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 phenotypic profiles in the cerebrospinal fluid and peripheral blood of multiple sclerosis patients.

Thirty-nine patients with clinically definite multiple sclerosis (MS) entered the study. Of 28 subjects with a relapsing-remitting course, 19 were classified in acute relapse, 9 in remission; 11 patients had a progressive course without remissions. Furthermore, 6 subjects with inflammatory neurological disease (IND), and 10 with non-inflammatory and non-neoplastic neurological disease (NIND) were investigated. We simultaneously studied cerebrospinal fluid (CSF) and peripheral blood (PB) T-, B- and NK-cell subsets, as defined by following monoclonal antibodies: anti-CD3, -CD4, -CD8, -CD19, -CD16, -HLA-DR and -IL-2-R. We found a significant increase of CD4+ T-cells compared with controls in CSF, with respect to PB, of MS patients, particularly in acute relapse. An increase of HLA-DR+ cell percentages in the CSF than in the PB in all MS groups, especially in attacks of MS but also in remission, was also observed, with a positive correlation between CD4+ T-cell and DR+ cell percentages both in the CSF as well as in the PB of relapsing MS patients. These findings, together with the increase of IL-2-R+ cells in the PB, particularly in relapsing MS, give further support for the presence of a systemic T-cell activation in MS.     

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.     

Comparison of ventricular and lumbar cerebrospinal fluid T cells in non-inflammatory neurological disorder (NIND) patients

The aim of the present study was to define the cellular composition of ventricular, as compared with lumbar, cerebrospinal fluid (CSF) in patients with non-inflammatory neurological disorders (NIND). We addressed this issue by determining the cellular composition of lumbar CSF from patients with normal pressure hydrocephalus (NPH) who were undergoing lumbar CSF drainage during evaluation for shunting procedures, and evaluating ventricular CSF from a subset of these who underwent subsequent placement of ventriculoperitoneal shunts. We determined the cellular composition of lumbar CSF from 18 patients with NPH, and found that the leukocyte differentials, and relative proportions of CD4+ and CD8+ central memory (TCM), effector memory (TEM) and naive cell (TNaive) populations, were equivalent to those found previously in studies of CSF from patients with NIND. We further evaluated cells in the ventricular CSF of five patients who had previously undergone lumbar drainage. Leukocyte differential counts, as well as CD4+ and CD8+ TCM, TEM, and TNaive proportions, were equivalent in matched ventricular and lumbar CSF samples. These observations support the hypothesis that leukocytes enter the CSF in a selective fashion, at its site of formation in the choroid plexus. The results implicate CSF T cells in the immune surveillance of the central nervous system.     

髓鞘少突胶质细胞糖蛋白抗体在多发性硬化中的意义

目的探讨髓鞘少突胶质细胞糖蛋白(MOG)抗体与多发性硬化(MS)临床表现及复发缓解型MS(RRMS)复发的关系。方法采用酶联免疫吸附法(ELISA)对60例MS、23例其他炎性神经疾病(OIND)、29例非炎性神经疾病(NIND)以及50例神经系统正常的对照(NC)患者血清及脑脊液(CSF)MOG抗体进行检测。结果MS患者CSFMOG抗体阳性率为28.3%(17/60),明显高于NC组[2%(1/50)]和NIND组[0%(0/29)],但与OIND组[21.7%(5/23)]比较差异无统计学意义。各组血清MOG抗体均为阴性。抗体阳性率急性活动期为31.8%(14/44),与稳定期[18.75%(3/16)]比较差异无统计学意义。CSFMOG抗体阳性的RRMS患者复发时间早于阴性患者,且其第1、2年复发率均高于阴性患者。结论在部分MS患者中枢神经系统内存在异常的MOG特异性B细胞免疫应答,且CSFMOG抗体对RRMS的复发有一定预测作用。     

Parkinson's disease and immunological abnormalities: increase of HLA-DR expression on monocytes in cerebrospinal fluid and of CD45RO+ T cells in peripheral blood

The etiology of Parkinson's disease is mainly unknown. Immune abnormalities have been described, but the cause of such abnormalities has not been resolved. We examined by two-colour flow cytometry HLA-DR antigen expression on monocytes from cerebrospinal fluid (CSF) and blood and, moreover, lymphocyte subpopulations (CD4⁺ CD45RO⁺, CD4⁺ CD45RA⁺, CD8⁺ CD11b^+high) in peripheral blood from patients with Parkinson's disease compared with age-matched patients with other neurological diseases (OND) and tension headache. We found higher HLA-DR expression on CSF monocytes compared with blood monocytes. This difference was restricted to Parkinson's disease patients. T helper cell analysis revealed a decreased percentage of CD45RA⁺"naive" and an increased percentage of CD45RO⁺"memory" T cell subset from CD4⁺ T cells in peripheral blood of patients with Parkinson's disease compared with patients with tension headache. The proportions of CD8⁺ CD11b^+high"suppressor" T cells remained unchanged, among the three patient groups compared. A selective loss of CD4⁺ CD45RA⁺ cells, previously observed in diseases like multiple sclerosis and Down's syndrome as compared with healthy controls suggests a common immunological abnormality in neurological disorders.     

Increased expression of CXCR3 and CCR5 on memory CD4+ T-cells migrating into the cerebrospinal fluid of patients with neuroborreliosis: the role of CXCL10 and CXCL11

The aim of this study was to evaluate the contribution of chemokine receptor CXCR3 and the corresponding ligands CXCL10 and CXCL11 to the recruitment of peripheral blood (PB) memory CD4+ T-cells into the cerebrospinal fluid (CSF) of patients with acute neuroborreliosis. Percentages of memory CD45RO+CD4+ T-cells expressing CXCR3 and CCR5 were significantly enriched in the CSF compared to the PB. Concentrations of CXCL10 and CXCL11 in the CSF of neuroborreliosis patients were significantly higher compared with the corresponding serum samples. Our results suggest that CXCL10 and CXCL11 create a chemokine gradient between the CSF and serum and recruite CXCR3-expressing memory CD4+ T-cells into the CSF of neuroborreliosis patients and that CCR5 also plays a role in this process.     

Cellular immunoregulatory mechanisms in the central nervous system: characterization of noninflammatory and inflammatory cerebrospinal fluid lymphocytes

Dual-label flow cytometric analysis of cerebrospinal fluid (CSF) and blood lymphocytes with combinations of monoclonal antibodies such as CD4 plus CD45R or Leu8, and CD8 plus CD11b was performed in 37 patients with noninflammatory neurological diseases (NINDs) to clarify the differences in cellular immunoregulatory mechanisms present in the central nervous system (CNS) and in the systemic circulation. In the CSF of patients with NINDs, the paucity of CD4+CD45R+ and CD8+CD11b+ cells was striking, whereas the same subsets accounted for substantial proportions in the blood. CD4+CD45R- and CD4+Leu8- cells as well as CD8+CD11b- cells increased in the CSF when compared with those in the blood. Seven patients with active multiple sclerosis (MS) and 10 patients with other inflammatory diseases in the CNS (CNS-infl) were also studied. Patients with active MS were characterized by a consistent increase in percentage of CD4+CD45R- cells in the CSF, whereas an increase of CD4- CD45R+ cells in the CSF was a feature of the patients with CNS-infl, when compared with patients with NINDs. These findings indicate that the CNS is routinely surveyed by particular subsets of lymphocytes different from those in the blood, and cellular immune reaction in the CNS varies according to the types of CNS inflammatory conditions.     

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.     

Preliminary studies of T lymphocyte subsets in patients with neurologic diseases

With immunofluorescence assay a preliminary study of T-cell subsets in the peripheral blood (PB) and cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS), viral encephalitis, Guillain-Barré syndrome (GBS), tuberculous meningitis, and other neurological diseases (OND) was carried out. The results revealed that the T4+ percentage and T4+/T8+ ratio of PB as well as CSF increased, T8+ decreased in cases of MS. The changes of CSF T subsets in cases of (GBS) were similar to those in cases of MS. There was no difference in PB T subsets between GBS and the normal controls. The T4+/T8+ ratio of PB and CSF reduced in cases of tuberculous meningitis, and such changes were also found in PB of viral encephalitis patients. Therefore, it was demonstrated that the immunoregulatory dysfunctions were present in these neurological disorders, and the characters of the changes were different between infectious and autoimmune diseases.     

CSF lymphocyte subsets in aseptic meningitis: dual-labelling analysis with flow cytometry

In order to characterize the CSF (cerebrospinal fluid) lymphocytes in CNS (central nervous system) inflammation, we examined paired samples of CSF and PB (peripheral blood) of 19 patients with acute aseptic meningitis, performing the dual labelling method on flow cytometry. Significantly higher percentages of CD3+ (T cell), CD4+ (helper-inducer), Leu3a+ Leu15- (cytotoxic-T) and HLA-DR+ CD3+ (activated-T) cells were identified in the CSF than in the PB of these patients. We observed significantly lower percentages of CD19+ (B cell), Leu2a+ Leu18+ (suppressor-inducer) and HLA-DR+ CD3- cells in the CSF than in the PB of these patients. Relative increases in helper-inducer, cytotoxic-T and activated-T cells in the CSF of aseptic meningitis are supposed to represent an active inflammatory process. However, whether these changes are specific or pathognomonic to any disease(s) remains to be solved.     

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.     

VEGF and mRNA VEGF expression in CSF from Behçet's disease with neurological involvement

Vascular endothelial growth factor (VEGF) stimulates angiogenesis, but is also pro-inflammatory and plays an important role in the development of neurological disease, where it can have both attenuating and exacerbating effects. Several studies have indicated that VEGF-A (VEGF) may play a role in the pathogenesis of neurological inflammatory diseases.To assess the role of VEGF in patients with Behçet's disease with neurological involvement, VEGF was measured in the cerebrospinal fluid (CSF) of 32 patients compared to a group of 12 patients with noninflammatory neurological diseases (NIND) and 14 patients with multiple sclerosis (MS). We have also studied the expression of mRNA-VEGF (VEGF-A) in CSF and in peripheral blood mononuclear cells.The mean VEGF_CSF was significantly increased in neuro-BD and MS patients compared to NIND patients. There was an association between neuro-BD-VEGF_CSF, and leukocyte count. A significant correlation was also observed between neuro-BD-VEGF_CSF and CSF_%CD4 cells. As a measure of the integrity of the blood-brain barrier Q_albumin was found correlated to VEGF_CSF. VEGF mRNA was significantly increased in neuro-BD patients compared to NIND patients.These results indicate that, VEGF may be associated with the increased percentages of CD4 cell subpopulation. The role of VEGF is within the inflammatory cascade in the mediation of blood-brain barrier disruption and not specific to Behçet's.     

Expression of CCR7 and CD45RA in CD4+ and CD8+ subsets in cerebrospinal fluid of 134 patients with inflammatory and non-inflammatory neurological diseases

We investigated CD45RA and CCR7 expression in CD4+ and CD8+ subsets of cerebrospinal fluid (CSF) lymphocytes, both immediately ex vivo and after stimulation, from 134 patients with a variety of inflammatory and non-inflammatory neurological diseases. Most inflammatory diseases had a higher CD4+:CD8+ ratio and higher percentage of effector memory T cells (T(EM)) than non-inflammatory controls, excluding active infection. Moreover, we found that patients with highly elevated cell counts in the CSF tended to have a lower percentage of central memory T cells (T(CM)) than patients with low or absent pleocytosis, with a concomitant increase in T(EM). We also found that samples with elevated IgG index or presence of oligoclonal bands had a significantly higher CD4+:CD8+ ratio than normal samples, consistent with increased CD4+ help for intrathecal IgG synthesis by B cells.     

Increase of CD4+TNF{alpha}+IL-2-T cells in cerebrospinal fluid of multiple sclerosis patients

Intracellular production of TNFalpha and IL-2 after stimulation with phorbol myristate/ionomycin was flowcytometrically measured in CD4(+) T cells from peripheral blood (PB) and cerebrospinal fluid (CSF) of 29 patients with multiple sclerosis (MS), and 16 with other inflammatory and 41 with other non-inflammatory neurological diseases. In CSF, the percentages of CD4(+)TNFalpha(+)IL-2(-)T cells were significantly higher in patients with MS than either of the controls, whereas no difference was found in CD4(+)TNFalpha(+)IL-2(+)T or CD4(+)TNFalpha(-)IL-2(+)T cells. The increase was more pronounced at relapse than in remission. No significant change was detected in PB. These findings suggested that CD4(+)TNFalpha(+)IL-2(-)T cells are intrathecally upregulated in MS.     

Elevated cerebrospinal fluid CD4/CD8 T cell ratio in myasthenia gravis

The proportions of CD2+, CD4+ and CD8+ lymphocytes were determined with the 3-layer indirect immunoperoxidase technique in the cerebrospinal fluid (CSF) of 31 patients with myasthenia gravis (MG) and 21 control subjects without autoimmune or central nervous system (CNS) diseases. None of the MG patients were using immunosuppressive drugs and all were thymectomized shortly after CSF sampling. Analysis of the reference population showed that the percentage of CD4+ lymphocytes and accordingly the CD4+/CD8+ T cell ratio is normally higher in CSF than in peripheral blood (PB). Compared to the controls, the mean percentage of CD4+ lymphocytes and the mean CD4+/CD8+ ratio in CSF were significantly higher in MG patients. In addition, the CD4+/CD8+ ratio was elevated in the CSF of 15 MG patients (48%) as a result of an elevation in the proportion of CD4+ and/or a decrease in CD8+ T cells. Among MG subjects the mean proportion of CD4+ lymphocytes was higher in the CSF of patients with also an elevated number of enlarged stimulated lymphoid cells in their CSF, which implies that these lymphocytes are often of the CD4+ phenotype. The percentage of CD4+ T cells in CSF was significantly higher in MG patients with a hyperplastic thymus or a thymoma than in those with an involuted thymus. Neither in MG patients nor in the reference population could an association be observed between CSF and PB lymphocyte subsets. In the controls this suggests that immunologic events of the CNS are normally not directly reflected in PB.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Assessment of HIV-intrathecal humoral immune response in AIDS-related neurological disorders

Intrathecal synthesis of IgG directed to HIV antigens was investigated by antibody specific index (ASI), affinity-mediated immunoblot (AMI) and Western blot (WB) assay in a group of 88 AIDS patients of which 28 with HIV-associated neurological disorders (HAND), 13 without associated neurological disorders (WAND) and 47 with non-HIV-associated neurological disorders (non-HAND). CD4+ count was above 50 cells/mm3 (CD4+>50) in 30 and below 50/mm3 (CD4+<50) in 58 patients, respectively. A significantly higher frequency for CSF complete anti-gag profile (p<0.001), and for HIV-specific oligoclonal patterns ("mixed" pattern=p<0.01) was observed in HAND as compared to patterns from the other clinical groups. A decrease in complete anti-env, anti-pol and anti-gag reactivity was present in CSF of patients with CD4+<50 as compared to those with CD4+>50. Our findings suggest that AIDS appears to be characterized by an anti-HIV intrathecal humoral immune response which is principally directed to env products with a prevalence of oligoclonal patterns and CSF complete anti-gag profile in HIV-associated neurological involvement.     

Monocyte phenotype in blood and cerebrospinal fluid: Compartment-specific pattern is unrelated to neurological disease

An immunocytochemical study of surface and cytoplasmic markers of the monocyte/macrophage lineage was undertaken on peripheral blood (PB) and cerebrospinal fluid (CSF) cells in patients with multiple sclerosis (MS) or other neurological diseases (OND) and in three healthy controls. Size and granularity were assessed by means of cytofluorimetric analysis. Fewer monocytes were positive for complement receptors 1 and 3, myeloperoxidase, KiM6, IOM-3 and CD14 in CSF than in PB. Only DR-positive monocytes were present with the same frequency in the two compartments. There was no difference in the monocyte phenotype between patients and healthy controls. Furthermore, there was no difference between patients with MS or OND. Cytofluorimetric analysis showed that in CSF, regardless of clinical status, few cells displayed the physical features of blood monocytes. A population of granular and large granular cells that was not normally present among PB mononuclear cells was recognized in the CSF. A large proportion of these cells was able to adhere to plastic and - when sorted - displayed the morphology and surface markers of monocyte lineage. The results of our study demonstrate compartment-specific differences in the monocyte phenotype between CSF and PB but suggest that these changes may be unrelated to the investigated neurological diseases.     

Leukocyte types in cerebrospinal fluid and peripheral blood enumerated immunoenzymatically in aseptic meningitis and the Guillain-Barré syndrome

To study celltype distribution simultaneously in peripheral blood (PB) and cerebrospinal fluid (CSF) from patients with aseptic meningitis (AM) (n = 14) and Guillain-Barré syndrome (GBS) (n = 9) we used an immunoenzymatic method that enabled the use of several monoclonal antibodies, also in CSF samples with normal cellcounts. In both patient groups a different cell-distribution in CSF compared to PB was found with regard to pan T cells (CD5+/anti-Leu1+), T cell subpopulations (CD4+/anti-Leu3+, CD8+/anti-Leu2+), B cells (OKB2+, OKB7+), monocytes/macrophages (CD11+/OKM1+) and HLA/DR expressing cells, whereas the distribution of HLA/DC+ cells was similar in CSF and PB. Thus, the CSF cell distribution does not reflect the distribution in PB. The proportion of T cells was higher and the proportion of B-cells was lower in CSF than in PB in both patient groups, which is a finding similar to that in patients with multiple sclerosis. The OKT9 marker, labelling proliferating cells expressing the transferrin receptor, was not useful as marker of local proliferation.