

Elevated levels of fragmented DNA nucleosomes in native and activated lymphocytes indicate an enhanced sensitivity to apoptosis in sporadic Alzheimer's disease. Specific differences to vascular dementia

Apoptotic cell death is thought to be the most likely mechanism of cell death contributing to neurodegeneration in Alzheimer's disease (AD). Here, we provide evidence that in sporadic AD cases the vulnerability of peripheral cells to undergo apoptosis is increased compared to non-demented elderly controls and, very importantly, to patients with subcortical vascular encephalopathy (SVE) as another, but demented control group. Quiescent 'native' and 'activated' lymphocytes from AD patients that were predisposed to commit apoptotic cell death by priming the cells with interleukin-2, are shown to accumulate apoptosing cells to a significantly higher extent in spontaneous and in oxidative stress-induced in vitro apoptosis. Our results demonstrate robust differences in cell death sensitivity between AD and vascular dementia. In none of the conditions investigated, lymphocytes from SVE patients were significantly different from non-demented controls. The comparable findings of a higher extent of apoptotic features in neurons and in peripheral blood cells of AD patients are remarkable and may suggest a rather general modulation of apoptotic mechanisms by the disease, which even can be picked up at the level of peripheral lymphocytes under specific in vitro conditions.     

Upregulation of the inhibitor of apoptosis proteins in activated T lymphocytes from patients with multiple sclerosis

The pathogenesis of multiple sclerosis (MS) may involve failure of programmed cell death (apoptosis) to eliminate potentially pathogenic, autoreactive T lymphocytes. This failure may be caused by multiple abnormalities of the cell death machinery. In this study, we investigated the expression of the inhibitors of apoptosis (IAP) proteins, cellular IAP-1, IAP-2, and X-linked IAP (XIAP), in T lymphocytes from patients with active relapsing-remitting MS and appropriate controls. The expression of IAP proteins was significantly higher in mitogen-stimulated intrathecal and peripheral T lymphocytes from MS patients when compared to corresponding expressions from inflammatory and non-inflammatory neurologic controls, and healthy individuals. IAP proteins were also expressed in resting (unstimulated) T lymphocytes predominantly from MS patients. The heightened expression of IAP proteins in MS patients correlated with T lymphocyte resistance to apoptosis, and was independent of cellular expression of the death receptor protein Fas. In contrast, cellular expression of the anti-apoptosis protein Bcl-2 was relatively similar between MS patients and the control groups. These findings suggest that over-expression of IAP proteins in mitogen-stimulated T lymphocytes is a feature of multiple sclerosis.     

Production and secretion of calcitonin gene-related peptide from human lymphocytes

Programmed cell death (apoptosis) is critical for the normal development and homeostasis of the immune system. There is increasing evidence that dysregulations of apoptotic pathways are associated with autoimmune disease, including multiple sclerosis (MS). Cellular commitment to apoptosis is partly regulated by the Bcl-2 family proteins, which includes the death antagonists Bcl-2 and Bcl-X(L), and death agonists Bax and Bad. Since the role of these proteins in the pathogenesis of MS is currently unknown, we analyzed their expression profile in peripheral and intrathecal lymphocytes from MS patients and appropriate controls. We observed a significant reduction in the expression ratios of pro-apoptotic to anti-apoptotic Bcl-2 members in both peripheral and intrathecal lymphocytes from MS patients when compared to corresponding ratios in patients with inflammatory or noninflammatory neurologic controls, or healthy individuals. The relative coexpression ratios of these pro- and anti-apoptotic Bcl-2 family proteins in MS were more significant than the expression of individual members. The low cellular expression ratios of pro-apoptotic proteins in MS were confirmed in vitro activated T lymphocytes. 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 or FLIP. Our findings suggest that the abnormal expression patterns of Bcl-2 family proteins in MS may promote apoptotic resistance of potentially pathogenic, autoreactive lymphocytes, and may allow for continuing cellular proliferation and tissue destruction within the central nervous system.     

Reduced expression of the inhibitor of apoptosis proteins in T cells from patients with multiple sclerosis following interferon-beta therapy

Treatment with interferon-beta reduces clinical exacerbations in multiple sclerosis (MS) through several immunomodulatory mechanisms that involve the augmentation of programmed cell death (apoptosis) of peripheral T lymphocytes. The recently identified family of inhibitor of apoptosis (IAP) proteins is a potent regulator of cell death. The expression of IAP-1, IAP-2, and X-linked IAP (XIAP) is upregulated in mitogen stimulated T lymphocytes from MS patients, and this expression correlates with MS disease activity. In this study, we sought to evaluate the effect of interferon-beta on cellular expression of IAP proteins and other apoptosis regulatory molecules. In a prospective study, we evaluated the expression of IAP proteins, the anti-apoptosis Bcl-2 protein, and the death receptor Fas in in vitro stimulated T lymphocytes from MS patients, before and serially after treatment with interferon-beta. We also investigated the long-term effects of interferon-beta on cellular expression of these proteins and T lymphocyte apoptosis in a cross-sectional study of MS patients receiving drug therapy for a mean of 4.8 years. Treatment with interferon-beta reduced the expression of IAP-1, IAP-2 and XIAP in stimulated T lymphocytes. This reduced expression correlated with increased T cell susceptibility to apoptosis and with clinical response to treatment. In contrast, interferon-beta therapy did not alter cellular expression of Bcl-2 protein or the death receptor Fas. This downregulatory effect of interferon-beta on cellular expression of IAP proteins was maintained following long-term therapy. Our findings suggest that interferon-beta therapy exerts a regulatory effect on peripheral T lymphocytes through an anti-apoptosis mechanism that involves the downregulation of cellular IAP proteins expression.     

Proliferation and programmed cell death of neuronal precursors in the mushroom bodies of the honeybee

We have studied proliferation and programmed cell death in the brain of the honeybee during metamorphosis. DNA fragmentation detection using the TUNEL method combined with 5-bromodeoxyuridine incorporation experiments reveal that in the mushroom bodies neurogenesis is terminated by extensive apoptosis. Proliferation of mushroom body neuroblasts is active until the fourth day of pupal development, ceasing abruptly within 1 day after the onset of apoptosis in the mushroom body proliferative clusters. Inside the mushroom bodies, apoptosis spreads from the apical ends of proliferative clusters, beneath the brain's surface, toward the basal ones. The distributions of apoptotic cells and those in the S phase of the cell cycle overlap significantly. Electron microscopic analysis gives further evidence that mushroom body neuroblasts themselves undergo programmed cell death. We suggest that programmed cell death may be the main factor controlling the final number of Kenyon cells produced during metamorphosis. The overlap in time and space between proliferation and apoptosis raises the question of whether the neuronal precursors switch to programmed cell death during the progression of the cell cycle, or afterwards.     

Upregulated survivin expression in activated T lymphocytes correlates with disease activity in multiple sclerosis

Programmed cell death (apoptosis) is critical for the normal development and homeostasis of the immune system. There is emerging evidence that failure of apoptosis to eliminate potentially pathogenic, autoreactive T lymphocytes may be involved in the pathogenesis of multiple sclerosis (MS). This failure is related to multiple abnormalities of apoptosis-regulatory molecules that involve survivin, a recently described cell cycle-regulated anti-apoptosis protein. In this study, we investigated the relationship between survivin expression in peripheral T lymphocytes and clinical features of MS. We detected a significant over-expression of survivin in mitogen stimulated T lymphocytes from patients with active MS when compared with corresponding expression in patients with stable MS or those with inflammatory and non-inflammatory neurologic disorders. This over-expression of survivin in patients with active MS correlated with cellular resistance to apoptosis and with features of disease activity, such as disease duration and the number of enhanced lesions on cranial magnetic resonance imaging. There was no correlation between cellular survivin levels and the expression of other apoptosis-inhibitory proteins, such as Bcl-2 and Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (FLIP). Our findings indicate that cellular over-expression of the novel anti-apoptosis protein survivin is a feature of clinically active MS.     

Down-regulation of survivin expression in T lymphocytes after interferon beta-1a treatment in patients with multiple sclerosis

BACKGROUND: Treatment with interferon beta reduces clinical exacerbations in multiple sclerosis (MS) through several immunomodulatory mechanisms that involve the augmentation of programmed cell death (apoptosis) of peripheral T lymphocytes. The expression of survivin, a cell cycle-regulated antiapoptosis protein, is up-regulated in mitogen-stimulated T lymphocytes from patients with MS, and this expression correlates with MS disease activity. OBJECTIVE: To evaluate the effect of interferon beta on the expression of survivin and other apoptosis regulatory molecules in peripheral T lymphocytes from patients with MS. PATIENTS AND METHODS: In a prospective, combined clinical and immunologic study, we evaluated the expression of survivin, Bcl-2 protein, and the death receptor Fas in mitogen-stimulated T lymphocytes from 26 patients with MS, before and serially after treatment with interferon beta-1a. We also investigated the long-term effects of interferon beta-1a on cellular expression of these proteins and T-lymphocyte apoptosis in a cross-sectional study of 19 patients with MS receiving long-term interferon beta-1a therapy. RESULTS: Treatment with interferon beta-1a reduced the expression of survivin in in vitro stimulated T lymphocytes. This reduced expression correlated with augmented T-cell susceptibility to apoptosis and with clinical response to treatment. In contrast, interferon beta-1a therapy did not significantly alter cellular expression of Bcl-2 protein or Fas. This down-regulatory effect of interferon beta-1a on cellular expression of survivin was maintained after long-term therapy. CONCLUSIONS: Our observations suggest that interferon beta exerts a regulatory effect on peripheral T lymphocytes through an antiapoptosis mechanism that involves the down-regulation of cellular survivin expression.     

Systemic IFN-beta treatment induces apoptosis of peripheral immune cells in MS patients

In multiple sclerosis (MS), an impaired apoptotic deletion of activated CNS-specific immune cells, leading to their pathogenic persistence, has been suggested to maintain chronic brain inflammation. We here investigated whether interferon-beta (IFN-beta) therapy induces apoptosis of peripheral immune cells. Serial blood samples from 127 relapsing-remitting MS patients were analyzed prior to the initiation of a weekly IFN-beta 1a therapy and 4, 26, and 52 weeks thereafter. Peripheral immune cells were investigated for apoptosis and for the expression of apoptosis-regulatory genes CD95, CD95 ligand, FLIP, Bcl-2, Bcl-X(L), Bag-1, and caspase 3 by quantitative real-time PCR. Biological efficacy of IFN-beta treatment was checked by quantification of Mx expression (ELISA and real-time PCR). We found a significant increase in the apoptosis rate of immune cells in response to IFN-beta treatment, compared to baseline levels. While Bcl-2 levels were permanently and Bag-1 levels transiently elevated upon therapy, other apoptosis-regulatory genes revealed no alterations. Upregulation of Mx expression confirmed the activity of IFN-beta in vivo. These findings indicate that immunomodulatory IFN-beta therapy involves the induction of apoptotic cell death with the observed RNA upregulation of Bcl-2 family members rather reflecting a possible compensatory mechanism. The increased apoptosis susceptibility of peripheral immune cells may contribute to the known reduction of brain inflammatory lesions during IFN-beta treatment.     

Programmed cell death of myelin basic protein-specific T lymphocytes is reduced in patients with acute multiple sclerosis

We investigated the apoptosis of myelin basic protein (MBP)-specific T lymphocytes in multiple sclerosis (MS) patients with acute (AMS) or stable (SMS) MS by evaluating the expression of apoptosis markers on peripheral cells. Cells of healthy controls (HC) were evaluated as well. Results showed that mitogen-stimulated apoptosis was comparable among patients and controls, whereas MBP-stimulated CD4+ and CD8+ 7-AAD+ and 7-AAD+ Fas+ cell (apoptotic cells) were significantly reduced in AMS patients. A reduction of the apoptotic rate of myelin-specific CD4+ and CD8+ T lymphocytes could be involved in the immune-mediated destruction of the myelin sheath seen in AMS patients.