The enigma of oligoclonal immunoglobulin G in cerebrospinal fluid from multiple sclerosis patients

The cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) contains immunoglobulin G (IgG) of restricted heterogeneity (oligoclonal IgG). Philip Paterson and Caroline Whitacre here review the core evidence that IgG in MS patients and normal subjects is synthezised intracerebrally and that most of it does- not have demonstrable antigenic specificity. They advance the view that the enigmatic oligoclonal IgG may be a product of B cells which are non-specifically activated by mitogenic stimuli originating within injured CNS tissue as a consequence of the MS process.     

Role of isoelectric focusing of cerebrospinal fluid immunoglobulin G in the early biological assessment of multiple sclerosis

A retrospective study was carried out at the Neurological and Neurosurgical Hospital of Lyon in order to evaluate the interest of detecting IgG oligoclonal bands by isoelectric focusing with IgG immunorevelation for the early diagnosis of multiple sclerosis (MS). Patients have been grouped according to their disorders: multiple sclerosis (281 cases), definite (182 cases) and possible (99 cases), others inflammatory neurological diseases (63 cases), various non-inflammatory neurological disorders (180 cases) and indefined neurological disorders (664 cases). The following examinations were performed: CSF cell count and cytology after concentration and cytocentrifugation, CSF and serum determination of albumin and IgG with CSF/serum ratios, agarose gel electrophoresis and isoelectric focusing of oligoclonal IgG. The technique used was isoelectric focusing using agarose gel, transfer onto PVDF membrane and then IgG immunorevelation with biotinylated anti-human IgG antibodies. Isoelectric focusing with IgG immunorevelation is the most sensitive (94%) and specific (96%) technique. Isoelectric focusing with immune detection can be recommended as the most efficient test (gold standard) for the detection of chronic CNS inflammation.     

Modulation of dendritic cell development by immunoglobulin G in control subjects and multiple sclerosis patients

Intravenous immunoglobulin (IVIg) preparations are reportedly effective in inhibiting the relapse of multiple sclerosis (MS), but few reports have investigated the effect of IVIg on dendritic cells (DCs), which are thought to be involved in such relapses. In the system that uses monokines to differentiate DCs from peripheral blood monocytes (Mo-DCs), we investigated the effect of immunoglobulin G (IgG) on these antigen-presenting cells. Using monocytes derived from healthy volunteers, IgG partially inhibited the expression of CD1a, a marker of immature DCs (imDCs), and CD40 and CD80, which are markers associated with T cell activation. In contrast, IgG enhanced the expression of CD83, a marker of mature DCs (mDCs). Furthermore, IgG markedly inhibited the expression of CD49d [very late activation antigen (VLA)-4 alpha4-integrin], the adhesion molecule required for mDCs to cross the blood-brain barrier. We obtained similar results on all the aforementioned cell surface molecules investigated in both healthy controls and MS patients. In addition, IgG treatment of cells from both healthy controls and MS patients inhibited the production of interleukin (IL)-12, a cytokine associated with mDC differentiation, but did not inhibit the production of IL-10. These results suggested the possibility that IgG treatment, apart from its known ability to regulate inflammation, may help to prevent relapses of MS by controlling DC maturation, consequently inhibiting invasion of immune cells into the central nervous system and affecting the cytokine profile.     

Fungal infection in cerebrospinal fluid from some patients with multiple sclerosis

Multiple sclerosis (MS) is the prototypical inflammatory disease of the central nervous system and spinal cord, leading to axonal demyelination of neurons. Recently, we have found a correlation between fungal infection and MS in peripheral blood of patients. The present work provides evidence of fungal infection in the cerebrospinal fluid (CSF) of some MS patients. Thus, fungal antigens can be demonstrated in CSF, as well as antibodies reacting against several Candida species. Comparison was made between CSF and blood serum for the presence of fungal antigens (proteins) and antibodies against different Candida spp. Analyses of both CSF and serum are complementary and serve to better evaluate for the presence of disseminated fungal infection. In addition, PCR analyses indicate the presence of DNA from different fungal species in CSF, depending on the patient analyzed. Overall, these findings support the notion that fungal infection can be demonstrated in CSF from some MS patients. This may constitute a risk factor in this disease and could also help in understanding the pathogenesis of MS.     

Natural killer cell subsets in cerebrospinal fluid of patients with multiple sclerosis

Changes in blood natural killer (NK) cells, important players of the immune innate system, have been described in multiple sclerosis (MS). We studied percentages and total cell counts of different effector and regulatory NK cells in cerebrospinal fluid (CSF) of MS patients and other neurological diseases to gain clearer knowledge of the role of these cells in neuroinflammation. NK cell subsets were assessed by flow cytometry in CSF of 85 consecutive MS patients (33 with active disease and 52 with stable MS), 16 with other inflammatory diseases of the central nervous system (IND) and 17 with non-inflammatory neurological diseases (NIND). MS patients showed a decrease in percentages of different CSF NK subpopulations compared to the NIND group. However, absolute cell counts showed a significant increase of all NK subsets in MS and IND patients, revealing that the decrease in percentages does not reflect a real reduction of these immune cells. Remarkably, MS patients showed a significant increase of regulatory/effector (CD56^bright/CD56^dim) NK ratio compared to IND and NIND groups. In addition, MS activity associated with an expansion of NK T cells. These data show that NK cell subsets do not increase uniformly in all inflammatory neurological disease and suggest strongly that regulatory CD56^bright and NK T cells may arise in CSF of MS patients as an attempt to counteract the CNS immune activation characteristic of the disease.     

Detection of viral DNA sequences in the cerebrospinal fluid of patients with multiple sclerosis

The role of viruses in the pathogenesis of multiple sclerosis (MS) is a subject of heated debate. The presence of six different neurotropic viruses was sought, including JC virus (JCV), varicella zoster virus (VZV), human herpesvirus 6 (HHV‐6), and Epstein‐Barr virus (EBV), in cerebrospinal fluid (CSF) samples collected from 51 patients with MS and 30 patients with other neurological diseases. Cell‐free or cell‐associated viral DNA in CSF samples was detected by real‐time PCR, and viral loads were determined. Magnetic resonance imaging (MRI) examinations were also performed to look for active lesions. Cell‐associated JCV DNA was detected in 3 of the 51 patients with MS and in 2 of the 30 patients with other neurological disease. Cell‐free JCV DNA was detected in one additional patient with MS. Cell‐free VZV DNA was detected in one patient without MS, cell‐free HHV‐6 was detected in one patient with MS, and cell‐free EBV was detected in one patient with MS. All other study patients had no detectable viral DNA in CSF samples and no double infections were found. The small percentage of patients with detectable viral DNA in CSF samples was comparable between patients with MS and those with other neurological disease, and presence of viral DNA was not a predictor of brain lesions. Additional observations suggest that cell trafficking from the periphery, rather than leakage through the blood–brain barrier, results in the transport of viruses to the CNS, where local immunosurveillance can control viral replication in immunocompetent individuals. J. Med. Virol. 82:1051–1057, 2010. © 2010 Wiley‐Liss, Inc.     

Multiple sclerosis: disturbed kappa: lambda chain ratio of immunoglobulin G in cerebrospinal fluid

Abnormal kappa: lambda chain ratios were found in cerebrospinal fluid from some patients with multiple sclerosis, but not in a variety of other neurological disorders. The increase in ratio was a property of immunoglobulin G.     

Association of CSF IgG concentration and immunoglobulin allotype in multiple sclerosis and optic neuritis

The cerebrospinal fluid (CSF) and serum from 64 patients with multiple sclerosis (MS) and 47 patients with monosymptomatic optic neuritis (ON) were analyzed for the distribution of allotypic determinants on IgG and compared to similar samples from 51 patients with other neurological diseases (OND) as well as to serum samples from 97 healthy controls. The results indicate a significantly increased frequency of the haplotypes Gm a;g and Gm a,x;g among MS patients (P = 0.024) with an associated increase in relative risk for MS among individuals with the Gm a,(x);g haplotypes compared to those individuals without them (P = 0.014). Among MS patients, those with the Gm a,(x);g haplotypes had significantly higher CSF levels of IgG than those without (P = 0.016); levels of serum IgG did not covary with Gm haplotype. Two-way analysis of variance indicates that familial cases have significantly higher levels of CSF IgG than nonfamilial cases (P less than 0.001) and that familial cases with the Gm a,(x);g haplotypes have the highest CSF IgG levels (P less than 0.005). There was no correlation between Gm haplotype and CSF or serum IgG levels in patients with ON or OND. The allotype effects were independent of age at onset and duration of disease. In all patients, regardless of disease classification, the phenotypes found in serum samples were identical to those found in CSF samples. The data presented support the hypothesis that the etiology of MS has as one of its parameters an immunoregulatory/immunogenetic factor. The successful analysis of these various parameters will provide useful information not only about MS but also about general principles of human immune responsiveness.     

Maintenance of B lymphocyte-related clones in the cerebrospinal fluid of multiple sclerosis patients

A longitudinal study of Ig V gene segments utilized by B cells from the cerebrospinal fluid (CSF) of two patients with multiple sclerosis (MS) was carried out using RT-PCR methodologies. One patient with a relapsing-remitting (RR)-MS was investigated at onset and at relapse, 1 year later. A patient with secondary-progressive (SP)-MS was tested 9 and 13 years after disease onset. Sequence analyses of V(H)DJ(H) segments bearing V(H)3 and V(H)4 that were obtained from Cgamma cDNA genes demonstrated a substantial proportion of shared clones in the samples taken at different times; these clones were identical or closely related, i.e. had the same third complementary determining region (CDR) of the H chain variable region gene (HCDR3) with different mutations in the V(H) segment. Collectively, these data demonstrate that in MS patients there is a strong selective pressure, which could be exerted by antigen (or autoantigen) stimulation, for the maintenance and partial diversification of certain V(H)DJ(H) Cgamma sequences.     

Tetanus toxoid reactive T lymphocytes in the cerebrospinal fluid of multiple sclerosis patients

Cerebrospinal fluid (CSF) lymphocytes of 6 multiple sclerosis (MS) patients were cultured with tetanus toxoid (TT) and irradiated autologous antigen presenting cells (APC) followed by propagation of the responding T-cells in interleukin-2 containing medium. TT-reactive cell lines were recovered from 4 of the 6 CSF samples, even though the patients had not been TT booster immunized in recent years. These findings suggest an active circulation of antigen reactive lymphocytes from the systemic immune compartment(s) into the CSF even without recent activation by booster immunization. Since immune reactions to TT are very unlikely to be pathogenic in MS, these findings also indicate that presence of CSF lymphocytes reactive to a particular antigen does not necessarily imply a causal role.     

Increased prevalence of varicella zoster virus DNA in cerebrospinal fluid from patients with multiple sclerosis

In order to investigate the possible involvement of viruses in Multiple Sclerosis (MS), the study evaluated the presence of viral genomic sequences in cerebrospinal fluid (CSF), as markers of viral replication within the central nervous system (CNS). A total of 85 CSF samples were collected from 38 MS patients, 28 patients with other neurological diseases and 19 subjects without neurological diseases. Using nested-PCR, the investigation focused on the presence of human herpes virus DNA, including herpes simplex virus 1 (HSV-1) and 2 (HSV-2), the Epstein-Barr virus (EBV), varicella zoster virus (VZV), human cytomegalovirus (HCMV), human herpes virus 6 (HHV-6) and JC virus (JCV). All the CSF samples from the individuals without neurological diseases were negative for viral DNA. Genomic sequences of HSV-1, HCMV, EBV, HHV6, and JCV were found in patients with MS and other neurological diseases without significant differences between the two groups. VZV DNA was detected more frequently (P < 0.05) in the MS group (31.6%), particularly among the relapsing-remitting MS patients (43.5%), compared with patients with other neurological diseases (10.7%). In addition, the results indicated that JCV and HHV-6 were replicating actively in the CNS of a small, but significant number of patients with MS and other neurological diseases. Most importantly, the study revealed a high frequency of VZV DNA in the CSF of patients with MS, suggesting a possible role of this virus in the pathogenesis of MS.     

Analysis of immunoglobulin G in multiple sclerosis brain: quantitative and isoelectric focusing studies.

Immunoglobulin G (IgG) in soluble and particulate fractions of discrete tissue samples from multiple sclerosis (MS) brain was analysed. Supernatant IgG/albumin rations and particulate-bound IgG levels were highest in samples dissected from MS plaques and adjacent white matter. Acid extracts of particulate fractions from the equivalent of 1 g of MS plaque tissue contained up to 15 micrograms IgG, an order of magnitude less than the amount in extracts from a subacute sclerosing panencephatitis (SSPE) brain, but 20 times more than those from control brain. By contrast, supernatant fractions from SSPE brain and some MS plaques contained comparable amounts (100-200 micrograms/g tissue) of IgG, which were 10 times greater than those from control brain. Samples were subjected to isoelectric focusing (IEF), and IgG was visualized by immunoperoxidase staining. The IEF patterns of IgG from control samples were diffuse but samples from demyelinated MS tissue displayed distinct oligoclonal bands of IgG. A number of common IgG bands were apparent in extracts of supernatant and particulate fractions from the same plaque. The IEF spectra of plaque samples from three MS brains were different. Furthermore, quantitative variations in certain IgG bands were observed in different plaques from the same brain.     

Concentrations of immunoglobulin free light chains in cerebrospinal fluid predict increased level of brain atrophy in multiple sclerosis

Recent studies showed that B cells play a major role in the pathogenesis of neurodegeneration in multiple sclerosis (MS). In this study, we aimed to determine the possible link between immunoglobulin free light chains (FLC) and brain atrophy in patients with MS. Ninety-two patients (32 males and 60 females) with MS were included. Kappa and lambda FLC concentrations in serum and cerebrospinal fluid (CSF) samples of MS patients were measured using ELISA assay. FLC quotients (Q-k and Q-λ, respectively) were calculated. In a cross-sectional group (n?=?92), the MRI data were acquired within 6 months from the date of the lumbar puncture. Twenty patients from this cohort performed a follow-up MRI after 1 year of observation. Brain volumes were calculated with SIENAX and the brain atrophy (percentage brain volume change (PBVC)) was assessed with SIENA. Spearman’s test was performed to assess correlations. We have shown statistically significant correlation of Expanded Disability Status Scale (EDSS) level with normalized brain volume (NBV, r?=???0.2721, p?=?0.0062), white matter volume (WMV, r?=???0.2425, p?=?0.015), and gray matter volume (GMV, r?=???0.216, p?=?0.0309). Multiple Sclerosis Severity Score (MSSS) score correlated with NBV (r?=???0.2521, p?=?0.0352) and WMV (r?=???0.315, p?=?0.0079). Neither EDSS, nor MSSS scores correlated with the age of patients and relapse rate during the first year and 5 years. In our study, we found statistically significant correlations of k-FLC in the CSF with NBV (r?=???0.311, p?=?0.003) and with GMV (r?=???0.213, p?=?0.0423). Q-k correlated only with NBV (r?=???0.340, p?=?0.006) and Q-λ were negatively correlated with WMV (r?=???0.366, p?=?0.003). We did not find correlations of k-FLC in CSF, λ-FLC in CSF, Q-k, and Q-λ with duration of MS course, EDSS, MSSS, number of relapses during the first year, and during the first 5 years of disease. Additionally, we subdivided the study population in accordance with level of k-FLC CSF, Q-k, and Q-λ on the 25th and 75th percentile subgroups (25-k-FLC_CSF/75-k-FLC_CSF; 25-λ-FLC_CSF/75-λ-FLC_CSF; 25-Q-k/75-Q-k; 25-Q-λ/75-Q-λ). We found statistically significant difference of NBV and GMV between 25-k-FLC_CSF and 75-k-FLC_CSF subgroups (p?=?0.0047, p?=?0.0297 respectively), NBV between 25-Q-k and 75-Q-k subgroups (p?=?0.038), and NBV and WMV between 25-Q-λ and 75-Q-λ subgroups (p?=?0.0446, p?=?0.0026 respectively). PBVC in the prospective group showed negative correlation with kappa FLC in the CSF (r?=???0.4853, p?=?0.0301) and Q-k (r?=???0.6132, p?=?0.0224), but not with other clinical, epidemiological data. In this study, we showed a strong negative correlation of k-FLC, Q-k, and Q-λ with brain atrophy in MS patients. Additionally, patients with high concentration of FLC had lower brain volumes. We did not find correlations of FLC with the relapse rate, age of patients, and MS time course. In the prospective group, the rate of atrophy was correlated with k-FLC and Q-k. We suggest that level of intrathecal production of FLC can be a good prognostic biomarker for MS.     

In vivo activated T lymphocytes in the peripheral blood and cerebrospinal fluid of patients with multiple sclerosis

We found an increase in peripheral-blood lymphocytes bearing the T-cell-specific activation antigen Ta1 in 20 of 35 patients with progressive multiple sclerosis, 4 of 18 patients with stable or improving multiple sclerosis, 1 of 17 patients with other neurologic diseases, and 1 of 14 normal controls (P less than 0.0002, Fisher's exact test). No increases in two other markers of T-cell activation, T113 and the interleukin-2 receptor, were found. In the cerebrospinal fluid, patients with progressive multiple sclerosis (pleocytosis, 3.9 +/- 1.6 cells per cubic millimeter) had 42 +/- 3.0 per cent Ta1+ cells. In contrast, patients with other inflammatory central nervous system diseases (36 +/- 13 cells per cubic millimeter) had 9.6 +/- 1.8 per cent Ta1+ cells (P less than 0.01). In patients with other neurologic diseases without inflammation (0.7 +/- 0.16 cells per cubic millimeter), the percentage of Ta1+ cells was equivalent to that in patients with multiple sclerosis (39 +/- 5.4 per cent), although the absolute number was lower. There was a positive correlation between the presence of Ta1+ cells in the spinal fluid and blood of patients with other neurologic diseases, but not patients with multiple sclerosis. Less than 1 per cent of lymphocytes from the spinal fluid of patients with multiple sclerosis expressed interleukin-2 receptors, as compared with 9.8 per cent of cells from subjects with other inflammatory neurologic diseases (P less than 0.01). These results suggest that the T cells in the spinal fluid of patients with multiple sclerosis may be activated by a different mechanism or in a different temporal sequence from that in patients with other nervous system diseases. Furthermore, the increase in Ta1+ cells in the peripheral blood of patients with multiple sclerosis demonstrates systemic immune activation in the disease; monitoring such cells may provide an objective measure of abnormal immunologic activity.     

High interindividual variability in the CD4/CD8 T cell ratio and natalizumab concentration levels in the cerebrospinal fluid of patients with multiple sclerosis

Strongly decreased leucocyte counts and a reduced CD4/CD8 T cell ratio in the cerebrospinal fluid (CSF) of natalizumab (NZB)‐treated multiple sclerosis (MS) patients may have implications on central nervous (CNS) immune surveillance. With regard to NZB‐associated progressive multi‐focal leucoencephalopathy, we aimed at delineating a relationship between free NZB, cell‐bound NZB, adhesion molecule (AM) expression and the treatment‐associated shift in the CSF T cell ratio. Peripheral blood (PB) and CSF T cells from 15 NZB‐treated MS patients, and CSF T cells from 10 patients with non‐inflammatory neurological diseases and five newly diagnosed MS patients were studied. Intercellular adhesion molecule‐1 (ICAM‐1), leucocyte function antigen‐1 (LFA‐1), very late activation antigen‐4 (VLA‐4), NZB saturation levels, and T cell ratios were analysed by flow cytometry. NZB concentrations were measured by enzyme‐linked immunosorbent assay (ELISA). Lower NZB saturation levels (P < 0·02) and a higher surface expression of ICAM‐1 and LFA‐1 (P < 0·001) were observed on CSF CD8 T cells. CSF T cell ratios (0·3–2·1) and NZB concentrations (0·01–0·42 µg/ml) showed a pronounced interindividual variance. A correlation between free NZB, cell‐bound NZB or AM expression levels and the CSF T cell ratio was not found. Extremely low NZB concentrations and a normalized CSF T cell ratio were observed in one case. The differential NZB saturation and AM expression of CSF CD8 T cells may contribute to their relative enrichment in the CSF. The reduced CSF T cell ratio appeared sensitive to steady‐state NZB levels, as normalization occurred quickly. The latter may be important concerning a fast reconstitution of CNS immune surveillance.     

Electrophoretic distribution of Kappa and Lambda immunoglobulin light chain determinants in serum and cerebrospinal fluid in multiple sclerosis

The electrophoretic distribution of Kappa and Lambda light chain antigenic determinants were studied in cerebrospinal fluid (CSF) and serum from cases with abnormal Immunoglobulin G patterns on agar gel electrophoresis of CSF. Electrophoretically homogeneous immunoglobulin fractions containing an excess of either Kappa or Lambda determinants were found in CSF but not in serum. The findings indicate the production of monoclonal immunoglobulins within the central nervous system in multiple sclerosis.     

Decreased level of kynurenic acid in cerebrospinal fluid of relapsing-onset multiple sclerosis patients

The present study was undertaken to measure cerebrospinal fluid (CSF) levels of kynurenic acid (KYNA) in patients with relapsing-onset multiple sclerosis (MS) during remission or not progressing for at least 2 months. In these patients the levels of CSF KYNA were found to be significantly lower compared with subjects with non-inflammatory neurological diseases, as well as those with inflammatory disease (median (interquartile range): 0.41 (0.3-0.5) pmol/ml, n=26 vs. 0.67 (0.5-1.1), n=23, P<0.01 and 1.7 (1.5-2.6), n=16, P<0.001, respectively). These results provide further evidence of the alterations in the kynurenine pathway during remitting-onset MS.     

B cells expressing CD5 are increased in cerebrospinal fluid of patients with multiple sclerosis.

By two-colour flow cytometric analysis, we found increased numbers of B cells co-expressing the pan-T cell marker CD5 and the B cell marker CD19 in cerebrospinal fluid (CSF) of 21 patients with multiple sclerosis (MS), compared with 17 control subjects with muscular tension headache. Only one patient with MS, but nine controls lacked CD5+ B cells in CSF. This difference was not observed in peripheral blood. Numbers of CD5+19+ B cells were increased in CSF compared with blood in MS, but not in the controls. In both groups, CD5+19+ B cells were not restricted to small resting lymphocytes, but were also found among larger-sized lymphocytes. The relative density of CD5 molecules and of CD19 molecules was lower in CD5+19+ than in CD5-19+ B cells and CD5+19- T cells. CD5+ B cells are assumed to be responsible for autoantibody production, and our results suggest a pathogenetic role of such cells, predominantly within the central nervous system, in MS.