Mitoxantrone does not restore the impaired suppressive function of natural regulatory T cells in patients suffering from multiple sclerosis. A longitudinal ex vivo and in vitro study

CD4+CD25+ regulatory T (T(reg)) cells play a major role in controlling autoimmunity by suppressing self-reactive T cells. Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the central nervous system, where T cells are of major importance. T(reg) cell frequency and function are potential therapeutic targets of MS treatment. Mitoxantrone (MX) is a potent immunosuppressant for the treatment of active MS. We investigated the long-term effects of MX on the suppressive function of T(reg) after mitogen and myelin basic protein (MBP) stimulation in 20 MX-treated patients. MX therapy did not restore the reduced suppressive activity of a mixture of CD25(intermediate) or CD25(high) expressing T(reg) (healthy controls, MBP: 37.3%; MS patients, MBP: -1.9 vs. 6.6% suppression after MX treatment for 6 months, p > 0.2). The frequency of T(reg) cells was not affected by MX. A single infusion of MX (10 mg/m(2) body surface) induced a selective and persistent reduction of approximately 30% of absolute and relative counts of B lymphocytes (p < 0.05). MX therapy does not influence T(reg) cell frequency or function. MX seems to exhibit its efficacy in MS mainly by a suppression of humoral immunity.     

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.     

Partial synergy of bisindolylmaleimide with apoptotic stimulus in antigen-specific T cells--implications for multiple sclerosis

In multiple sclerosis (MS), induction of T cell apoptosis constitutes a promising therapeutic strategy. Recently, bisindolylmaleimide has been shown to be an effective treatment of experimental autoimmune encephalomyelitis, presumably due to enhancement of CD95-mediated T cell apoptosis. Therefore, we studied the effects of bisindolylmaleimide on human (auto)antigen-specific T cells. We observed a synergistic effect of bisindolylmaleimide with apoptotic stimulus assessed via caspase activity and annexin V-binding, but no potentiation of DNA fragmentation or cell death. Thus, bisindolylmaleimide might be useful for modulating T cell apoptosis, yet more potent substances have to be generated re-establishing immunological control over auto-reactive T cells.     

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.     

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.     

Serum CD95 of relapsing remitting multiple sclerosis patients protects from CD95-mediated apoptosis

Failure of CD95-mediated apoptosis as a potential negative regulatory mechanism of T cell expansion may be involved in T cell-mediated autoimmune diseases such as multiple sclerosis (MS). Recently, soluble CD95 has been shown to be elevated in MS patients with active disease. Here, we report that the sera of MS patients inhibit CD95 ligand-induced apoptosis of susceptible target cells in a concentration-dependent manner and dependent on the amount of serum CD95 levels. Thus, MS sera contain biologically active inhibitors of T cell apoptosis that may allow for prolonged abnormal immune responses.     

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.     

Impact of aging: sporadic, and genetic risk factors on vulnerability to apoptosis in Alzheimer's disease

The identification of specific genetic (presenilin-1 [PS1] and amyloid precursor protein [APP] mutations) and environmental factors responsible for Alzheimer's disease (AD) has revealed evidence for a shared pathway of neuronal death. Moreover, AD-specific cell defects may be observed in many other nonneuronal cells (e.g., lymphocytes). Thus, lymphocytes may serve as a cellular system in which to study risk factors of sporadic, as well as genetic AD in vivo. The aim of our present study was to clarify whether lymphocytes bearing genetic or sporadic risk factors of AD share an increased susceptibility to cell death. Additionally we examined whether a cell typespecific vulnerability pattern was present and how normal aging, the main risk factor of sporadic AD, contributes to changes in susceptibility to cell death. Here, we report that lymphocytes affected by sporadic or genetic APP and PS1 AD risk factors share an increased vulnerability to cell death and exhibit a similar cell type-specific pattern, given that enhanced vulnerability was most strongly developed in the CD4+ T-cell subtype. In this paradigm, sporadic risk factors revealed the highest impact on cell type-specific sensitivity of CD4+ T cells to apoptosis. In contrast, normal aging results in an increased susceptibility to apoptosis of both, CD4+ and CD8+ T cells.     

Higher expression of BDNF receptor gp145trkB is associated with lower apoptosis intensity in T cell lines in multiple sclerosis

Conflicting data exist on expression of gp145trkB, the high affinity receptor for brain-derived neurotrophic factor (BDNF), on peripheral blood immunocompetent cells in multiple sclerosis (MS). We analyzed expression of gp145trkB by western blotting and flow cytometry in myelin basic protein (MBP)- and ovalbumin (OVA)-T cell lines prepared from 12 patients with relapsing-remitting MS and 12 normal healthy subjects (NHS) and correlated it with activation-induced apoptosis. We found a higher percentage of gp145trkB-expressing MBP-T cells in MS patients than in NHS (p=0.011). gp145trkB was mainly expressed by CD8(+) T cells to a higher extent in MS patients than in NHS (p=0.04). MBP-T cell lines from MS patients showed significantly lower apoptosis intensity than those from NHS (p=0.011). We found also a significant negative correlation between gp145trkB expression and apoptosis intensity in MS patients only (p=0.02). OVA-T cell lines showed a gp145trkB expression similar to that of MBP-T cell lines, with a higher expression in MS patients than NHS, and similar correlations with apoptosis intensity in MS. These findings suggest that gp145trkB is mainly expressed on T cell lines from MS patients and that the BDNF/gp145trkB axis is involved in the regulation of peripheral T cell apoptosis in MS.     

Death ligands and autoimmune demyelination.

Death ligands induce apoptosis, which is a cell suicide program leading mainly to selective elimination of an organism's useless cells. Importantly, the dying cell is an active participant in its own demise ("cellular suicide"). Under physiological conditions, apoptosis is most often found during normal cell turnover and tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance, development of the nervous system, and endocrine-dependent tissue atrophy. However, apoptotic processes have also been suggested to contribute to the pathology of the autoimmune demyelinating disease multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis. Here, apoptosis plays a double role. On one hand, impaired apoptosis may result in increased numbers or persistence of activated myelinspecific T cells. On the other hand, local tissue damage involves apoptosis of oligodendrocytes and neurons, leading to the clinical symptoms. In this article, an overview is given of the current knowledge of the roles of apoptosis-mediating and immune regulatory death ligands of the tumor necrosis factor (TNF) family (TNF, lymphotoxin-beta, OX40L [CD134L], CD154 [CD40L], CD95L, CD70 [CD27L], CD153 [CD30L], 4-1BBL [CD137L], TRAIL, TWEAK, BAFF, GITRL) in the pathogenesis of MS and of their implications for related therapeutic strategies.     

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.     

Hyaluronate receptor (CD44) and integrin alpha4 (CD49d) are up-regulated on T cells during MS relapses

A longitudinal study of peripheral blood T cell adhesion molecule expression was performed in 24 relapsing-remitting MS patients. There were 15 relapses in 11 patients during 15 months of observation. In comparison with remission, expression of hyaluronate receptor (CD44) was highly significant, and expression of integrin alpha4 (CD49d, VLA-4) significantly up-regulated during relapses. CD44 and CD49d are putative activity markers and CD44 a potential novel therapeutic target in MS.     

Upregulation of TRAIL expression on human T lymphocytes by interferon beta and glatiramer acetate

We measured the in vivo and in vitro effects of interferon (IFN)beta and glatiramer acetate (GA) on the expression of the regulatory molecule, tumor necrosis factor related apoptosis inducing ligand (TRAIL), in patients with multiple sclerosis (MS). We confirmed the prior observation that TRAIL is enhanced on anti-CD3 activated T cells by the in vitro addition of IFNbeta. T cells from IFNbeta-treated patients stimulated with anti-CD3 only, had higher levels of TRAIL than untreated patients, suggesting that in vivo IFNbeta exposure has an effect on TRAIL expression in association with T cell activation. In vitro IFNbeta-induced TRAIL upregulation on anti-CD3 or phytohemagglutinin-activated T cells was comparable for IFNbeta-treated and non-treated MS patients and controls, indicating that IFN receptors were neither saturated nor down-regulated by current IFNbeta therapy. Although GA in vivo or in vitro did not induce TRAIL, the IFNbeta +GA combination in vitro enhanced TRAIL expression to higher levels than IFNbeta alone on CD4+ T cells obtained from MS patients, regardless of GA treatment status, and healthy donors, and on GA reactive T cell lines derived from GA-treated patients or controls. Whether any observed therapeutic effects of GA/IFNbeta combination therapy will correlate with TRAIL expression and function remains to be determined.     

Impact of aging

The identification of specific genetic (presenilin-1 [PS1] and amyloid precursor protein [APP] mutations) and environmental factors responsible for Alzheimer’s disease (AD) has revealed evidence for a shared pathway of neuronal death. Moreover, AD-specific cell defects may be observed in many other nonneuronal cells (e.g., lymphocytes). Thus, lymphocytes may serve as a cellular system in which to study risk factors of sporadic, as well as genetic AD in vivo. The aim of our present study was to clarify whether lymphocytes bearing genetic or sporadic risk factors of AD share an increased susceptibility to cell death. Additionally we examined whether a cell type-specific vulnerability pattern was present and how normal aging, the main risk factor of sporadic AD, contributes to changes in susceptibility to cell death. Here, we report that lymphocytes affected by sporadic or genetic APP and PS1 AD risk factors share an increased vulnerability to cell death and exhibit a similar cell type-specific pattern, given that enhanced vulnerability was most strongly developed in the CD4⁺ T-cell subtype. In this paradigm, sporadic risk factors revealed the highest impact on cell type-specific sensitivity of CD4⁺ T cells to apoptosis. In contrast, normal aging results in an increased susceptibility to apoptosis of both, CD4⁺ and CD8⁺ T cells.     

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.     

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.     

Apoptosis of inflammatory cells in immune control of the nervous system: role of glia

The elimination of inflammatory cells within the central nervous system (CNS) by apoptosis plays an important role in protecting the CNS from immune-mediated damage. T cells, B cells, macrophages, and microglia all undergo apoptosis in the CNS. The apoptotic elimination of CNS-reactive T cells is particularly important, as these cells can recruit and activate other inflammatory cells. T-cell apoptosis contributes to the resolution of CNS inflammation and clinical recovery from attacks of experimental autoimmune encephalomyelitis (EAE), an animal model of the demyelinating disease multiple sclerosis (MS). T-cell apoptosis in the CNS in EAE occurs in both an antigen-specific and an antigen-nonspecific manner. In antigen-specific T-cell apoptosis, it is proposed that T cells that recognize their antigen in the CNS, such as CNS-reactive T cells, are deleted by the process of activation-induced apoptosis after activation of the T-cell receptor. This may result from the ligation of T-cell death receptors (such as CD95 (Fas) or tumor necrosis factor (TNF) receptor 1) by CD95 ligand (CD95L) or TNF expressed by the same T cell or possibly by microglia, astrocytes or neurons. Inadequate costimulation of the T cell by antigen-presenting glial cells may render T cells susceptible to activation-induced apoptosis. T cells expressing CD95 may also die in an antigen-nonspecific manner after interacting with glial cells expressing CD95L. Other mechanisms for antigen-nonspecific T-cell apoptosis include the endogenous release of glucocorticosteroids, deprivation of interleukin-2, and the release of nitric oxide by macrophages or glia. Apoptosis of autoreactive T cells in the CNS is likely to be important in preventing the development of autoimmune CNS diseases such as MS.     

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.     

Apoptosis of T cells in peripheral blood and cerebrospinal fluid is associated with disease activity of multiple sclerosis

Apoptotic elimination of pathogenic T cells is considered to be one of regulatory mechanisms in multiple sclerosis (MS). To explore the potential relationship between Fas-mediated apoptosis and the disease course of MS, we examined apoptosis, defined by annexin V (AV) binding, and Fas (CD95) expression in CD4+ and in CD8+ T cells in MS patients by using five-color flow cytometry. The percentage of AV+CD4+CD3+ cells and CD95+AV+CD4+CD3+ cells in peripheral blood and cerebrospinal fluid (CSF) were significantly decreased in active MS patients compared with inactive MS patients. A significantly lower proportion of CD95+AV+CD8+CD3+ cells in CSF was observed in active MS patients compared with inactive MS patients, but not in peripheral blood. These results indicate that the resistance of T cells to Fas-mediated apoptosis is involved in exacerbation of MS and/or that Fas-mediated apoptosis of T cells is associated with remission of MS.