Analysis of lymphocyte subpopulations in cerebrospinal fluid and peripheral blood in patients with multiple sclerosis and inflammatory diseases of the nervous system

We analysed different subsets of lymphocytes from peripheral blood (PB) and cerebrospinal fluid (CSF) by flow cytometry in order to determinate alterations in patients with multiple sclerosis (MS) in acute relapse and viral inflammatory neurological disease (IND). We found increased levels of adhesion molecules (LFA-1 and β₁ integrin) in the CSF of patients with MS and IND compared to NIND. CD4 ⁺/CD8 ⁺ ratio was significantly higher in CSF of MS as compared with all groups analysed and compared with PB. We detected a significantly higher expression of the interleukin-2 receptor in PB of MS patients when compared with other groups. In patients with IND a significant higher expression of the interleukin-2 receptor was found in the CSF compared with MS and NIND. Our findings indicate that the activation of T lymphocytes primarily occurs in the peripheral immune compartment in MS and the increase of adhesion molecules in CSF is related to inflammatory disorders and not only to MS.     

Cytokine secretion and nitric oxide production by mononuclear cells of patients with multiple sclerosis

Several experimental findings suggest a potential role of excessive nitric oxide (NO) production by macrophages, microglia and astrocytes in the pathogenesis of demyelinating lesions in MS. We assessed the production of nitrites by peripheral blood mononuclear cells (PBMCs) of 15 MS patients (10 F and 5 M) with the R–R form (EDSS: 1–3.0) and in 15 age-matched control subjects. 9 out of the 15 MS patients showed active lesions in MRI at the time of examination. 7 patients were also monitored at the onset, during and following a clinical relapse. Secretion of cytokines by PBMCs was assessed at the basal time and after 24 h of incubation with lipopolysaccharide (LPS). The production of nitrites in the supernatants of PBMCs stimulated and not stimulated with lipopolysaccharide was evaluated. The secretion of IL1β, IFN-γ, TNF-, IL-6 IL-10 and TGF-β by PBMCs was detected using ELISA methods. The production of NO, both basal and stimulated, was significantly higher in the patients with active lesions than in those without active lesions (p<0.01). No significant difference was evident between the basal and LPS-stimulated production of NO between control subjects and MS patients without active lesions. During relapses there was a significant increase in NO production by PBMCs compared to the clinical stable stage of the disease (p<0.0001). This increase was significantly greater in the early stage of relapse than in the late stage (p<0.04). A decline of NO levels was observed during recovery. Steroid treatment induced a significant decrease in the PBMC NO production of MS patients during exacerbations (p<0.01). The levels of IL-1β, IFN-γ and TNF- are significantly higher in the supernatants of the PBMCs which produced greater amounts of NO (p<0.02, p<0.03, p<0.01, respectively). On the other hand, NO levels were negatively related to IL-10 and TGF-β production (R=−75, p<0.0001 and R=−0.79, p<0.0001, respectively). The increase production of NO by peripheral blood mononuclear cells demonstrated in our study to be associated with increased production of proinflammatory cytokines could therefore be considered to be a marker of mononuclear cell activation in the peripheral blood of MS patients and, indirectly, of disease activity. Its increased secretion during T cell and monocyte homing in the CNF could contribute to the damage to the blood–brain barrier and the subsequent cytokine-mediated cytotoxic effect to myelin and oligodendrocytes in the white matter of MS patients.     

Serum and cerebrospinal fluid nitrite and nitrate levels in relapsing-remitting and secondary progressive multiple sclerosis patients

Nitric oxide (NO) has been implicated in immune mediated cellular cytotoxicity and inflammatory processes including multiple sclerosis (MS). We aimed to assess NO production in MS patients and to delineate its involvement in different stages. The stable end-products of NO; nitrite(NO₂⁻) and nitrate(NO₃⁻) were analysed both in serum and CSF (cerebrospinal fluid) of patients with MS and non-inflammatory neurological diseases. Nitrite levels were quantified by calorimetric assay based on the Griess reaction. Nitrate levels were examined spectrophotometrically. MS patients exhibited significantly increased serum and CSF levels of NO₂⁻+NO₃⁻ compared with the control subjects. CSF NO₂⁻+NO₃⁻ levels were raised significantly in MS patients with both relapsing remitting (RR) and secondary progressive (SP) course. There was no significant difference between RR and SP MS patients with regard to NO metabolites. No significant correlation was found between NO metabolites and disability score, disease progression index, MRI (magnetic resonance imaging) activity and development of cortical atrophy on MRI. This study provides further evidence for excessive NO production both in CSF and peripheral blood of MS patients. Excessive CSF NO₂⁻+NO₃⁻ levels being more increased than the levels in sera supports pathological inflammatory process within CNS (central nervous system) in both stages of MS. Another implication for the role of NO and INOS inhibitors in the treatment of MS patients with both RR and SP courses was also suggested.     

CD4 and CD8 lymphocyte subsets in cerebrospinal fluid and peripheral blood from patients with multiple sclerosis, meningitis and normal controls

Objectives - To study the distribution of CD4⁺ and CD8⁺ T-cell subsets in cerebrospinal fluid (CSF) and peripheral blood from patients with multiple sclerosis (MS), meningitis, other neurological diseases and healthy controls.
Material and methods - The expression of markers for naive and memory cells (CD45RA⁺ and CD45RO⁺), and helper/inducer cells (CD29⁺) on CD4⁺ cells as well as CD45RO⁺ and killer/effector (S6F1⁺) on CD8⁺ cells was investigated in cerebrospinal fluid (CSF) and peripheral blood from patients with multiple sclerosis (n=28), meningitis (n = 13), other neurological diseases (n = 16), and healthy controls (n = 16) by 2-color flow cytometry.
Results - The majority of T cells in the CSF of the 4 groups exhibited the phenotype of memory cells (CD45RO⁺) on both CD4⁺ and CD8⁺ cells. The proportion of helper/inducer (CD29TD4⁺ in CD4⁺) cells was also larger in the CSF compared to peripheral blood in the 3 patient groups and controls investigated. In contrast, CD8⁺ cells with killer/effector (S6F1+) phenotype were fewer in CSF compared to peripheral blood in all 4 groups. There were no significant differences between patients and controls regarding the distribution of these activation markers in the CSF or peripheral blood.
Conclusion - Our observations support the notion that activated T cells of both CD4⁺ and CD8⁺ phenotype selectively pass the blood-brain barrier under both pathological and normal conditions.     

Serum and CSF levels of MCP-1 and IP-10 in multiple sclerosis patients with acute and stable disease and undergoing immunomodulatory therapies

The two chemokines, monocyte chemoattractant protein (MCP)-1 and gamma-interferon inducible protein (IP)-10, are thought to be involved in the pathogenesis of multiple sclerosis (MS). We measured MCP-1 and IP-10 levels in serum and CSF samples from 38 acute and 25 stable MS patients and from 40 controls. The latter consisted in patients with other inflammatory neurological diseases (OIND) or with non-inflammatory neurological diseases, and healthy controls. CSF MCP-1 levels exceeded those found in serum in all the patients studied as well as in healthy controls. CSF MCP-1 levels were significantly lower in acute MS [468+/-(S.E.M.) 18 pg/ml] than in stable MS (857+/-104 pg/ml). When detectable, serum and CSF IP-10 levels were significantly higher in acute MS (serum 331+/-66 pg/ml; CSF 118+/-16 pg/ml) than in stable MS (serum 69+/-7 pg/ml; CSF 25+/-2 pg/ml). Among OIND patients, those with HIV-1-associated dementia showed high serum and CSF levels of both MCP-1 and IP-10. Those with encephalitis showed high serum and CSF levels of IP-10 and CSF mononuclear pleiocytosis. We also evaluated the effects of 6-methylprednisolone or IFN-beta1a therapy on circulating MCP-1 and IP-10 levels. Neither MCP-1 nor IP-10 post-therapy levels varied significantly from baseline values. Our findings suggest that (a) MCP-1 could be constitutively produced within the brain; (b) MCP-1 and IP-10 CSF levels in acute MS vary significantly from those in stable MS, and these variations are inverse; and (c) current MS therapies do not modify circulating levels of MCP-1 and IP-10.     

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.     

T-cell subsets in multiple sclerosis: relationships between peripheral blood and cerebrospinal fluid

We contemporarily studied cerebrospinal fluid (CSF) and peripheral blood (PB) T-cell subsets, defined by monoclonal antibodies, in 29 patients with multiple sclerosis (MS) and 10 patients with other neurological diseases (OND). All subjects showed a clear-cut prevalence of CSF T-cells. Similarly, T-helper and T-suppressor subsets tended to show higher percentages in CSF in almost all subjects except relapsing MS, who were characterized by low percentages of T-suppressors in PB and even much lower percentages in CSF. Helper/suppressor ratios were found to be almost similar in the two body compartments of OND patients, lower in CSF than in PB of chronic progressive MS, always higher in CSF than in PB of relapsing MS. MS patients in remission showed both patterns of progressive MS and OND patients. Our results demonstrate that the loss of PB T-suppressor in relapsing MS is not due to a migration of such cells into CSF. Furthermore, regarding T-lymphocyte subsets, a typical CSF/PB pattern characterizes relapsing MS from other patients.     

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.     

Characterization of natural killer cells in paired CSF and blood samples during neuroinflammation

Natural killer (NK) cells from paired CSF and blood samples of patients with multiple sclerosis (MS), other neuroinflammatory diseases (IND), and non-inflammatory neurological diseases (NIND) were characterized using flow cytometry. NK cell frequency in CSF was overall decreased compared to blood, particularly in MS patients. In contrast to blood NK cells, during neuroinflammation, CSF NK cells display an immature phenotype with bright expression of CD56 and CD27 and reduced CX3CR1 expression. Our findings suggest that, as for central memory T cells, CSF may represent an intermediary compartment for NK cell trafficking and differentiation before entering the CNS parenchyma.     

多发性硬化患者外周血单个核细胞转录因子Sp3基因的缺陷表达

目的：研究我国多发性硬化（MS）病人外周血单个核细胞（PBMC）转录因子Sp3基因表达情况及其与临床相关性。方法：采用反转录－聚合酶链反应（RT－PCR）技术，经2对引物扩增，检测了33例MS、30例非免疫 性其他神经系统疾病对照、30例神经系统其他自身免疫性疾病对照和30名健康对照组者PBMC的Sp3基因表达。结果：Sp3基因表达缺如，MS患者组（45％，15／33）明显高于其他3组（依次为16％，5／30，6％，2／30；10％，3／10，P均（＜0．01），可自Sp3表达阴性MS患者的DNA中扩增出相应Sp3片段。Sp3表达阴性MS患者改良的伤残状态量表（EDSS）评分显著高于表达阳性者。脑脊液的IgG24h合成率和血清可溶性白细胞介素－2受全稍高，4例MS患者治疗前病情重和治疗后病情轻时相比，未见其Sp3基因表达有明显变化。结果：汉 MS病人PBMC中有Sp3表达缺陷，Sp3阴性患者残疾程度和免疫功能紊乱程度明显高于阳性者，作 为转录调节因子Sp3的表达缺乏可能与免疫控制的异常启动有关。     

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.     

High cerebrospinal fluid concentration of glial fibrillary acidic protein (GFAP) in patients with normal pressure hydrocephalus

The concentration of glial fibrillary acidic protein (GFAP) in lumbar cerebrospinal fluid (CSF) was measured in 12 patients with normal pressure hydrocephalus (NPH) 11 patients with primary degenerative dementia (PDD), 8 patients with various other neurological diseases, and 18 patients without signs of organic nervous disease (controls).

Mean CSF GFAP concentration was significantly higher in NPH patients: 96 ± 23 ng/ml (SEM) when compared with PDD patients: 8.2 ± 1.9 ng/ml (P < 0.01), or with controls: 4.3 ± 0.7 ng/ml (P < 0.01). Only 2 NPH patients had a GFAP concentration within the range of the control group (2–14 ng GFAP/ml CSF). No significant differences were found between the PDD patients and the control group, or between the group of patients with other neurological diseases and the control group.

In addition, a rostro-caudal gradient of GFAP in CSF could be demonstrated. In 6 NPH and 2 PDD patients both ventricular and lumbar CSF samples were investigated. In all cases the ventricular GFAP concentration was higher than the lumbar concentration. The difference was statistically significant (P < 0.01).

Our results suggest that determination of CSF GFAP concentration might be of diagnostic value in discrimination between NPH patients and patients with enlarged ventricles associated with degenerative brain disease.     

IP-10 and MCP-1 levels in CSF and serum from multiple sclerosis patients with different clinical subtypes of the disease

Interferon-gamma-inducible Protein-10 (IP-10) and Monocyte Chemotactic Protein-1 (MCP-1) levels were measured by enzyme-linked immunosorbent assay (ELISA) in the CSF and in the serum from 74 patients affected by different clinical forms of Multiple Sclerosis (MS), including 39 patients with Relapsing Remitting (RR) MS in an active phase, 14 patients in a stable phase of the disease, 12 patients with Secondary Progressive (SP) MS and 9 patients with Primary Progressive (PP) MS. IP-10 and MCP-1 levels were also determined in 19 subjects with no neurological diseases or major systemic disorders, 18 patients with non-inflammatory neurological diseases, as well as in 15 patients with other inflammatory neurological diseases.IP-10 levels were significantly elevated in CSF and serum from RR and SP, but not PP-MS patients. On the contrary, MCP-1 levels were decreased in CSF and serum of all MS patients. CSF concentrations of IP-10 and MCP-1 did not significantly correlate neither with each other, nor with CSF mononuclear cell count, albumin quotient or CSF IgG index. No correlation between disease duration, clinical course or EDSS score and chemokine levels was found.IP-10 and MCP-1 undergo modifications in different subtypes of the disease: IP-10 levels in CSF and serum samples are markedly increased when inflammation is prominent, and not in PP--MS patients, where inflammation is less evident. MCP-1 decrease in CSF and serum from MS patients could be related to the regulation of T-cell polarization.     

Genetic analysis of inflammation, cytokine mRNA expression and disease course of relapsing experimental autoimmune encephalomyelitis in DA rats

Genetic analysis of experimental autoimmune encephalomyelitis (EAE) can provide clues to the etiology of multiple sclerosis (MS). Identifying the susceptibility genes of DA rats may be particularly rewarding since they are prone to develop a remarkably MS-like chronic and demyelinating disease. As a first step in this direction, we investigated the role of DA genes within and outside the major histocompatibility complex (MHC) for susceptibility to severe protracted and relapsing EAE (SPR-EAE). This form of EAE developed in DA rats but not in LEW, ACI and BN rats after immunization with syngeneic spinal cord and complete Freund's adjuvant. Studies of crosses between DA and BN rats revealed that non-MHC genes determine susceptibility to SPR-EAE. A role for MHC-genes was also established using MHC-congenic rat strains, in which the DA MHC haplotype (av1) associated with relapsing EAE. Again, non-MHC genes were decisive since a high incidence of SPR-EAE only occurred in rats with DA non-MHC genes. Analysis of cytokine mRNA expression and infiltrating cells in the spinal cords of congenic strains revealed that the av1 haplotype associated with a high CD4/CD8 ratio and expression of mRNA for interferon-γ (IFN-γ), but not for transforming growth factor-β (TGF-β) or interleukin-10 (IL-10). In contrast, the other MHC haplotypes (h, l, u) associated with low CD4/CD8 ratios and mRNA expression for TGF-β and IL-10, but not for IFN-γ. DA non-MHC genes determined the intensity of inflammation since the number of cells expressing MHC class II, CD4 and interleukin-2 receptor (IL-2R) was higher in DA rats than in LEW.1AV1 and PVG.1AV1 rats which also carry the av1 haplotype. We conclude that the MHC haplotype of DA rats favors a prolonged proinflammatory autoimmune response associated with relapses, while the DA background intensifies inflammation correlating with a high incidence of relapsing disease.     

A comparison of regulatory cells in spinal fluid and blood in patients with multiple sclerosis and other neurologic diseases

Multiple sclerosis is a disease in which immune abnormalities are present both in the CNS and peripheral blood. Whether these changes are primary or secondary to the disease process is not known. We tested T-cell clones derived from activated lymphocytes in the blood and CSF of MS patients and controls for their capacity to regulate T-cell responses to alloantigens. A wide spectrum of regulatory functions were observed, ranging from marked enhancement to almost complete suppression. Clones from different patient populations and anatomic sites were equivalent in their regulatory functions with the net effect of clones in each compartment being suppression. However, certain clones from CSF and peripheral blood had the capacity to stimulate autologous T cells. Percentages of such clones in the peripheral blood of MS patients were significantly higher than in controls, while percentages in MS and other neurologic diseases (OND) CSF were equivalent. Our data suggest that (1) functional suppressor cells are not lost from the blood or CSF or MS and OND patients, (2) lymphocytes that have entered the CNS in patients with MS and other CNS diseases have equivalent regulatory functions, (3) MS may be an illness in which peripheral immunologic events are important in perpetuating the disease process, and (4) responses to autologous antigens may also play a role in this perpetuation.     

Retinol measurements and retinoid receptor gene expression in patients with multiple sclerosis

Treatment with interferon (IFN)-beta1a has been associated with decreased disease activity in patients with multiple sclerosis (MS). In several biological systems, type 1 IFNs and retinoids have been demonstrated to have synergistic effects. In these studies, we measured blood and cerebrospinal fluid (CSF) retinol levels and na?ve and memory T-helper cell subset percentages in samples from a group of patients with MS. We also examined retinol receptor expression in peripheral blood cells from MS patients with or without a history of prior treatment with IFN-beta1a. The mean plasma retinol level for untreated relapsing-remitting (RR) MS patients was lower than for patients with noninflammatory neurological disease. Among IFN-beta1a-treated RR patients, mean levels were slightly higher than for RR patients not on treatment Lower plasma retinol levels among the MS patents studied were associated with higher CSF retinol index measurements--a measure that was calculated to correct for nonspecific leakage of retinol from blood into CSF. Far the MS samples examined, there was a borderline statstically significant direct correlation between CSF retinol index measurements and CSF memory T-helper cell percentages. Examination of peripheral blood from untreated RR patents for retinoid receptor mRNA expression revealed the expression of the retinoic add receptor (RAR)-alpha, RAR-gamma, and retinoic X receptor (RXR)-alpha receptor subtypes. For RR patients on IFN-beta1a therapy, expression of the some RAR subtypes was noted as well as expression of RXR-beta and RXR-gamma. These studies suggest an association between plasma retinol levels and clincal disease activity in patents with MS and that treatment with IFN-beta1a may be associated with activation of specific retnoid receptor subtypes.     

Presence of detectable levels of soluble HLA-G molecules in CSF of relapsing-remitting multiple sclerosis: relationship with CSF soluble HLA-I and IL-10 concentrations and MRI findings

We have investigated the presence of non-classical soluble HLA-G molecules (sHLA-G) in cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients and the possible relationships between CSF levels of sHLA-G, classical soluble HLA-I (sHLA-I) molecules, IL-10 amounts and Magnetic Resonance Imaging (MRI) findings were evaluated. We studied by ELISA technique the sHLA-I, sHLA-G and IL-10 levels in CSF of 50 relapsing-remitting (RR) MS patients stratified according to clinical and MRI evidence of disease activity. Thirty-six patients with other inflammatory neurological disorders (OIND) and 41 with non-inflammatory neurological disorders (NIND) were used as controls. CSF mean levels were significantly higher in MS and OIND than in NIND for sHLA-I (p<0.001) and in MS than in controls for sHLA-G (p<0.001), with no differences among the various groups for IL-10 mean concentrations. An increase in CSF sHLA-I was found in MS patients with Gd-enhancing lesions (p<0.01), while sHLA-G and IL-10 were more represented in MS patients without lesional activity on MRI scans (p<0.02). In MRI-inactive MS, CSF IL-10 mean concentrations were significantly greater in patients with CSF-detectable levels of sHLA-G than in those without any evidence of CSF sHLA-G expression (p<0.05). Our findings suggest that CSF classical sHLA-I and non-classical sHLA-G levels may modulate MS activity as assessed by MRI acting in opposite directions. The association observed between sHLA-G and IL-10 when Gd-enhancing lesion resolved indicates a potential immunoregulatory role for IL-10 in the control of MS disease activity by shifting the sHLA-I/sHLA-G balance towards sHLA-G response.     

Lymphocyte subpopulations in the cerebrospinal fluid and peripheral blood in multiple sclerosis

Subpopulations of lymphocytes in the CSF and peripheral blood were studied in 30 patients with MS, 16 with other neurologcial diseases (OND) and 15 control subjects without any neurological abnormalities. In patients with relapse of MS, the absolute numbers of total lymphocytes, alpha-naphthyl acid esterase (ANAE) positive, E-rosette forming and bearing the "avid"FcIgG receptor lymphocytes were significantly increased in the CSF as compared with stable or slowly progressive MS patients, patients with other OND and control subjects.
The relative number of ANAE-positive cells was higher, and "avid"FcIgG receptor bearing cells lower in the CSF of all patients with MS than in the two other groups. The significance of the finding is unclear. The imbalance between lymphocyte subpopulations may reflect a primary defect in MS, or may be secondary, due to the presence of circulating immune complexes. In peripheral blood no substantial differences in lymphocyte behavior were observed between MS patients and other groups.