CSF cells in multiple sclerosis: monoclonal antibody analysis and relationship to peripheral blood T-cell subsets

Mononuclear cells were analyzed in CSF and blood of 102 patients with MS. In CSF, the majority (78%) of cells were T lymphocytes (T3+), and the ratio of inducer (T4+) to suppressor/cytotoxic (T8+) cells was 2:1. No characteristic alterations in CSF phenotypes could be related to changes in circulating T8 cells or to disease activity. In a group of 75 patients, CSF cell count was higher in patients with low numbers of circulating T8 cells than in those with normal T8 cells. Thus, decreases in suppressor cells in the blood of MS patients are associated with CSF pleocytosis but not with fluctuations in the ratio of different subsets in CSF. Furthermore, large numbers of T8 cells are not sequestered in CSF when these cells are decreased in peripheral blood.     

Characterization of T cell lines derived from glatiramer-acetate-treated multiple sclerosis patients

We analyzed the effects of glatiramer acetate (GA) therapy on in vitro proliferative responses and cytokine production by lymphocytes derived from multiple sclerosis patients receiving this therapy. We confirmed that lymphocytes derived from GA na?ve patients show a high frequency of response when initially exposed to GA in vitro; this frequency decreased following GA therapy. The frequency of lymphocytes responding to whole MBP stimulation did not change with GA therapy. GA- and MBP-specific T cell lines generated from these patients by repeated cycles of in vitro stimulation did not cross react. Some (23%) whole MBP-reactive T cell lines did cross react with MBP peptide 83-99. The mean levels of interferon (IFN) gamma secretion and the mean ratio of IFN-gamma/IL-5 were lower for GA-reactive cell lines, derived from patients both prior to and during GA therapy, compared to MBP-reactive T cell lines. The proportion of IFN-gamma(+) cells in unfractionated lymphocyte preparations derived from the GA-treated patients did not differ from that found for healthy controls. Our findings indicate that GA-reactive T cell lines derived from GA-treated MS patients continue to show a relative Th2 cytokine bias consistent with a bystander suppressor function. GA treatment is not associated with a cytokine phenotype shift in the total T cell or MBP-reactive T cell populations.     

Catecholamine levels in peripheral blood lymphocytes from multiple sclerosis patients

Circumstantial evidence suggests the involvement of sympathoadrenergic mechanisms in the progress of multiple sclerosis (MS).We studied peripheral blood lymphocytes from MS patients. The levels of dopamine (DA), norepinephrine (NE), epinephrine (E) and their metabolites in extracts of lymphocytes from 58 MS patients and 19 healthy controls were measured by using capillary electrophoresis. The MS patients were divided into clinical subgroups: a laboratory-supported definitive (first-attack) MS group, and a relapsing-remitting (RR) group in remission.The peripheral blood lymphocyte level of epinephrine was significantly higher in the first-attack MS patients (p=0.028) than in the controls. However, the norepinephrine levels were significantly (p=0.027) lower in the RR patients in remission. The catecholamines are known to be able to affect the lymphocyte activity, both by stimulation and by immunosuppression. Our results suggest that the catecholamines are important regulators of lymphocyte activation in MS, and of potential importance as concerns new diagnostic and therapeutic methods.     

Interferon gamma and interleukin 4 producing T cells in peripheral blood of multiple sclerosis patients undergoing immunomodulatory treatment

Intracellular cytokine flow cytometry was used to analyse the percentages of interferon (IFN) gamma and interleukin (IL)-4 producing T cells in the peripheral blood of multiple sclerosis patients, before and after immunomodulatory treatment, and of healthy controls. After six months of treatment, different doses of IFN beta1a (Avonex or Rebif) decreased CD4+ (Th1, Th2) and CD8+ (Tc1) cells to a similar extent, without affecting the Th1/Th2 ratio. These T cell subsets were unmodified after nine months of glatiramer acetate (Copaxone) treatment, and after six day courses of high dose 6-methylprednisolone. The data suggest that IFN beta1a produces sustained downmodulation of IFN gamma and IL-4 producing T cells in vivo, which may contribute to its therapeutic efficacy; that glatiramer acetate possibly acts without altering non-specific cellular immunity; and that glucocorticoid induced lymphocytopenia does not affect the percentages of Th1, Th2, and Tc1 cells; at least in the periphery, none of the treatments caused a Th1 to Th2 shift that could account for their respective therapeutic effects.     

Gene expression changes in peripheral blood mononuclear cells from multiple sclerosis patients undergoing beta-interferon therapy

OBJECTIVE: Multiple sclerosis (MS) is a disabling idiopathic inflammatory disorder with evidence of immune dysfunction. Current therapies for MS include preparations of beta-interferon (beta IFN). We studied the gene expression patterns in peripheral blood mononuclear cells from relapsing-remitting MS patients undergoing weekly beta IFN-1a therapy (Avonex; 30 mg intramuscular) to identify biomarkers for beta IFN responsiveness. METHODS: Oligonucleotide microarrays were used for the comparative analysis of gene expression patterns from longitudinal PBMC samples taken from five patients undergoing beta IFN therapy. RESULTS: On the basis of two-fold changes in expression levels and statistical analyses we selected a candidate diagnostic set of 136 genes that were differentially expressed between pretreatment and IFN-beta-1a-treated MS patients. When we applied this gene set to cluster the specimens according to their expression profiles, the pretreatment samples clustered in one branch, and acute and chronic samples following treatment clustered in another branch. However, the chronic samples from the single clinical non-responder clustered with the pretreatment branch, suggesting that a possible reversal of beta IFN-induced gene expression may be contributing to the poor clinical response. CONCLUSIONS: These 136 genes represent potential targets for new MS therapeutics and the basis for lack of beta IFN response.     

Enumeration of T, B and natural killer peripheral blood cells of patients with multiple sclerosis and controls.

The median percentages of peripheral blood immunoglobulin-positive (Ig+) lymphocytes (8%, n = 46), CD8+ (12%, n = 49) and CD57+ cell numbers (5%, n = 37) of patients suffering from multiple sclerosis (MS) were significantly (p less than 0.05) lower than the values of age- and sex-matched healthy individuals (Ig+ cells: 13%, n = 46; CD8+ cells: 17%, n = 49; CD57+ cells: 9%, n = 37). Comparison of calculations on decreased peripheral blood cell counts and increased brain cell counts in MS patients revealed that sequestration of blood cells into the MS brain is a possible explanation of these findings.     

Leptin enhances the release of cytokines by peripheral blood mononuclear cells from acute multiple sclerosis patients

Objective To explore the effect of leptin on cytokine production by PBMCs obtained from MS patients either in acute (relapse) or in stable (nonrelapse) phase of disease. Methods PBMCs were collected from 25 untreated acute MS patients, 11 stable MS patients and 20 healthy controls. PBMCs were cultured either with RPMI-1640 alone or with leptin (1.25 nmol /ml) , phytohemagglutinin (PHA) (100μg/ml) , and leptin PHA. 72 h later the supernate of the culture medium were collected and stored at -70℃. The pro-inflammatory cytokine (IFN-γ) concentration were determined using an enzyme-linked immunosorbent assay (ELISA) , and the anti-inflammatory cytokine (IL-4) concentration were investigated by radioimmunity methods. Results Our data showed that leptin induced IFN-γ production by PBMCs of patients in an acute phase of disease but not in a stable phase or in healthy controls. Moreover, we found that PHA induced IL-4 production by PBMCs of patients in an acute phase of disease, but leptin inhibited this ability of PHA. Conclusiotl Leptin can affect on pro- and anti-inflammatory cytokine production by PBMCs collected from MS patients, may be this connected with leptin increase the susceptiveness of MS.     

Detection of human herpesvirus 6 variant A in peripheral blood mononuclear cells from multiple sclerosis patients

Several authors report that human herpesvirus 6 (HHV-6) variants have different epidemiologies, in vivo tropism and pathogenic potentials. However, it is not well known what pathogenic roles its neurotropism might have in the variant type. As some active plaques of multiple sclerosis (MS) brain tissue harbor HHV-6 DNA divergent from the prototype virus, the possibility that the variant strain may play a role in the pathogenesis of MS has been suggested. Therefore, we tried to investigate the role of HHV-6 variants in the pathogenesis of MS. As HHV-6 is predominantly a T-cell-tropic virus, we examined HHV-6 DNA sequences in peripheral blood mononuclear cells (PBMC) from 34 MS patients, 6 with idiopathic transverse myelitis, 2 with optic neuritis and 20 healthy controls. Nested polymerase chain reaction was used to detect the HHV-6 genome. To discern HHV-6 variants A and B, amplification products were digested by restriction enzyme. We found that 7 of 34 MS patients and 2 of 6 patients with idiopathic transverse myelitis had the HHV-6 genome. On the contrary, there was no HHV-6 genome in the control group. All genomic sequences were of HHV-6 variant A (HHV-6A). Our results suggest that the detection of HHV-6A in the PBMC of patients with MS may raise the possibility of a relationship between latent HHV-6A infection and the pathogenesis of MS.     

Leptin enhances the release of cytokines by peripheral blood mononuclear cells from acute multiple sclerosis patients

Objective To explore the effect of leptin on cytokine production by PBMCs obtained from MS patients either in acute （relapse） or in stable （nonrelapse） phase of disease. Methods PBMCs were collected from 25 untreated acute MS patients, 11 stable MS patients and 20 healthy controls. PBMCs were cultured either with RPMI-1640 alone or with leptin （ 1.25 nmol/ml）, phytohemagglutinin （PHA） （ 100 μg/ml）, and leptin ＋ PHA. 72 h later the supemate of the culture medium were collected and stored at -70℃. The pro-inflammatory cytokine （IFN-γ） concentration were determined using an enzyme-linked immunosorbent assay （ELISA）, and the anti-inflammatory cytokine （IL-4） concentration were investigated by radioimmunity methods. Results Our data showed that leptin induced IFN-γ production by PBMCs of patients in an acute phase of disease but not in a stable phase or in healthy controls. Moreover, we found that PHA induced IL-4 production by PBMCs of patients in an acute phase of disease, but leptin inhibited this ability of PHA. Conelusion Leptin can affect on pro- and anti-inflammatory cytokine production by PBMCs collected from MS patients, may be this connected with leptln increase the susceptiveness of MS.     

Infrequent detection of human herpesvirus 6 DNA in peripheral blood mononuclear cells from multiple sclerosis patients

Several studies have suggested an association between human herpesvirus 6 (HHV-6) infection and multiple sclerosis. As HHV-6 is predominantly a T-cell tropic virus, we examined the frequency of detection of HHV-6 genome in peripheral blood mononuclear cells from relapsing–remitting (n = 32) and chronic progressive (n = 14) patients and from healthy (n = 17) and neurological (n = 7) controls. Two sensitive polymerase chain reaction assays were used to target different regions within the HHV-6 genome. Depending on the polymerase chain reaction assay used, the detection of HHV-6 genome ranged from 11.7 to 23.5% (controls), 3.1 to 23.0% (relapsing–remitting), and 14.2 to 28.5% (chronic progressive). Although these observations do not exclude a pathogenic role for HHV-6 in multiple sclerosis, they indicate a lack of correlation between HHV-6 infection of peripheral blood mononuclear cells and the development of multiple sclerosis.     

Surface phenotypes of peripheral blood mononuclear cells in multiple sclerosis

Using monoclonal antibodies the subpopulations of mononuclear cells were studied in peripheral blood in patients with multiple sclerosis in active phase of the disease. The results suggested the following conclusions: 1. In the active phase of multiple sclerosis (during exacerbations and slowly progressive course) a rise occurs in the proportions of Ia-positive and M1-positive cells without changes in the proportions of T4 and T8 lymphocytes and in their mutual ratio. 2. No differences were observed in the percent of peripheral blood mononuclear cells between all patients in the active phase of the disease and those with slowly progressing disease. 3. During immunomodulatory treatment (alternating administration of prednisone and levamisole) a rise was observed in the percent of suppressor cells T8/Ia-positive, and a fall in the T4/T8 suppressor cell ratio.     

Restricted T cell receptor delta chain genes repertoire in peripheral blood of multiple sclerosis patients

Multiple sclerosis (MS) is a chronic inflammatory disease characterized by focal demyelination of central nervous system (CNS). Susceptibility to MS is thought to be affected by multiple genes including HLA and T cell receptor (TCR) genes. In view of the recent evidence, that in addition to α/β T lymphocytes also γ/δ T cells may have autoreactive potential, TCR delta repertoire in peripheral blood of MS patients has been studied. TCR delta repertoire, as assessed by Vδ-Jδ rearrangements, has been analysed in 13 MS cases and in 30 healthy individuals by seminested PCR technique. Oligonucleotide primers specific for six Vδ regions and for Jδ1 gene were used for amplification of Vδ-Jδ junctional region responsible for the diversity of γ/δ TCR. In the majority of MS patients PAGE analysis of Vδ1-Jδ1, Vδ3-Jδ1 and Vδ5-Jδ1 rearrangements showed single-band or two-band pattern. The most striking result has been observed in Vδ5-Jδ1 rearrangement, where in nine cases studied single band and in four patients two bands have been found. In all but one MS cases multi-band pattern of Vδ2-Jδ1 rearrangement was obtained. None of the 13 MS patients showed single-band rearrangement pattern of Vδ4-Jδ1 and Vδ6-Jδl. Contrary to the MS group almost all healthy individuals produced smear-like or multi-band pattern of Vδ1-Vδ5 to Jδ1 rearrangements. On the basis of the banding pattern produced by Vδ-Jδ rearrangement in MS, it can be suggested that T lymphocytes had undergone clonal expansion in vivo, most likely due to stimulation by antigen related to CNS. In particular a very consistent single-band pattern of Vδ5-Jδ1 rearrangement observed in almost all MS patients studied, argues very strongly for a significant role of γ/δ T cells with Vδ5 rearrangement in the pathogenesis of MS. However, it cannot be excluded that the observed patterns of TCR δ gene rearrangement in MS patients may represent secondary changes to CNS damage.     

Interferon-beta induces brain-derived neurotrophic factor in peripheral blood mononuclear cells of multiple sclerosis patients

Interferon-beta (IFN-beta) achieves its beneficial effect on multiple sclerosis (MS) via anti-inflammatory properties. In this study, we assessed the expression of the brain-derived neurotrophic factor (BDNF) in peripheral blood mononuclear cells (PBMC) from relapsing-remitting multiple sclerosis (RRMS) patients treated or not with IFN-beta. Intracellular BDNF was measured by Western blot and ELISA and compared with serum BDNF. We found higher levels of BDNF in PBMC of IFN-beta-treated versus non-treated patients, whereas serum levels of BDNF were similar. We hypothesize that the increased intracellular BDNF secondary to IFN-beta is not released in the periphery. This release is probably not tissue specific but in MS patients, BDNF could be specifically delivered by PBMC at the site of re-activation, i.e. within the central nervous system.     

T cells from multiple sclerosis patients recognize immunoglobulin G from cerebrospinal fluid

Idiotopic sequences are created after V, D and J recombinations and by somatic mutations during affinity maturation of immunoglobulin (Ig) molecules, and may therefore be potential immunogenic epitopes. Idiotope-specific T cells are able to activate and sustain the B cells producing such idiotopes. It is therefore possible that idiotope-specific intrathecal T cells could help maintain the persisting intrathecal synthesis of oligoclonal IgG observed in patients with multiple sclerosis (MS). This study was undertaken to examine T-cell responses to cerebrospinal fluid (CSF) IgG. Peripheral blood mononuclear cells (PBMC) from 14 of 21 MS patients and four of 17 control patients with other neurological diseases proliferated upon stimulation with autologous CSF IgG, while five and three, respectively, responded to serum IgG. By comparison, responses to myelin basic protein were recorded in only four MS and three control patients. Data from a limited number of patients indicate that the CSF IgG responsive cells were CD4+ and human leucocyte antigen DR restricted, that PBMC also respond to CSF IgG from other MS patients and that the CSF may contain T cells responding to autologous CSF IgG. This suggests that CSF IgG, or substances bound to this IgG, may represent T-cell immunogens, which could contribute to the intrathecal immune response in MS.     

多发性硬化症患者外周血LIGHT的表达研究

目的 观察多发性硬化症(MS)患者外周血肿瘤坏死因子超家族(TNFSF)成员LIGHT水平的变化,并探讨其作用机制.方法 选择北京大学人民医院神经内科白2009年11月至2011年5月收治的25例MS患者,22例脑梗死患者和问期健康对照(HC)者27例作为研究对象,应用酶联免疫吸附分析(ELISA)和实时—聚合酶链反应(PCR)检测其血浆LIGHT水平和外周血单个核细胞(PBMC) LIGHT、HVEM mRNA的表达,应用扩展的残疾状况量表(FDSS)评定MS患者的病情严重程度.结果 与HC组(41.2 pg/mL)和腩梗死组(79.55 pg/mL)比较,MS组血浆LIGHT 水平(133.2 pg/mL)明显增高,差异有统计学意义(P＜0.05);与HC组(0.3相对单位)和脑梗死组(0.44相对单位)比较,MS组LIGHT mRNA水平(0.75相对单位)明显增高,差异有统计学意义(P＜0.05).结论 MS患者存在着外周LIGHT表达的失凋,该异常表达可能在其CNS内炎性发病过程中发挥重要和复杂的作用.     

Immunoglobulin-producing cells in CSF and blood from patients with multiple sclerosis and other inflammatory neurological diseases enumerated by Protein-A plaque assay

Using the Protein-A plaque assay, numbers of IgG + IgA + IgM producing cells determined in patients with multiple sclerosis (MS) were 0.1–5% in CSF and 0.1–0.7% in peripheral blood; interestingly, 7 of 11 MS patients had IgM producing cells in CSF. In patients with aseptic meningitis (AM), the corresponding values were 0.04–7.5% in CSF and 0.4–2.4% in peripheral blood. There were more Ig producing cells in peripheral blood from patients with AM and MS than in healthy subjects. cocorrelation between numbers of IgG producing cells in CSF and the concentrations of intrathecally produced IgG (CSF IgG index) was registered in patients with AM: the same was true for IgA. The Protein-A plaque method, adopted for 20 × 10³ lymphocytes, makes possible enumeration of Ig-producing cells in CSF and discrimination among cells secreting different Ig classes, thereby being a powerful tool for studying immune reactions in the CNS-CSF compartment.     

Production of viral antibodies in vitro by CSF cells from mumps meningitis and multiple sclerosis patients

Cerebrospinal fluid (CSF) cells from 4 mumps meningitis and 11 multiple sclerosis (MS) patients were cultured in vitro for 7 days with and without pokeweed mitogen (PWM) stimulation. The cells produced varying amounts of IgG without stimulation and no significant increase of IgG synthesis was observed after PWM stimulation. Antibodies against mumps, measles, rubella, herpes simplex, and adeno viruses were measured in the supernatants of the cultures by a sensitive enzyme immunoassay. In the mumps meningitis patients, the largest amount of antibody was against mumps virus but low amounts of antibodies with other specificities were also synthesized by CSF cells of one patient. The most commonly detected specificities in MS patients were against measles and rubella viruses, whereas antibodies against adeno and mumps viruses were detected in only one CSF cell supernatant. No antibodies produced against herpes simplex virus in vitro were detected in any of the supernatants. The amounts of viral antibodies produced in vitro and intrathecally were only partially correlated.     

Interleukin-1 and interleukin-2 production by peripheral blood mononuclear cells in multiple sclerosis patients

The production of interleukin-1 (IL-1) and interleukin-2 (IL-2) by peripheral blood mononuclear cells (PBM) was assessed in multiple sclerosis (MS) patients in relapse, chronic progressive MS patients, patients with other neurological diseases (OND) and healthy subjects. Production was defined as the level of IL-1 and IL-2 in PBM supernatants. Neither spontaneous nor LPS-induced IL-1 production differed significantly in MS, OND patients or healthy individuals. On the other hand PHA-induced PBM IL-2 production was significantly less in MS patients in relapse (130 +/- 10.0 U/ml) than in chronic progressive MS patients (172 +/- 9.8 U/ml), OND patients (192 +/- 11.5 U/ml) and healthy subjects (215 +/- 13.8 U/ml) (P less than 0.02). Spontaneous IL-2 production was also diminished in MS patients in relapse (31 +/- 7.2 U/ml) as compared to chronic progressive MS patients (46 +/- 8.8 U/ml) and healthy subjects (49 +/- 11.1 U/ml) (P less than 0.01). Anti-Tac monoclonal antibody was used to study IL-2 receptor expression on the same sample of PBM that was used for IL-2 study. MS patients in relapse had significantly higher levels of IL-2 receptor-positive unstimulated PBM (6.0 +/- 2.2%) as compared to chronic progressive MS (2.0 +/- 0.9%), OND (2.5 +/- 1.1%) and healthy subjects (1.5 +/- 0.7%) (P less than 0.002). We postulate that reduced apparent IL-2 production by PBM of MS patients in relapse may result from immediate IL-2 binding to receptor expressed on activated T lymphocytes and internalization of IL-2-receptor complex.