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.     

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.     

Administration of monthly-pulse cyclophosphamide in multiple sclerosis patients. Effects of long-term treatment on immunologic parameters

Eleven patients with chronic progressive multiple sclerosis received monthly pulses of cyclophosphamide (CY) for approximately one year. During the final 9 months the monthly dose ranged between 1000 mg/m2 and 2000 mg/m2. This resulted in a marked (47% or greater) reduction in CD4 (T helper/inducer) cells a less striking (22%) decrease in CD8 (T suppressor/cytotoxic) cells and a decline in the CD4/CD8 ratio. The magnitude of the decrease in CD4 cells correlated with the total dose received (r = 0.88, P less than 0.05). B cells were reduced 50% and FcR+ lymphocytes were reduced 48% without comparable reduction in natural killer cells or antibody-dependent cellular cytotoxicity. Proliferative responses to PHA were suppressed. Two patients improved, seven stabilized and two continued to worsen. Monthly pulses of CY can achieve substantial and differential reduction in immune parameters and appear to slow the disease progression in some MS patients.     

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.     

Intracellular cytokine profile in T-cell subsets of multiple sclerosis patients: different features in primary progressive disease

OBJECTIVE: To evaluate the expression of cytokines in both CD4+ and CD8+ T cells derived from peripheral blood of untreated multiple sclerosis (MS) patients with either relapsing-remitting (RR), secondary progressive (SP) or primary progressive (PP) MS and healthy controls (HC). BACKGROUND: MS is an immune-mediated disease and cytokines hove been hypothesized to contribute significantly to disease progression. Compared to the relapse-onset (RR, SP) form of the disease, PPMS patients have different clinical, immunological and pathological features. Surprisingly, the ability of their circulating T cells to produce immunoregulatory cytokines has not been extensively studied so far. METHODS: Seventy-two MS patients (24 RR, 26 SP, 22 PP) and 34 HC were studied. Stimulated peripheral blood derived CD4+ and CD8+ T MS patients express significantly more CD4+ and CD8+ T cells were analyzed for IFN-gamma, IL-2, TNF-alpha, IL-4, IL-10 and IL-13 production. RESULTS: cells producing IFN-gamma compared to HC. Compared to the other forms of the disease, PPMS patients display a significant decrease in CD4+ T cells producing IL-2, IL-13 and TNF-alpha and a significant increase in CD8+ T cells producing IL-4 and IL-10. CONCLUSIONS: The data presented here demonstrate that patients with PPMS express less pro- and more anti-inflammatory cytokine producing T cells compared to the relapse-onset form of the disease, confirming the view on PPMS as a distinct disease entity.     

CD8+ T cell activation correlates with disease activity in clinically isolated syndromes and is regulated by interferon-beta treatment

An increased percentage of blood CD8+ T cells from patients with clinically isolated syndromes (CIS) suggestive of multiple sclerosis (MS) was found to express CD26 and CD69. The percentage of CD26 or CD69 positive CD8+ T cells was higher in patients with MRI evidence of disease dissemination in space or with active MRI lesions than in the remaining patients. Treatment of MS with interferon (IFN)-beta resulted in a decrease in the percentage of CD26 and CD71 positive CD8+ T cells and an increase in the percentage of CD8+ T cells that expressed interleukin (IL)-10 and IL-13. CD8+ T cell activation in MS may be linked to disease activity already at disease onset, and is regulated by treatment with IFN-beta.     

Correlation of immunological studies and disease progression in chronic progressive multiple sclerosis

Thirty untreated patients with clinically definite chronic progressive multiple sclerosis were matched with 10 patients with clinically stable definite multiple sclerosis and 16 patients with other neurological diseases. A group of 12 normal control (NC) volunteers was matched to these groups. All patients with chronic progressive multiple sclerosis and normal control subjects were analyzed for the concanavalin A suppressor assay, mitogen stimulation, and phenotyping of peripheral blood mononuclear cells. In addition, serum was analyzed for interleukin-2 levels. Results of mitogen stimulation studies did not distinguish the groups. Concanavalin A-induced suppression was significantly decreased in the patients with chronic progressive multiple sclerosis (p less than 0.01). Phenotyping of fresh cells showed an elevated CD4: CD8 ratio in the patients with chronic progressive multiple sclerosis. Neither phenotyping nor concanavalin A-induced suppression correlated with or predicted the degree of disability, but the serum levels of interleukin-2 correlated inversely with disability (p less than 0.01) and directly with a poor prognosis after 18 months of observation (p less than 0.05). Serum levels of interleukin-2 decreased as the disease progressed.     

Activated suppressor cell function in multiple sclerosis--clinical correlations

Activated suppressor cell function mediated by either freshly isolated peripheral blood mononuclear cells (MNCs), freshly isolated CD8+ lymphocytes or by CD8+ cell lines, has previously been found to be reduced compared to controls in multiple sclerosis (MS) patients with progressive disease (MS-P). In this study, we found that suppressor activity mediated by CD8+ cell lines, derived from MS patients with stable disease (MS-S) patients and maintained in culture for 14 days, was significantly greater (45 +/- 6%) compared to that mediated by MS-P patients' CD8+ cells (11 +/- 4%, P less than 0.005). The MS-S suppressor values were, however, suggestively reduced compared to controls (60 +/- 6%, P less than 0.05). MNC-mediated suppressor values for the MS-S group (61 +/- 5%) did not differ from the control group (67 +/- 6%). Values for the MS-P group (7 +/- 6%) were significantly reduced compared to MS-S and control groups. Cytotoxic activity mediated by CD8+ cell lines showing defective suppressor function did not differ from control values. The cell lines in MS and control did not differ with respect to their rate of proliferation in the presence of IL-2 and OKT3. Suppressor function in this assay was ablated if exogenous IL-2 was removed from the culture media. These data suggest that defective activated suppressor function is characteristic of the progressive form of MS, although a suppressor defect is also partially expressed in stable MS patients when CD8+ cell lines are studied.     

Relationship between clinical activity and lymphocyte subsets in patients with Guillain-Barré syndrome]

T lymphocytes are probably of pathogenic importance in Guillain-Barré syndrome (GBS). Blood samples from 31 patients with GBS and from 25 healthy controls were studied with flow cytometry. Subsets of the CD4 population were determined: CD4+CD29+ (helper-inducer) cells and CD4+CD45RA+ (naive or suppressor-inducer) cells. Recently, deviations of circulating CD8 populations have been noticed. Therefore, subsets of the CD8 population were also determined: CD8+CD11b dull (suppressor-effector) cells and CD8+ CD11b bright (cytotoxic-effector/NK) cells. During the active stage of GBS, significantly increased proportions of CD4+HLA-DR+ (activated CD4+) cells and CD4+CD29+ cells were found compared to those in control samples (p < 0.0005, p < 0.05, respectively). The proportion of CD8+CD11b dull cells was significantly lower than that in the control samples (p < 0.005). Sequential analysis showed a decrease in the proportion of CD4+HLA-DR+ cells and CD4+CD29+ cells from the active to the convalescent stage (p < 0.05, p < 0.005, respectively). The proportion of CD8+CD11b dull cells was low in GBS patients with high disease severity.     

Long-term favorable response to interferon beta-1b is linked to cytokine deviation toward the Th2 and Tc2 sides in Japanese patients with multiple sclerosis

To address the immune mechanism of the long-term beneficial effects of interferon beta (IFN-beta), we measured the intracellular cytokine production patterns of IFN-gamma, IL-4 and IL-13 in peripheral blood CD4+ and CD8+ T cells, which previously displayed alterations during the early course of IFN-beta treatment, in 15 Japanese patients after long-term IFN-beta administration. The patients were treated with IFN-beta-1b 8 x 10(6) units given subcutaneously every other day for a mean period of 34.5 +/- 5.5 months (range: 26-43 months). During the follow-up period, 6 patients experienced 33 relapses, while the other 9 were relapse-free. The results revealed the following cytokine alterations: (1) type 2 cytokine, such as IL-4 and IL-13, were significantly increased in producing cell percentages in both CD4+ (p = 0.0356 and p = 0.0007, respectively) and CD8+ (p = 0.0231 and p = 0.0170, respectively) T cells while IFN-gamma, a representative type 1 cytokine, was significantly decreased in the absolute producing cell numbers (p = 0.0125 in CD4+ T cells and p = 0.0022 in CD8+ T cells) even after approximately 3 years of IFN-beta administration; (2) the intracellular IFN-gamma / IL-4 ratio tended to decrease in both CD4+ and CD8+ T cells (p = 0.0535 and p = 0.0783, respectively), reflecting a strong downmodulation of type 1 cytokine producing cells; and importantly (3) alterations such as the decreased intracellular IFN-gamma / IL-4 ratio in CD4+ T cells and increased percentage of CD8+ IL-13+ T cells compared with the pretreatment levels were only statistically significant in MS patients without relapse during IFN-beta therapy (p = 0.0152 and p = 0.0078, respectively). Therefore, we consider that cytokine deviation toward the Th2 and Tc2 sides is linked to a long-term favorable response to IFN-beta, while a higher intracellular IFN-gamma / IL-4 ratio is associated with treatment failure.     

Fluctuations in T helper subpopulations in relapsing-remitting multiple sclerosis

Patients with active multiple sclerosis (MS) have been reported to have a depletion of CD4+ CD45R+ cells, the immature resting CD4+ subpopulation. Using Leu 3a(anti-CD4) and Leu 18(anti-CD45R), the frequencies and absolute numbers of CD4+ CD45R+ and CD4+ CD45R- subsets were measured in 30 patients with MS and 17 healthy controls. These subsets were monitored every 6 weeks over a 6 month period. CD4+ CD45R- cells were found to be increased in relapse compared to remission (p less than 0.005) while CD4+ CD45R+ levels were not significantly altered in relapse. However, the CD4+ subset ratio (CD4+ CD45R-/CD4+ CD45R+) was significantly higher in relapse compared with remission (p less than 0.002). Furthermore, these findings were upheld when data from the same 6 patients in relapse and remission was compared. Increased disease activity was not associated with changes in any of the other parameters measured (total T cells, total CD4+ cells, suppressor cells or activated T cells). These results suggest that relapse in MS is accompanied by the conversion of CD4+ CD45R+ resting cells to CD4+ CD45R- primed cells.     

Decrease in memory (CDw29-high) cerebrospinal fluid cells from acute MS patients

The CD45R (2H4) and CDw29 (4B4) molecules are probably involved in the regulation of immunological memory: CD45R functionally defines naive or virgin cells (prior to activation by exposure to antigen), while CDw29 characterizes previously activated (memory) cells. By means of two-colour fluorescence analysis, we studied CD4 and CD8, CD45R and CD4 and CD8 CDw29 cells from the cerebrospinal fluid (CSF) of 27 acute (MSa), and 10 stable (MSs) multiple sclerosis (MS) patients. We also compared 17 patients affected with various non-inflammatory (OND) diseases of the central nervous system (CNS). The most striking finding was the increased percentage of CD4+CD29-, CD4+CD45R- and CD8+CD45R- cells and the decreased percentage of CD4-CDw29+ and CD8- CDw29+ subsets in MSa patients compared to OND and MSs populations. These data suggest a decrease in memory cells (CDw29+) during the acute phase of MS. The modulation of CDw29 receptor could play a role in the genesis of acute MS attacks.     

Expression of TH1/TH2-related chemokine receptors on peripheral T cells and correlation with clinical disease activity in patients with multiple sclerosis

Th1 cells play an important role in the pathogenesis of multiple sclerosis (MS), a disease likely linked to an autoimmune process. We measured the levels of chemokines in serum or cerebrospinal fluid (CSF) samples by ELISA, and also studied the expression of Th1-related CXCR3/CCR5 chemokine receptors and Th2-related CCR4/CCR3 chemokine receptors on blood cells from MS patients using three-color flow cytometry. The Bonferroni correction was used for the statistical analysis. The levels of CXCL10, CCL3, and CCL5 in the CSF samples for the MS groups were significantly higher than those for the control group. However, the levels of CCL2 in both the CSF and serum samples for the remission group were significantly higher than those for the active group. The percentage of CXCR3-expressing CD4+ T cells in patients with MS was significantly elevated compared with the healthy controls. Moreover, MS patients in an active phase showed a more increased CD4+CXCR3+/CD4+CCR4+ ratio than patients in a remission phase. The increased percentage of CD4+CXCR3+ cells in the blood was associated with relapses in MS. This study suggested that the CD4+CXCR3+/CD4+CCR4+ ratio could be a sensitive maker of immune dysfunction in MS.     

Increased peripheral blood CD5+ B cells predict earlier conversion to MS in high-risk clinically isolated syndromes

Clinically isolated syndrome patients (CIS) with oligoclonal IgG bands (OCGB) are at high risk for clinically definite multiple sclerosis (MS). However, the outcome for individual patients is unpredictable and the search for reliable blood markers predicting early conversion to multiple sclerosis (MS) has clinical relevance. CD5+ B cells (CD5+Bc) are involved in some autoimmune diseases. This study investigated whether high blood CD5+Bc percentage can predict CIS conversion to MS. Fifty-five consecutive CIS showing OCGB were prospectively studied. Every patient underwent a brain MRI study and a flow cytometry analysis of CD5+Bc percentage. Conversion to MS was studied during follow-up. The CD5+Bc percentage was assessed in 40 controls and a cut-off value of 3.5% (mean+2 SD) was calculated. A blood CD5+Bc percentage above this value predicted earlier conversion to MS in the whole group (hazard ratio [HR]: 3.40; 95% confidence interval [CI]: 1.69-6.68; p=0.0005) and in CIS patients fulfilling three or more Barkhof-Tintoré criteria plus OCGB, who showed higher risk for MS (HR: 3.79; 95% CI: 1.86-15.32; p=0.0018). Multivariate analysis also showed a predictive value for high blood CD5+Bc count (HR: 4.3; 95% CI: 1.9-9.5; p<0.0001). It was concluded that high percentages of CD5+Bc independently associate with increased risk of early conversion to MS in CIS patients with OCGB and Barkhof-Tintoré criteria.     

多发性硬化患者外周血和脑脊液淋巴细胞亚群的观察

用碱性磷酸酶抗酸酶法检查了４６例多发性硬化活动期患者外周血和脑脊液的淋巴细胞亚群。结果显示：活动期ＭＳ者外周血ＣＤ＾＋４，ＣＤ＾＋９细胞较对照组减少，ＣＤ＾＋２５细胞，ＣＤ＾＋４／ＣＤ＾＋８比值较对照组升高。ＣＳＦ中ＣＤ＾４，ＣＤ＾＋２５细胞，ＣＤ＾＋４／ＣＤ＾＋８比值较对照组升高，ＣＤ＾＋８细胞降低，且ＣＳＦ中淋巴亚群均高于自身外周血中的相应细胞。     

Increased T cell expression of CD154 (CD40-ligand) in multiple sclerosis

CD154 (CD40-ligand, gp39), expressed on activated T cells, is crucial in T cell-dependent immune responses and may be involved in the pathogenesis of multiple sclerosis (MS). We studied cerebro-spinal fluid and peripheral blood T cell expression of CD154 in MS by flow cytometry. Patients with secondary progressive MS (SPMS) had constitutive CD154 expression on CD4 and CD8 T cells in blood. Constitutive CD154 expression was not observed in patients with relapsing-remitting MS (RRMS) or clinically isolated syndromes (CIS) suggestive of demyelinating disease. After ex vivo activation CD154 was, however, expressed on a higher percentage of T cells from patients with CIS or RRMS than from healthy control subjects. These results suggest involvement of CD154 in the pathogenesis of MS, and the shift from a relapsing-remitting to a secondary progressive disease course may be associated with constitutive, systemic CD154 expression.     

Soluble CD4 and CD8 in the peripheral blood of patients with multiple sclerosis and HTLV-1-associated myelopathy

We evaluated the presence of soluble (s) CD4 and sCD8, released from activated T cells, in the sera of patients with multiple sclerosis (MS) and human T lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM) using an enzyme-linked immunosorbent assay (ELISA). In addition, peripheral blood T cell subsets in patients with MS and HAM were analyzed by single and two color flow cytometry. The serum level of sCD8 was significantly elevated in MS patients as compared with controls (p less than 0.001). Sera from patients with an exacerbation of acute relapsing MS showed a higher sCD8 level than the patients in remission or controls (p less than 0.01 and p less than 0.001, respectively). The serum levels of both sCD4 and sCD8 were also significantly elevated in patients with HAM (p less than 0.001 and p less than 0.001, respectively). In addition, a significantly increased serum level of soluble interleukin-2 receptor (sIL-2R) was found in patients with HAM as compared with that of controls (p less than 0.001). These observations suggest that CD8 cells may be activated in the peripheral blood of patients with MS and sCD8 may be related to clinical activity, but that both CD4 and CD8 cells may be activated in the peripheral blood of patients with HAM.     

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.     

Analysis of peripheral blood lymphocyte phenotype and function during dexamethazone treatment of progressive multiple sclerosis

Five patients with chronic progressive multiple sclerosis (MS) and three control patients with lumbar disc herniation were treated with dexamethazone during 14 days. The effect on peripheral blood T-cell subsets and on the proliferative response of peripheral blood mononuclear cells (PBMC) to pokeweed mitogen (PWM) and anti-mu antibody was analyzed. Before treatment, the proportion of CD3+ and CD4+ PBMC was similar in MS and control patients, but the proportion of CD8+ and DR+ PBMC was lower and the PBMC were less responsive to anti-mu stimulation in MS patients compared to controls. Steroid treatment induced reversible granulocytosis and lymphocytosis. CD3+ and CD4+ cells increased and DR+ cells decreased in MS patients but not in controls. Proliferation of anti-mu stimulated PBMC increased in MS-patients during the two weeks of treatment, but decreased in controls. The enhancement in the MS patients of pre-existing immune abnormalities suggests that a cautious attitude is warranted in the use of steroid treatment in chronic progressive MS.     

Differences in systemic and central nervous system cellular immunity relevant to relapsing–remitting multiple sclerosis

In order to elucidate the differences between systemic and central nervous system (CNS) immunity that are relevant to exacerbations of multiple sclerosis (MS), paired peripheral blood and cerebrospinal fluid (CSF) samples obtained from 36 non-treated patients with relapsing-remitting MS (RRMS) were simultaneously examined using flow cytometry to determine the percentages of functional lymphocyte subsets, as well as enzyme-linked immunosorbent assays for measuring soluble immune mediators.Active RRMS patients (n = 27) were characterized by an increase in CD4+ CXCR3+ Th1 cells in blood as compared with inactive patients (n = 9), and this parameter was inversely correlated with plasma levels of IL-10 and IL- 12p70. In contrast, an increase in the percentage of CD4+ CD25+ cells and a decrease in the percentage of CD8+ CD11a(high) cells were features of CSF samples from those with active RRMS. Further, CSF CD4+ CD25+ cells had a close association with leukocyte counts as well as albumin and CXCL10 levels in the CSF, and, thus, could be useful as a measure for inflammatory reactions in the CNS. On the other hand, CD8+ CD11a(high) cells may function as immunoregulatory cells, as their percentage in the CSF showed a positive correlation with CSF levels of the anti-inflammatory cytokine IL-4. These findings suggest that MS relapses occur in a combination with altered cell-mediated immunity that differs between the peripheral blood and CSF compartments, while measurement of lymphocyte subsets may be helpful for monitoring disease status.