EAE in beta-2 microglobulin-deficient mice: axonal damage is not dependent on MHC-I restricted immune responses

There is accumulating evidence that CD8-positive (CD8+) T-cells and MHC-I expression may also play a role in neurodegeneration associated with multiple sclerosis (MS). We investigated the role of MHC-I and CD8+ T-cells by studying experimental autoimmune encephalomyelitis (EAE) in beta-2 microglobulin knockout mice induced by myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 or whole rat myelin basic protein (rMBP). For both encephalitogens and even after reconstitution of the immune system with MHC-I-positive bone marrow and transfer of mature CD8+ T-cells (iMHC-I+ CD8+ beta2m-/- mice), the disease course in beta2m-/- mice was significantly more severe with a 10-fold increased mortality in the beta2m-/- mice as compared to wild-type C57BL/6 mice. EAE in beta2m-/- mice caused more severe demyelination after immunization with MOG than with rMBP and axonal damage was more marked with rMBP as well as MOG even in iMHC-I+ CD8+ beta2m-/- mice. Immunocytochemical analysis of spinal cord tissue revealed a significant increase in macrophage and microglia infiltration in beta2m-/- and iMHC-I+ CD8+ beta2m-/- mice. The different pattern of T-cell infiltration was underscored by a 2.5-fold increase in CD4-positive (CD4+) T-cells in beta2m-/- mice after induction of MOG 35-55 EAE. We conclude that lack of functional MHC-I molecules and CD8+ T-cells aggravates autoimmune tissue destruction in the CNS. Enhanced axonal damage speaks for pathways of tissue damage independent of CD8+ T-cells and neuronal MHC-I expression.     

Increased percentage of "double phenotype" form T lymphocytes in blood of patients with multiple sclerosis

We studied the percentage of double positive (CD4+CD8+) form of T-cells in a group (total 77) multiple sclerosis (MS) patients, measured by means of monoclonal antibodies anti-CD3, CD4/FITC, CD8/RPE and flow cytometry FACScan (Becton Dickinson). In our study we have shown that the percentage of the double positive T cells is higher (p < 0.05) in the peripheral blood of patients with acute exacerbation of MS (n = 21), and in the course of chronic progressive MS (n = 27) comparing to MS remission (n = 29) and other neurological diseases (n = 25) groups. In the study we have shown that the percentage of double positive T-cells in peripheral blood depends mainly on disease activity.     

Current trends in multiple sclerosis research: an update on pathogenic concepts

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) of presumed autoimmune origin which develops in a genetic susceptible individual triggered by additional environmental factors. In this review we will provide an update of basic pathogenic concepts. In addition, we will discuss newly evolving concepts in MS pathogenesis such as pathogenic heterogeneity, importance of axonal loss and the role of CD8+ T lymphocytes in tissue injury. In the last part of this review we will briefly describe currently approved MS treatments and summarize some promising therapeutic approaches that are currently under evaluation.     

T-cell subsets in the cerebrospinal fluid and peripheral blood of multiple sclerosis patients treated with high-dose intravenous methylprednisolne

To determine the effects of high-dose intravenous methylprednisolone (MP) on lymphocytes and lymphocyte subpopulations in the cerebrospinal fluid (CSF) and peripheral blood (PB) in multiple sclerosis (MS) patients, we studied 67 patients with definite MS treated with MP. They were classified according to the disease course: 32 chronic progressive (CP) patients, 25 relapsing-remitting (RR) patients, and 10 patients with a chronic progressive disease course accompanied by relapses and remissions (CP + RR). MS patients were treated with 1000 mgr intravenous MP daily for 10 consecutive days. Before and after MP treatment we simultaneously studied CSF and PB CD3 +, CD4 +, CD8 +, CD20 +, and Ial + cells subsets. Kurtzke's Expanded Disability Status Scale (EDSS) was used for clinical evaluation. Progression rate was defined as the ratio of EDSS to disease duration. Thirteen patients with lumbar disk herniation were investigated as controls. Before MP, we found in MS patients, especially in the CP group, significantly lower CD4 + T-cell percentages in the PB with respect to controls (P<0.05). The percentage of CD4 + T-cells in the CSF of MS patients was significantly higher compared with PB (p = 0.0001), and tended to be higher than in controls (p = 0.072). The CSF mononuclear cell counts were significantly correlated with higher percentages of CSF CD3 + (r = 0.40) and CD4 + (r = 0.47) T-cells and lower CSF CD8 + (r = -0.33) T-cell percentages. B-cell percentages in the CSF were significantly elevated compared with controls for all MS groups. No relation could be obtained between T- or B-cell subsets and EDSS or progression rate. After MP, a significant decrease in PB CD8 + T-cell percentage and simultaneously an increase of the percentage CD8 + T-cells in CSF was noted in the entire MS group and in the CP and RR MS patients. Except for the CP + RR MS patients, CD4 + T-cell percentages in the PB or CSF showed insignificant changes. Our findings support the view that in MS MP might affect the inflammatory process of demyelination by a selective and dissociative effect on T-suppressor/cytotoxic cells in the PB and CSF.     

Immunization with neurofilament light protein induces spastic paresis and axonal degeneration in Biozzi ABH mice

Axonal damage is the major cause of irreversible neurologic disability in patients with multiple sclerosis. Although axonal damage correlates with antibodies against neurofilament light (NF-L) protein, a major component of the axonal cytoskeleton, the possible pathogenic role of autoimmunity to axonal antigens such as NF-L has so far been ignored. Here we show that Biozzi ABH mice immunized with NF-L protein develop neurologic disease characterized by spastic paresis and paralysis concomitant with axonal degeneration and inflammation primarily in the dorsal column of the spinal cord. The inflammatory central nervous system lesions were dominated by F4/80+ macrophages/microglia and relatively low numbers of CD4+ and CD8+ T-cells. In splenocyte cultures, proliferation to NF-L was observed in CD4+ T-cells accompanied by the production of the proinflammatory cytokine interferon-gamma. Elevated levels of circulating antibodies recognizing recombinant mouse NF-L were present in the serum, and immunoglobulin deposits were observed within axons in spinal cord lesions of mice exhibiting clinical disease. These data provide evidence that autoimmunity to NF-L protein induces axonal degeneration and clinical neurologic disease in mice, indicating that autoimmunity to axonal antigens, as described in multiple sclerosis, may be pathogenic rather than acting merely as a surrogate marker for axonal degeneration.     

The role of CD4+ T-cells in the development of MS

OBJECTIVE: Multiple sclerosis (MS) is a chronic, progressive central nervous system (CNS) disease with unknown cause. Considerable evidence supports an autoimmune origin with an important role for cellular immune responses in its pathogenesis. METHODS: We have reviewed the current literature dealing with lymphocyte responses and their interactions as it relates to MS and present supporting evidence from animal models. RESULTS: Issues regarding CD4+ T-cell subpopulations, their functional differentiation and regulatory interactions as they relate to their presumed role in MS-related pathology have been updated with references to the current literature. DISCUSSION: The evidence reviewed supports an important role of CD4+ T-cells in the immunopathogenesis of MS. The successful outcome of blocking CD4 cells entry into the CNS of animals with experimental demyelinating disease and humans with MS is a strong support for other evidence of an important role of these cell populations in the pathogenesis of MS. The understanding of the specific roles of CD4+ T-cells in the development of MS is crucial for better disease management and the prevention of neurological disability.     

Role of perforin secretion from CD8+ T-cells in neuronal cytotoxicity in multiple sclerosis

Objectives: Multiple sclerosis (MS) is the most prevalent autoimmune disease of the central nervous system, and is characterized by inflammation and myelin damage. The immune system initiates the autoimmune response, although the mechanisms of neuronal damage have not been elucidated. The purpose of the present study was to investigate autoreactive CD4+ and CD8⁺ T lymphocytes, in conjunction with other inflammatory cells and cytokines in active MS lesions.

Methods: EAE animal models was established by plantar injections of MBP (200 μg per rat). Purified CD4+ or CD8+ T-cells were isolated from heparinized peripheral blood (EAE animals and control animals) via negative selection. To examine effects of presence of autoreactive CD4+ and CD8+ T lymphocytes, we carried out ELISA, Western blot analysis and TUNEL. In addition, we examined the direct effects of various factors on neuronal cell death using MTT assay.

Results: The data revealed that CD8+ T-cells were more toxic to neurons compared to CD4+ T-cells, in both the MBP and EAE conditions. Bax was greater increased when neurons were co-cultured with CD8+ T-cells in the MBP group. There is a significant increase in IL-17 secretion by CD4+ T-cells in both the MBP group and EAE group. Neuronal viability were affected by Perforin (1.5 μg/mL).

Conclusion: The present study extends previous research by demonstrating the role of CD8+ T-cells in MS and supports perforin secretion by CD8+ T-cells as a potential therapeutic factor. Furthermore, we determined that CD4⁺ T-cells can enhance CD8⁺ T-cell neuronal cytotoxicity via induction of intense inflammation.     

Mechanisms of axonal degeneration in EAE--lessons from CNTF and MHC I knockout mice

The major pathological hallmarks of multiple sclerosis (MS) comprise inflammation, demyelination with associated gliosis and axonal damage, which most likely correlates with persisting disability. Axonal damage can occur by several mechanisms. This article focuses on myelin disintegration and direct immune attack on axons by CD8-positive T-cells as two possible scenarios for axonal injury. As protoypic models, we investigated experimental autoimmune encephalomyelitis (EAE) in ciliary neurotrophic factor gene knockout mice (CNTF-/- mice) with severe myelin pathology and EAE in beta-2 microglobulin gene knockout mice (beta2m-/- mice) lacking CD8-positive T-cells. The results from these studies indicate that the trigger attack for axonal injury even in a well-defined experimental design can be multi-faceted. No single factor seems to be absolutely necessary for the initiation of the process, but they rather act in concert and orchestrate tissue destruction, inflammation and regeneration. Some mechanisms of primary or secondary axonal damage may be shared between inflammatory and degenerative diseases of the nervous system, thereby establishing a link which might be of importance for future therapeutic strategies.     

Relapses in multiple sclerosis are associated with increased CD8+ T-cell mediated cytotoxicity in CSF

MS is thought to be mediated by CD4(+) T-helper cells. To investigate the importance of CD8(+) cytotoxic T-cells in MS we analyzed peripheral blood T-cells by DNA microarray, and plasma and CSF levels of granzymes from MS patients and controls. Cytotoxic gene expression was decreased in peripheral T-cells from RRMS patients whereas plasma levels of granzymes were unchanged. However, granzyme levels were elevated in the CSF of RRMS patients at relapse compared with controls and remission. Thus, CD8+ T-cell-mediated cytotoxicity is confined to the CSF/CNS compartment in RRMS patients and may be involved in the immunopathogenesis of clinical relapses.     

The CD4+ T-cell subset lacking expression of the CD28 costimulatory molecule is expanded and shows a higher activation state in multiple sclerosis

Multiple sclerosis (MS) is a chronic debilitating disease, in which T-cells are considered to play a pivotal role. CD28 is the quintessential costimulatory molecule on T-cells and its expression declines progressively with repeated stimulations, leading to the generation of CD28(-) T-cells. Our aim was to examine whether CD4(+)CD28(-) T-cells were enriched in MS patients, and characterize the phenotype of this subset in MS patients and healthy controls (HC). All these changes could provide these CD4(+)CD28(-) T-cell characteristics that might be involved in the pathogenesis of MS, turning this T-cell subset into a potential target for future therapeutic strategies.     

CD4+ lymphocyte subsets in the cerebrospinal fluid of multiple sclerosis and non-inflammatory neurological diseases

Summary We studied paired cerebrospinal fluid (CSF) and peripheral blood (PB) samples from 18 inactive multiple sclerosis (MS) patients and 10 with non-inflammatory neurological diseases. By means of a dual-colour cytofluorimetric micromethod we were able to count 1500 cells on average in each CSF sample. We found a significant reduction of CD45RA+ and CD4+CD45RA+ cells in the CSF of MS patients. Similarly, CD45RA+ and CD4+CD45RA+ CSF/PB ratios were lower compared with controls. The reduction of suppressor-inducer T-cells did not correlate with CD8+ cell levels in the CSF. The CD4+ subset ratio (CD4+CD45RA–/CD4+CD45RA+) was significantly increased in the CSF of MS patients. Our data suggest that the reduction of CD4+CD45RA+ cells in the PB is not due to a segregation of such cells in the CSF. Conversely, CSF changes reflect changes in the PB similar to these found for other T-cell subsets.     

Glatiramer acetate (GA) therapy induces a focused, oligoclonal CD8+ T-cell repertoire in multiple sclerosis

We have demonstrated that GA therapy induces a differential upregulation of GA-specific, cytotoxic/suppressor CD8+ T-cell responses in MS patients. We utilized a novel combination of flow sorting and anchored PCR to analyze the evolving clonal composition of GA-specific CD4+ and CD8+ T-cells. TCRβ chain analysis revealed the development of an oligoclonal GA-specific CD8+ repertoire with persistence of dominant clones over long periods. Interestingly, some sequences resembled published oligoclonal CD8+ TCR sequences from MS lesions. In contrast, GA-specific CD4+ responses were polyclonal and showed continual evolution of their repertoire. This clonotypic and functional analysis provides mechanistic insights into GA therapy.     

Sialoadhesin deficiency ameliorates myelin degeneration and axonopathic changes in the CNS of PLP overexpressing mice

PLP overexpressing mice display demyelination and axonopathic changes, accompanied by an elevation of CD8+ T-lymphocytes and CD11b+ macrophages in the CNS. By crossbreeding these mutants with RAG-1-deficient mice lacking mature lymphocytes, we could recently demonstrate a pathogenetic impact of the CD8+ cells. In the present study, we investigated the pathogenetic impact of CD11b+ macrophages by crossbreeding the myelin mutants with knockout mice deficient for the macrophage-restricted adhesion molecule sialoadhesin (Sn). In the wild-type mice, Sn is barely detectable on CD11b+ cells, whereas in the myelin mutants, almost all CD11b+ cells express Sn. In the double mutants, upregulation of CD8+ T-cells and CD11b+ macrophages is reduced and pathological alterations are ameliorated. These data indicate that in a primarily genetically caused myelin disorder of the CNS macrophages expressing Sn partially mediate pathogenesis. These findings may have substantial impact on treatment strategies for leukodystrophic disorders and some forms of multiple sclerosis.     

T-cells expressing natural killer (NK) receptors are altered in multiple sclerosis and responses to alpha-galactosylceramide are impaired

Multiple sclerosis (MS) is an autoimmune disorder characterised by clinical relapse and remission and pathological demyelination with varying inflammation. Because it is suggested that T-cells expressing natural killer cell receptors (NKR) play important roles in regulating human autoimmune diseases, we have quantified populations of T-cells expressing the NKR CD56, CD161 and CD94 in the peripheral blood of MS patients, in healthy control subjects (HS) and in patients with other neurological diseases (OND). CD161(+) T-cells and CD94(+) T-cells were significantly decreased in MS patients with primary progressive disease and secondary progressive disease respectively whereas CD56(+) T-cell numbers were unchanged. In contrast NKT-cells that express the invariant Valpha24-Jalpha18(+) T-cell receptor identified here by specific receptor antibody and CD1d-tetrameric PBS57-loaded complexes, were increased in MS patients compared with HS. Reductions in CD161(+) T-cells and CD94(+) T-cells relative to HS were also observed in the OND group and this was particularly prominent in Parkinsonian patients. A striking functional finding was that while NKT-cells in unfractionated peripheral blood from healthy subjects expanded in number and produced IFN-gamma upon stimulation with alpha-galactosylceramide, NKT-cells from MS patients did not. Thus we have identified alterations in a number of potentially important lymphocyte sub-populations warranting further investigation in the immune response in MS.     

Immunopathogenesis of multiple sclerosis

Multiple sclerosis (MS) is a suspected autoimmune disease in which myelin-specific CD4+ and CD8+ T cells enter the central nervous system (CNS) and initiate an inflammatory response directed against myelin and other components of the CNS. Acute MS exacerbations are believed be the result of active inflammation, and progression of disability is generally believed to reflect accumulation of damage to the CNS, particularly axonal damage. Over the last several years, the pathophysiology of MS is being appreciated to be much more complex, and it appears that the development of the MS plaque involves a large number of cell populations, including CD8+ T lymphocytes, B cells, and Th17 cells (a population of helper T cells that secrete the inflammatory cytokine IL-17). The axonal transection and degeneration that is thought to represent the basis for progressive MS is now recognized to begin early in the disease process and to continue in the progressive forms of the disease. Molecules important for limiting aberrant neural connections in the CNS have been identified, which suppress axonal sprouting and regeneration of transected axons within the CNS. Pathways have also been identified that prevent remyelination of the MS lesion by oligodendrocyte precursors. Novel neuroimaging methodologies and potential biomarkers are being developed to monitor various aspects of the disease process in MS. As we identify the pathways responsible for the clinical phenomena of MS, we will be able to develop new therapeutic strategies for this disabling illness of young adults.     

Increase in dopaminergic, but not serotoninergic, receptors in T-cells as a marker for schizophrenia severity

Schizophrenia is characterized by a slow deteriorating mental illness. Although the pathophysiology mechanisms are not fully understood, different studies have suggested a role for the immune system in the pathogenesis of schizophrenia. To date, an altered expression or signaling of neurotransmitters receptors is observed in immune cells during psychiatric disorders. In the present study, we investigated the expression of different serotonin and dopamine receptors in T-cells of schizophrenic and control patients. We used flow cytometry to determine the pattern of expression of dopamine (D2 and D4) and serotonine receptors (SR1A, SR1C, SR2A, SR2B), as well as serotonin transporter (ST), in T-cell subsets (CD4 and CD8). Expression of serotonin receptors and ST in T-cells of schizophrenic patients were not different from controls. However, the percentages of CD4+D4+ and CD8+D4+ were increased in schizophrenic patients as compared to controls. In addition, increased percentages of CD8+D2+ cells were also observed in schizophrenic patients, albeit this population revealed lower CD4+D2+ cells in comparison to controls. Interestingly, a relationship between clinical symptoms and immunological parameters was also observed. We showed that the Brief Psychiatric Rating Scale (BPRS), the Positive and Negative Syndrome Scale (PANSS) and the Abnormal Involuntary Movement Scale (AIMS) were positively related to CD8+D2+ cells, though AIMS was inversely related to CD4+D4+ cells. In conclusion, the alteration in the pattern of cell population and molecules expressed by them might serve as a promising biomarker for diagnosis of schizophrenia.     

Memory and naive CD4+ lymphocytes in multiple sclerosis

Summary Helper-inducer (CD29+CD4+) and suppressor (CD45RACD4+) T-cells have been recently renamed as memory and naive T-cells, respectively. We measured cells expressing these phenotypes in peripheral blood of 46 definite multiple sclerosis (MS) patients [32 relapsing-remitting (RR-MS), 14 secondary progressive (P-MS)] and controls. CD25+ (interleukin-2-receptor-positive) cells were also evaluated in the same groups of patients. RR-MS patients showed increased levels of CD29+CD4+ and CD25+ cells compared with controls. This finding was more evident in RR-MS patients during the attack than during the stable phase of the disease. In P-MS patients we found a reduction of CD45+CD4+ cells compared with either RR-MS patients or control subjects. Our results show that RR-MS and P-MS are characterized by two different T-cell subpopulations. This finding supports the hypothesis that during the evolution from RR-MS and P-MS changes occur in the immunological status of the patients.     

CD95/Fas expression on peripheral blood T lymphocytes in patients with multiple sclerosis: effect of high-dose methylprednisolone therapy

Recent data indicate that the apoptotic process, mediated by the CD95/Fas cell surface receptor, is impaired in activated lymphocytes of patients with relapsing-remitting multiple sclerosis. Using flow cytometric-immunophenotyping, we analyzed the expression of CD95/Fas on peripheral blood CD4+ and CD8+ T lymphocytes (PBL) in 10 MS patients in relapse, and the effect of pulse corticosteroid therapy on the apoptosis of autoreactive lymphocytes. The proportions of CD8+ and CD8+CD95+ T lymphocytes were significantly higher in MS patients in relapse before than after pulse corticosteroid therapy. Conversely, the proportions of CD4+ and CD4+CD95+ T cells were significantly lower before than after therapy, but not significantly different from healthy persons. The different expression of CD95/Fas on peripheral blood CD8+ T lymphocytes in relapsing RRMS and in healthy controls suggests a possible involvement of apoptosis in the pathogenesis of MS. Our results also show that pulse corticosteroid therapy influences the CD95/Fas expression on CD8+ and CD4+ T lymphocytes in patients with RRMS.     

Association of Th1/Th2-related chemokine receptors in peripheral T cells with disease activity in patients with multiple sclerosis and neuromyelitis optica

We evaluated 30 patients with clinically definite multiple sclerosis (MS) and 8 patients with neuromyelitis optica (NMO) to investigate correlations between Th1/Th2 balance, disease activity, effects of interferon (IFN)-β treatment, and expressions of chemokine receptors CXCR3 and CCR4 on CD4+ and CD8+ T cells in peripheral blood. MS and NMO patients in the relapsing phase showed a significantly increased CD4+CXCR3+/CD4+CCR4+ ratio and CD8+CXCR3+/CD8+CCR4+ ratio compared with respective patients in the remission phase. After IFN-β treatment, the CD4+CXCR3+/CD4+CCR4+ ratio and CD8+CXCR3+/CD8+CCR4+ ratio were significantly decreased compared with the relapsing phase and slightly lower than in the remission phase. The CD8+CXCR3+/CD8+CCR4+ ratio showed a more marked change associated with disease activity than CD4+ T cells in MS and NMO patients. Moreover, in patients in the relapsing phase of NMO, the CD4+CXCR3+/CD4+CCR4+ ratio and CD8+CXCR3+/CD8+CCR4+ ratio were significantly higher than in MS patients in the relapsing phase. We confirmed marked changes in the CD8+CXCR3+/CD8+CCR4+ ratio according to disease activity and treatment of MS and NMO. Furthermore, this ratio was more strongly linked to immune and inflammatory activity in NMO patients than in MS patients, and may represent an important factor in differentiating the pathogenesis of MS and NMO.