CSF Of Multiple Sclerosis Patients Research Articles

Identification of soluble TREM-2 in the cerebrospinal fluid and its association with multiple sclerosis and CNS inflammation

Triggering receptor expressed on myeloid cells 2 (TREM-2) is a membrane-bound receptor expressed by microglia and macrophages. Engagement of TREM-2 on these cells has been reported to reduce inflammatory responses and, in microglial cells, to promote phagocytosis. TREM-2 function is critical within the CNS, as its genetic deficiency in humans causes neurodegeneration with myelin and axonal loss. Blockade of TREM-2 worsened the mouse model for multiple sclerosis. In the present study, a soluble form of TREM-2 protein has been identified by immunoprecipitation and by ELISA. Soluble TREM-2 protein (sTREM-2) was detected in human CSF, and was compared among subjects with relapsing-remitting multiple sclerosis (RR-MS; n = 52), primary progressive multiple sclerosis (PP-MS; n = 21), other inflammatory neurologic diseases (OIND; n = 19), and non-inflammatory neurologic diseases (NIND; n = 41). Compared to NIND subjects, CSF sTREM-2 levels were significantly higher in RR-MS (P = 0.004 by ANOVA) and PP-MS (P < 0.001) subjects, as well as in OIND (P < 0.001) subjects. In contrast, levels of sTREM-2 in blood did not differ among the groups. Furthermore, TREM-2 was detected on a subset of CSF monocytes by flow cytometry, and was also highly expressed on myelin-laden macrophages in eight active demyelinating lesions from four autopsied multiple sclerosis subjects. The elevated levels of sTREM-2 in CSF of multiple sclerosis patients may inhibit the anti-inflammatory function of the membrane-bound receptor suggesting sTREM-2 to be a possible target for future therapies.

Treatment of Inflammatory Autoimmune Disease Using a Small Molecule Cytokine Inhibitor

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Macrophage migration inhibitory factor (MIF), a ubiquitous inflammatory cytokine, is elevated in the CSF of MS patients during relapse. We have shown that MIF−/− (KO) mice show markedly suppressed clinical signs of experimental autoimmune encephalomyelitis (EAE) relative to wild type (WT) controls. The purpose of this study was to identify the timing in which MIF is important for progression of disease. Using adoptive transfer, we showed that activated lymphocytes from KO mice could transfer EAE to WT recipients, but that WT cells were unable to transfer EAE to KO recipients, suggesting that the CNS environment was protective in the absence of MIF. We used a small molecule inhibitor of MIF (INH) at two different times to selectively test the induction of EAE in the absence of MIF. Mice receiving INH at Day 0 were not protected, but mice treated after disease onset had less severe EAE. Despite the reduced severity of disease, lymphocytes from INH‐treated mice secreted substantial IFN‐γ and IL‐17. INH‐treated mice had a larger population of CD4+25+foxP3 lymphocytes. These findings suggest that MIF is important for disease progression but is not critical for T cell activation; an inhibitor could be a novel therapeutic approach for treating ongoing MS and other autoimmune diseases. (Supported by NIH grant AI 064320 and NMSS grant RG3272)

Immunopathogenesis of multiple sclerosis: concepts and controversies

Hundred and fifty years after the discovery of multiple sclerosis (MS), neither the etiology nor the mechanism of disease is fully charted, and current treatment has only modest effect. The conceptual understanding of MS rests on the animal model experimental autoimmune encephalomyelitis (EAE). Based on 70 years experience with EAE, it is widely believed that MS is an inflammatory attack on myelin and neurons orchestrated by myelin specific T cells. However, evidence supporting a key role for myelin specific T cells in MS is weak, the model fails to explain how immune self-tolerance is broken, and the peculiar B cell response of MS is poorly reflected in EAE. The pathogenesis of MS should therefore be studied in tissue samples and cells from MS patients, as close to the diseased organ as possible. Studies on lymphocytes from CSF of MS patients suggest that viral infections may be involved in T cell activation, and that intrinsic collaboration between T and B cells could sustain the immune response. These observations could explain the perpetuating immune response in MS in the absence of an overt antigen.

Corticotropin-releasing hormone and the blood-brain-barrier

Increased blood-brain-barrier (BBB) permeability precedes any clinical or pathologic signs and is critical in the pathogenesis of multiple sclerosis (MS) and brain metastases. CD4+ TH1 cells mediate demyelination in MS, but how they get sensitized and enter the brain to induce brain inflammation remains obscure. TH2 cytokines associated with allergic disorders have recently been implicated in MS, while genes upregulated in MS plaques include the mast cell-specific tryptase, the IgE receptor (Fc-epsilon-RI) and the histamine-1 receptor. Mast cell specific tryptase is elevated in the CSF of MS patients, induces microvascular leakage and stimulates protease-activated receptors (PAR), leading to widespread inflammation. BBB permeability, MS and brain metastases appear to worsen in response to acute stress that leads to the local release of corticotropin-releasing hormone (CRH), which activates brain mast cells to selectively release IL-6, IL-8 and vascular endothelial growth factor (VEGF). Acute stress increases BBB permeability that is dependent on CRH and mast cells. Acute stress shortens the time of onset of experimental alleric encephalomyelitis (EAE) that does not develop in W/W mast cell deficient or CRH -/- mice. Brain mast cell inhibition and CRHR antagonists offer novel therapeutic possibilities.

Anti‐heat shock protein 90β antibodies decrease pre‐oligodendrocyte population in perinatal and adult cell cultures. Implications for remyelination in multiple sclerosis

Lesions in the CNS of patients with multiple sclerosis (MS) often fail to remyelinate, resulting in neurological dysfunction. A key factor seems to be the inefficiency of oligodendrocyte precursor cells (OPCs). We recently reported antibodies against heat shock protein 90beta (Hsp90beta) in MS patients that recognized the antigen on the OPC surface. This study investigates the mechanism and result of anti-Hsp90beta antibody attack. These antibodies induced OPC death in culture in a complement-dependent fashion. Anti-Hsp90beta antibody-induced, complement-mediated OPC death only operated in these cells and caused a significant reduction in the number of O4-positive pro-oligodendrocytes (pre-oligodendrocytes). Adult cultured OPCs also expressed Hsp90beta on their cell surface and were attacked by anti-Hsp90beta antibodies leading to a significant decrease in the pre-oligodendrocyte population. In the presence of low levels of anti-Hsp90beta antibody--i.e. in the range seen in the CSF of MS patients--the complement concentration was critical to reduce the pre-oligodendrocyte population (via attack to OPCs). Higher concentrations of anti-Hsp90beta antibodies and complement became extinct the pre-oligodendrocytes. Complement 1-esterase inhibitor prevented these effects in the pre-oligodendrocyte population. These findings demonstrate, for the first time in vitro, a feasible mechanism to decrease the production of new oligodendrocytes, thus limiting the possibility of remyelination.

Expression of the immune-tolerogenic major histocompatibility molecule HLA-G in multiple sclerosis: implications for CNS immunity

HLA-G is a non-classical major histocompatibility complex (MHC) class I antigen with highly limited tissue distribution under non-pathological conditions. Although capable of acting as a peptide-presenting molecule, its strong immune-inhibitory properties identify HLA-G as a mediator of immune tolerance with specific relevance at immune-privileged sites such as trophoblast or thymus. To assess the role of HLA-G in CNS immunity, we investigated its expression in brain specimens from patients with multiple sclerosis (n = 11), meningitis (n = 2) and Alzheimer's disease (n = 2) and non-pathological CNS controls (n = 6). Furthermore, cultured human microglial cells and CSF of patients with multiple sclerosis and controls were assessed. Furthermore, CSF from MS patients and controls, as well as cultured human microglial cells were assessed. Using several HLA-G specific mAb and immunohistochemistry, HLA-G protein was found strongly expressed in brain specimens from patients with multiple sclerosis while it was rarely detectable in the non-pathological control specimens. In multiple sclerosis brain specimens, HLA-G immunoreactivity was observed in acute plaques, in chronic active plaques, in perilesional areas as well as in normal appearing white matter. In all areas microglial cells, macrophages, and in part endothelial cells were identified as the primary cellular source of expression. HLA-G was also found in other disease entities (meningitis, Alzheimer's specimens) where expression correlated to activation and MHC class II expression on microglial cells. Importantly, ILT2, a receptor for HLA-G, was also found in multiple sclerosis brain specimens thus emphasizing the relevance of this inhibitory pathway in vivo. HLA-G mRNA and protein expression and regulation could also be corroborated on cultured human microglial cells in vitro. Further, expression of HLA-G in the CSF of multiple sclerosis patients and controls was analysed by flow cytometry and ELISA. Monocytes represented the main source of cellular HLA-G expression in the CSF. Corresponding to the observations with the tissue specimens, CSF mean levels of soluble HLA-G were significantly higher in multiple sclerosis than in non-inflammatory controls (171 +/- 31 versus 39 +/- 10 U/ml; P = 0.0001). The demonstration of HLA-G and its receptor ILT2 on CNS cells and in areas of microglia activation implicate HLA-G as a contributor to the fundamental mechanisms regulating immune reactivity in the CNS. This pathway may act as an inhibitory feedback aimed to downregulate the deleterious effects of T-cell infiltration in neuroinflammation.

Co-purification of soluble membrane cofactor protein (CD46) and human herpesvirus 6 variant A genome in serum from multiple sclerosis patients

The association of human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) has been supported by several immunological and molecular studies. Recently, membrane cofactor protein (CD46) has been identified as the cellular receptor for the A and B variants of HHV-6. Elevated levels of soluble CD46 (sCD46) have been reported in the serum and CSF of MS patients. The aim of this study was to investigate a possible correlation between elevated levels of soluble CD46 and the presence of serum HHV-6 DNA in MS patients. An immunoaffinity column comprised of immobilized monoclonal antibodies to CD46 was developed to isolate sCD46 from cell free body fluids of MS patients and controls. After immunoaffinity purification, DNA was extracted from anti-CD46 column eluates and subjected to PCR amplification. Of the 42 MS samples tested, 4 serum samples were HHV-6 positive, 3 of which were typed as HHV-6A. The co-purification of sCD46 and HHV-6 DNA from MS sera indicates that HHV-6 is tightly connected to its receptor, CD46, in the serum of MS patients.

Study of Herpesvirus saimiri immortalization of γδ T cells derived from peripheral blood and CSF of multiple sclerosis patients

Human γδ T cells are an integral part of the innate immune system and have been difficult to study owing primarily to their relatively low abundance and their fastidious culture properties associated with short in vitro lifespan. Their increased presence within multiple sclerosis (MS) white matter plaques compared to peripheral blood (PB) suggests a specific interaction with central nervous system (CNS) tissues. This fact, together with their innate ability to lyse human oligodendrocytes in culture implicate them possibly in the pathogenesis of MS. To further investigate their potential role in MS, we studied whether γδ T cells could be effectively immortalized using Herpesvirus saimiri (HVS), so that they could be studied in longer-term cultures. Effective culture conditions were established resulting in efficient HVS growth transformation of multiple PB and CSF γδ T cell lines and clones that could exist in IL-2-dependent culture for periods in excess of 2 years. Phenotypic and functional comparison studies with parental nontransformed γδ T cells were performed to characterize the changes that possibly induced by viral transformation. Using panels of transformed γδ T cell clones representing discrete γδ TcR subtypes, there was no apparent correlation between intracytoplasmic cytokine expression or tumor cell cytotoxicity with a specific TcR. All transformed γδ T cells analyzed, regardless of their compartment of origin, strongly expressed intracytoplasmic IFN-γ and TNF-α, but little IL-2 or anti-inflammatory IL-4 or IL-10. These results indicate that HVS transformation of γδ T cells can be used to generate lines and clones from both the CSF and PB compartments for further study and elucidation of their potential role in MS pathogenesis.

Oligoclonal bands and antibody responses in multiple sclerosis.

Multiple Sclerosis (MS) is an inflammatory disease of the Central Nervous System with multifocal areas of demyelination. Although its etiology and pathogenesis remain controversial, several lines of evidence indicate that MS is mediated by a misdirected immune response against one or several myelin proteins. The involvement of diverse leukocyte subsets and their products in MS is still the subject of considerable debate. The emphasis on T cells has derived mainly from the detection of activated T cells in MS plaques and analogies with animal models of MS. Because of these observations cell-mediated immunity has dominated the research field of MS to this day. However, in recent years the role of B cells, plasma cells and immunoglobulins in MS have been re-examined, and current findings indicate that humoral immunity also plays a major role in disease pathogenesis. B cells and their products could exert several potential effects during the course of MS. Firstly, autoantibodies against specific myelin antigens could mediate damage to myelin membranes. Secondly, some studies suggest that natural autoantibodies could enhance remyelination. Thirdly, antibodies directed against myelin components can participate in anti-idiotypic networks, which may regulate the course of MS. Increased intrathecal immunoglobulin synthesis reflected by raised IgG indices and an oligoclonal pattern is the most common abnormality detected in MS patients. The introduction of more sensitive procedures for protein detection has allowed demonstrating oligoclonal bands (OCBs) in up to 95 % of patients with clinically definite MS. Although the presence of OCBs in CSF of MS patients is now well established as a sensitive laboratory test to support the clinical diagnosis, OCBs may be present in other disorders, including those not directly related to infection or abnormal immune response. Nevertheless, the pathogenesis of OCBs in MS is still obscure, and despite extensive research their antigenic target(s) have yet to be established. Therefore, a critical task is to identify the specificity of such target(s), thus providing significant clues about MS etiology. For this purpose, novel molecular immunologic strategies have been recently developed to offer alternative approaches to identify putative antigens recognized by antibodies present in MS patients. The elucidation of the mechanisms and target(s) responsible for the onset of MS has obvious implications for the further development of specific therapies.

Oligoclonal expansion of memory CD8+ T cells in cerebrospinal fluid from multiple sclerosis patients.

Multiple sclerosis is a chronic inflammatory demyelinating disease of the CNS. Although the aetiology of multiple sclerosis is still unknown, it is widely believed that T cells play a central role in its pathogenesis. To identify and characterize disease-relevant T cells, we analysed CD4+ and CD8+ T cells freshly isolated from the CSF and peripheral blood of 36 multiple sclerosis patients for their T-cell receptor variable beta (TCRBV) chain repertoire. In most patients, we found significant overexpression of individual TCRBV chains on CD8+ T cells from CSF compared with peripheral blood. In contrast, only a few multiple sclerosis patients showed differences between the two compartments in TCRBV expression on CD4+ T cells. The overexpression of specific TCRBV chains on CD8+ T cells was found to be stable over several months in selected patients and involved mainly T cells with a memory phenotype. In two patients studied, individual TCRBV chain overexpression was found to be caused by the expansion of T cell populations with identical or highly similar rearranged T-cell receptor beta- and alpha-chain sequences, which were not found among peripheral blood CD8+ T cells. Our findings demonstrate selective enrichment of memory CD8+ T cells in the CSF of multiple sclerosis patients, suggesting a role for these CD8+ T cells in the pathogenesis of multiple sclerosis. Our study provides a basis for future trials to identify disease-associated antigens and disease pathogenesis in multiple sclerosis.

Intrathecal antibody production against Chlamydia pneumoniae in multiple sclerosis is part of a polyspecific immune response.

Chronic intrathecal immunoglobulin (Ig) production is a hallmark of multiple sclerosis characterized by the presence of oligoclonal IgGs and, in addition, polyspecific recognition of different pathogens such as measles, rubella and herpes zoster virus. While the antigen specificity of the oligoclonal IgGs in multiple sclerosis is largely unknown, the oligoclonal IgGs arising during CNS infectious diseases are reactive against the specific pathogen. Recently, a link between Chlamydia pneumoniae and multiple sclerosis has been claimed. To test the possible role of C. pneumoniae in multiple sclerosis, we analysed (i) whether there is intrathecal IgG production against C. pneumoniae in multiple sclerosis and (ii) if the oligoclonal IgGs in the CSF of multiple sclerosis patients recognize C. pneumoniae. By studying paired serum-CSF samples from 120 subjects (definite multiple sclerosis, 46; probable multiple sclerosis, 12; other inflammatory neurological diseases, 35; other neurological diseases, 27) by enzyme-linked immunosorbent assay, we found that 24% of all patients with definite multiple sclerosis, but only 5% of patients with other inflammatory or non-inflammatory diseases, produced IgGs specific for C. pneumoniae intrathecally (definite multiple sclerosis versus other inflammatory neurological diseases: P = 0.027). The presence of intrathecal IgGs to C. pneumoniae was independent of the duration of disease and relatively stable over time. The major CSF oligoclonal IgG bands from multiple sclerosis patients with an intrathecal Ig production to C. pneumoniae did not react towards purified elementary bodies and reticulate bodies of C. pneumoniae on affinity-mediated immunoblot following isoelectric focusing (IEF-western blots). In contrast, the IgGs in the CSF of control patients with neuroborreliosis strongly reacted with their specific pathogen, Borrelia burgdorferi, by IEF-western blot analysis. Concomitant analysis of the CSF of 23 patients with a nested polymerase chain reaction for C. pneumoniae was negative in all cases. Together, our findings strongly suggest that the immune response to C. pneumoniae is part of a polyspecific intrathecal Ig production, as is commonly observed with other pathogens. This argues against a specific role for C. pneumoniae in multiple sclerosis.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in cerebrospinal fluid differ in multiple sclerosis and Devic's neuromyelitis optica.

Matrix metalloproteinases (MMPs) are increased in the CSF of patients with multiple sclerosis. Devic's neuromyelitis optica (DNO) is a demyelinating syndrome that involves the optic nerve and cervical cord but differs pathologically from multiple sclerosis. Therefore, we hypothesized that the type of inflammatory reaction that causes MMPs to be elevated in multiple sclerosis would be absent in patients with DNO. CSF was collected from 23 patients with relapsing-remitting or secondary progressive multiple sclerosis, all of whom were experiencing acute symptoms, from seven patients with DNO, and from seven normal volunteers. Diagnoses were made according to current criteria on the basis of clinical manifestations, imaging results and CSF studies. IgG synthesis was increased in the CSF of multiple sclerosis patients but not in that of DNO patients. Zymography, reverse zymography and ELISA (enzyme-linked immunosorbent assay) were used to measure gelatinase A (MMP-2), gelatinase B (MMP-9) and tissue inhibitors of metalloproteinases (TIMPs). Zymograms showed that multiple sclerosis patients had elevated MMP-9 compared with DNO patients and controls (P: < 0.05). TIMP-1 and TIMP-2 levels were similar in all three groups. We conclude that multiple sclerosis patients have higher MMP-9 levels in the CSF than patients with DNO, which supports the different pathological mechanisms of these diseases.

A pilot study on the antibodies to HHV-6 variants and HHV-7 in CSF of MS patients

In the possible role for human herpesviruses (HHV) in the pathogenesis of multiple sclerosis (MS) neither clear distinction between the two variants of HHV-6, nor the involvement of HHV-7 have been described. Therefore, we quantitated HHV-6 variant specific and HHV-7 reacting antibodies in the CSF of 13 patients with MS or other neurological disorders by ELISA. Predominance in the positivity of IgG (67%) and IgM (44%) to HHV-6B over that of IgG (44%) with no detectable IgM to HHV-6A, and no antibodies to HHV-7 were found in the CSF of MS patients. None of these antibodies were found in the CSF of controls. This suggests that, intrathecal chronic active or primary HHV-6B infection might contribute to MS progression, while the local effects of HHV-6A and HHV-7 seem to be less important.

Are there indicators of remyelination in blood or CSF of multiple sclerosis patients?

Are there indicators of remyelination in blood or CSF of multiple sclerosis patients?

Identification of peptides binding to IgG in the CSF of Multiple Sclerosis patients

Identification of peptides binding to IgG in the CSF of Multiple Sclerosis patients

Evaluation of interleukin 10 and interferon‐α in the cerebrospinal fluid and serum of patients with multiple sclerosis

The activities of IL‐10, a cytokine produced by TH2 cells, monocytes and B‐cells, and IFN‐α, a product of activated macrophages/monocytes, were investigated in the CSF and serum samples of 25 subjects with clinically definite multiple sclerosis (MS), of whom 14 were in the active and nine in the stable phase, and of 15 controls with other noninflammatory neurological diseases (OND). Elevated CSF IL‐10 and IFN‐α levels were found in MS patients in the stable phase with respect to patients in the active phase (p &gt; 0.001), while no significant differences were observed in the mean serum levels between MS patients (both in the active and stable phase) and controls. Finally, a significant correlation was detected between IL‐10 and IFN‐α in the CSF of MS patients in remission. This study suggests that IL‐10 may downregulate MS progression.

Organ-specific autoantigens induce interferon-gamma and interleukin-4 mRNA expression in mononuclear cells in multiple sclerosis and myasthenia gravis.

T cells recognizing the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) are increased in multiple sclerosis (MS), and there are elevated numbers of T cells recognizing the nicotinic acetylcholine receptor (AChR) in myasthenia gravis (MG). However, the cytokine repertoires in these diseases are largely unknown. We adopted in situ hybridization with radiolabeled complementary DNA oligonucleotide probes to enumerate mononuclear cells that expressed the T-helper type 1 (Th1) cell-related interferon-gamma (IFN-gamma) and Th2-associated interleukin-4 (IL-4) after short-term culture in the presence of autoantigen. High numbers of IFN-gamma and IL-4 mRNA-expressing cells in response to MBP and PLP were detected in patients with untreated MS, and to AChR in MG. The levels of IFN-gamma and IL-4 mRNA-positive cells in MS after culture in the presence of AChR, and in MG after culture in the presence of MBP or PLP, did not differ from those detected after culture without antigen. The CSF of MS patients contained four- to eightfold more myelin protein-reactive IFN-gamma and IL-4 expressing cells. The findings imply that MS and MG are associated with mixed Th1- and Th2-like cell responses directed to organ-specific target antigens.

Elevated levels of the T-cell activation antigen soluble CD27 in CSF of multiple sclerosis patients

Elevated levels of the T-cell activation antigen soluble CD27 in CSF of multiple sclerosis patients

Elevated levels of the T-cell activation antigen soluble CD27 in CSF of multiple sclerosis patients

Elevated levels of the T-cell activation antigen soluble CD27 in CSF of multiple sclerosis patients

Cytokine accumulations in CSF of multiple sclerosis patients

We identified the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF), and interleukin-6 (IL-6) by specific radioimmunoassays in the CSF of patients with multiple sclerosis (MS) and other neurologic diseases (OND). There was a high incidence of detectable IL-1 beta in patients with active MS compared with inactive MS or OND patients. TNF was also more frequently present in active MS than in OND CSF. By contrast, most MS CSF did not contain detectable IL-6. There was no correlation between the degree of CSF pleocytosis and the level of individual cytokines, suggesting that cytokine accumulations may be derived from CNS, and not CSF, cells. As IL-1 beta and TNF experimentally induce astrogliosis, demyelination, temperature elevation, lassitude, and sleep, and results raise the possibility that these cytokines may contribute to a variety of manifestations in MS and in other disease states.