Monocyte-derived cytokines in multiple sclerosis.

MS is an inflammatory, presumably autoimmune, disease mediated by the activation of T cells, B cells and monocytes (MO). Inflammation is thought to occur early during the relapsing-remitting phase of MS (RRMS), whereas in the later phases of MS such as secondary progressive MS (SPMS), inflammation tends to diminish. Our objective was to compare the types and amounts of proinflammatory and regulatory cytokines produced by MO from relapsing-remitting patients with or without treatment with IFN-beta (RRMS+ therapy, RRMS- therapy), respectively, from secondary progressive patients (SPMS) and from healthy controls (HC). MO were isolated by a density-gradient technique and three different techniques (RNase protection assay, ELISA and intracellular cytokine staining) were used to assess cytokine levels. An increase in IL6, IL12 and TNF-alpha was observed by all three methods for RRMS- therapy and for SPMS patients compared to HC and RRMS+ therapy patients. We conclude that proinflammatory and regulatory monokines can be derived from MO of MS patients and that these levels are modulated by IFN-beta therapy. Although it is believed that inflammation tends to diminish in SPMS patients, our data show that inflammatory cytokines continue to be released at high levels, suggesting that IFN-beta or IL10 treatment may be beneficial for this group.

Altered expression of costimulatory molecules in myasthenia gravis.

To characterize the involvement of costimulatory pathways in the pathogenesis of myasthenia gravis (MG), a multiparameter flow cytometry assay was adopted to enumerate blood mononuclear cells (MNC) expressing CD28, CD80, CD86, CD40, and CD40L molecules in patients with MG and healthy subjects. Patients with MG had lower percentages of CD8(+)CD28(+) cells, augmented percentages of CD4(+)CD80(+), CD4(+)CD86(+), CD8(+)CD80(+), CD8(+)CD86(+), CD14(+)CD80(+), and CD14(+)CD86(+) cells, and similar levels of cells expressing CD40 and CD40L and of B cells expressing CD80 and CD86 compared to the controls. Patients with early onset of MG (<40 years) had lower percentages of CD3(+)CD86(+), CD4(+)CD86(+), CD8(+)CD86(+) T cells and CD20(+)CD86(+) B cells compared to those with late onset (>40 years). There was a positive correlation between the patients' age and percentages of CD86(+) cells. The data indicate that the CD28/CD80-CD86 costimulatory pathway is involved in MG. The high percentages of CD80 and CD86 positive T cells and monocytes may reflect persistent activation of T and B cells, whereas the low CD28 expression may be the result of chronic exposure to CD80 and CD86. These molecules could be the focus for new and improved immunomodulating therapies of MG.

High numbers of perforin mRNA expressing CSF cells in multiple sclerosis patients with gadolinium-enhancing brain MRI lesions.

Enhanced expression of pro- and anti-inflammatory cytokines is a common finding in MS, but attempts to correlate cytokine expression with disease activity have produced conflicting results. In this paper, gadolinium-(Gd-)enhancing lesions on brain MRI were used as markers for active inflammation in patients with MS not treated with any immunomodulatory drugs. In parallel, in situ hybridization was used to detect blood and cerebrospinal fluid (CSF) mononuclear cells (MNC) expressing cytokine mRNA. An association was observed between numbers of perforin mRNA expressing CSF MNC and numbers of Gd-enhancing brain MRI lesions. Perforin mRNA expressing CSF MNC were not detected in any of the patients lacking active lesions on brain MRI. The expression of tumor necrosis factor-alpha, interleukin-10 (IL-10) and IL-12 mRNA in CSF MNC did not differ between MS patients with and without active MRI lesions. Based on the present finding, a role for perforin in the disruption of the blood-brain barrier in MS can be hypothesized.

High levels of IL-10 secreting cells are present in blood in cerebrovascular diseases.

Ischemic stroke is associated with altered systemic immune responses both early after the onset and in the recovery phase. Interleukin (IL)-10, a Th2 related cytokine, has multiple effects on different cell types, including T and B lymphocytes, monocytes, neutrophils and mast cells. IL-4 is another Th2 cytokine that inhibits the synthesis of pro-inflammatory cytokines by Th1 clones. We used enzyme-linked immunospot assays to detect and enumerate blood mononuclear cells (MNC) secreting IL-10 and IL-4 spontaneously as well as after stimulation with myelin basic protein (MBP), considered to be an autoantigen of possible pathogenic importance in, for example, multiple sclerosis, to evaluate the involvement of anti-inflammatory cytokines in ischemic stroke. All patients with ischemic stroke and cerebral hemorrhage had strongly elevated numbers of IL-10 secreting blood MNC compared with healthy individuals. Numbers of MBP-reactive IL-10 secreting blood MNC were also elevated in a proportion of the patients with stroke and hemorrhage. Levels of IL-4 secreting blood MNC did not differ in ischemic stroke versus healthy individuals. The anti-inflammatory IL-10 could play a pivotal role in ischemic stroke as well as cerebral hemorrhage.

Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis.

Myelin-directed autoimmunity is considered to play a key role in the pathogenesis of multiple sclerosis (MS). Increased production of both pro- and anti-inflammatory cytokines is a common finding in MS. Interleukin-17 (IL-17) is a recently described cytokine produced in humans almost exclusively by activated memory T cells, which can induce the production of proinflammatory cytokines and chemokines from parenchymal cells and macrophages. In situ hybridisation with synthetic oligonucleotide probes was adopted to detect and enumerate IL-17 mRNA expressing mononuclear cells (MNC) in blood and cerebrospinal fluid (CSF) from patients with MS and control individuals. Numbers of IL-17 mRNA expressing blood MNC were higher in patients with MS and acute aseptic meningoencephalitis (AM) compared to healthy individuals. Higher numbers of IL-17 mRNA expressing blood MNC were detected in MS patients examined during clinical exacerbation compared to remission. Patients with MS had higher numbers of IL-17 mRNA expressing MNC in CSF compared to blood. This increase in numbers of IL-17 mRNA expressing MNC in CSF was not observed in patients with AM. Our results thus demonstrate increased numbers of IL-17 mRNA expressing MNC in MS with higher numbers in CSF than blood, and with the highest numbers in blood during clinical exacerbations.

Metalloproteinases and their tissue inhibitors in multiple sclerosis.

Matrix metalloproteinases (MMPs) comprise a family of proteolytic enzymes. MMPs are capable of disrupting the blood-brain barrier (BBB), mediating the destruction of extracellular matrix and myelin components. MMPs are also involved in the processing of a variety of cell surface molecules, including the proinflammatory cytokine TNF-alpha. Each of these mechanisms are thought to be important in the pathogenesis of multiple sclerosis (MS). We investigated mRNA expression of MMP-3, MMP-9 and two tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in parallel in blood mononuclear cells (MNC) from patients with MS and controls, using in situ hybridization. Numbers of MMP-9 mRNA-expressing cells in blood were higher in patients with MS compared to other neurological diseases (OND), other inflammatory neurological diseases (OIND) and healthy subjects (P<0.0001 for all comparisons). Patients with MS had also higher levels of MMP-3 and TIMP-1 mRNA expressing blood MNC compared to patients with OND and healthy subjects. A positive correlation was observed for MMP-9 and TIMP-1 mRNA expression in MS. These results demonstrate that MMPs and TIMPs are upregulated in MS and may contribute to the pathogenesis of the disease.

Differential requirements for CD28 and CD40 ligand in the induction of experimental autoimmune myasthenia gravis.

The interactions of CD28-B7 and CD40-CD40 ligand (CD40L) pathways in T cell costimulation and autoimmune disease are incompletely understood. We sought to address this issue by investigation of the genesis of acetylcholine receptor (AChR)-induced antibody-mediated experimental autoimmune myasthenia gravis (EAMG) in CD28- and CD40L-deficient mice (CD28-/-, CD40L-/-). Compared to wild-type mice, the CD28-/- mice became less susceptible, and CD40L-/- mice were completely resistant to EAMG induction. Analysis of T helper functions, reflected by cytokine responses, revealed a switch to a Th1 profile in CD28-/- mice. Consistently, levels of serum AChR-specific antibodies of the IgG1 isotype were decreased in CD28-/- mice. In the CD40L-/- mice, both Th1 and Th2 cytokine responses were diminished, and T cell-dependent AChR-reactive B cell responses were more severely impaired than in the CD28-/- mice. Thus, CD28 and CD40L are differentially required for induction of EAMG.

No evidence for elevated numbers of mononuclear cells expressing MCP-1 and RANTES mRNA in blood and CSF in multiple sclerosis.

The perivascular accumulation of mononuclear cells (MNC) in brain white matter is critical in the development of active lesions in multiple sclerosis (MS). Chemokines contribute to leukocyte recruitment by increasing the adhesiveness of integrins expressed on leukocytes and by promoting migration through endothelium and extracellular matrix. By using an in situ hybridization technique, it was possible to enumerate blood and CSF MNC expressing mRNA for the two CC chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES (regulated upon activation, normal T cells, expressed and secreted) in MS patients and controls. No differences in numbers of blood MNC expressing MCP-1 or RANTES could be found in MS patients compared to healthy individuals or patients with acute aseptic meningoencephalitis (AM). High numbers of CSF MNC expressing MCP-1 and RANTES were found in some MS patients, but also in patients with AM. This shows that elevated numbers of MCP-1 and RANTES mRNA expressing CSF MNC are not specific for the inflammatory process in MS. We conclude that there is no evidence for a systemic dysregulation of the CC chemokines MCP-1 and RANTES in MS.

Linomide (roquinimex) affects the balance between pro- and anti-inflammatory cytokines in vitro in multiple sclerosis.

OBJECTIVES: Multiple sclerosis (MS) is characterized by high levels of circulating mononuclear cells (MNC) that respond to myelin proteins like myelin basic protein (MBP) in vitro by expressing mRNA of both pro-inflammatory cytokines, e.g. interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT) that may make MS worse, and anti-inflammatory cytokines like IL-4, IL-10 and transforming growth factor-beta (TGF-beta) that may act beneficially. Substances that down-regulate cytokines such as TNF-alpha or promote IL-10 or TGF-beta can be anticipated to affect MS beneficially. MATERIAL AND METHODS: In situ hybridization to detect and enumerate IFN-gamma, TNF-alpha, LT, IL-4, IL-10 and TGF-beta mRNA expressing blood MNC after stimulation with myelin basic protein (MBP), control antigens and without antigen in presence and absence of Linomide (roquinimex, LS-2616) was employed. In parallel, ELISPOT assay to detect MBP- and PHA-reactive IFN-gamma secreting blood MNC+/-Linomide was used. RESULTS: Here we report that Linomide, a synthetic immunomodulator, at concentrations effective in vivo reduces the number of MBP-reactive TNF-alpha and increases MBP-reactive IL-10 and TGF-beta mRNA expressing MNC from MS patients' blood when analysed in vitro. Compared to dexamethasone, Linomide up-regulated levels of blood MNC expressing mRNA of TGF-beta after culture in presence of MBP. CONCLUSIONS: Changes of cytokine balance towards a production of anti-inflammatory cytokines could be a desirable effect to be evaluated in future drug studies of Linomide-like substances. At present, Linomide is not evaluable in MS clinical trials due to side-effects.

The phosphodiesterase i.v. inhibitor rolipram in vitro reduces the numbers of MBP-reactive IFN-gamma and TNF-alpha mRNA expressing blood mononuclear cells in patients with multiple sclerosis.

The inflammatory nature of multiple sclerosis (MS) implicates the participation of cytokines as immune response mediators. Targeting the cytokine balance by downregulating proinflammatory cytokines and/or upregulating immunosuppressive cytokines could benefit patients with MS. This article reports on the in vitro effects of the phosphodiesterase i.v. inhibitor Rolipram on the production of pro- and anti-inflammatory cytokines in MS and, for reference, in myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultured in the presence of the organ-specific autoantigens myelin basic protein (MBP) or acetylcholine receptor (AChR), and in the absence of antigens, with and without Rolipram. In situ hybridization with synthetic oligonucleotide probes was used to detect and enumerate blood MNC expressing IFN-gamma, TNF-alpha, LT, TGF-beta, IL-4, and IL-10 mRNA. Numbers of MNC-secreting IFN-gamma and IL-4 in blood blood were examined by ELISPOT assays. Rolipram reduced the numbers of MBP-reactive IFN-gamma- and TNF-alpha mRNA-expressing blood MNC in MS, and numbers of AChR-reactive IFN-gamma-, TNF-alpha-, and LT mRNA-positive cells in MG. In contrast, expression of the Th2 cell related IL-4 and the anti-inflammatory IL-10, and TGF-beta was not affected. These data support a role for Rolipram in the treatment of diseases such as MS.

Interleukin-12 and perforin mRNA expression is augmented in blood mononuclear cells in multiple sclerosis.

Cytokines are suggested to orchestrate an abnormal immune response in multiple sclerosis (MS). The regulatory cytokine interleukin (IL)-12 induces T-helper (Th) cell switch to the Th1 type and the production by cytotoxic T cells of perforin, a cell lysis-inducing factor. It has been suggested that Th1-like cytokines may promote the development of MS, and the production of perforin to induce oligodendrocyte damage. In-situ hybridization with radiolabelled synthetic oligonucleotide probes was used to detect and enumerate mononuclear cells (MNC) expressing IL-12 and perforin mRNA in blood and cerebrospinal fluid (CSF) from patients with MS and controls. Plasma and CSF levels of IL-12 (p70) were evaluated by ELISA. Higher numbers of IL-12 and perforin mRNA-expressing MNC were registered in blood in MS and also in controls with aseptic meningoencephalitis (AM) compared to healthy subjects. There were a few patients with other non-inflammatory neurological diseases who also had high levels of IL-12 or perforin mRNA expressing blood MNC. A parallel elevation was observed for IL-12 (p70) concentrations in plasma. In the MS patients' CSF, there was a further augmentation of IL-12 mRNA expressing MNC. To evaluate autoantigen-induced IL-12 and perforin mRNA expression, blood MNC were cultivated +/- myelin basic protein (MBP), a candidate autoantigen in MS. Higher numbers of MBP-reactive IL-12 and perforin mRNA expressing blood MNC were detected in MS than controls. The augmentation of both IL-12 and perforin in MS might reflect ongoing inflammatory processes in MS and could represent targets for future treatments.

IL-15 mRNA expression is up-regulated in blood and cerebrospinal fluid mononuclear cells in multiple sclerosis (MS).

IL-15, produced by monocytes and epithelial cells, is a novel cytokine with actions similar to IL-2. IL-15 induces T cell proliferation, B cell maturation and natural killer (NK) cell cytotoxicity, and is a chemoattractant for T cells. We investigated the expression of IL-15 mRNA in blood and cerebrospinal fluid (CSF) mononuclear cells (MNC) in MS, an inflammatory disease of the central nervous system where cytokines are involved. MS patients had higher numbers of IL-15 mRNA-expressing blood MNC than patients with aseptic meningo-encephalitis (AM) and healthy controls. In CSF, MS patients had even higher numbers of IL-15 mRNA-expressing cells than in blood. This discrepancy between IL-15 mRNA expression between blood and CSF MNC was not seen in AM patients. Patients examined during the secondary chronic-progressive phase of MS had higher numbers of IL-15 mRNA-expressing blood MNC compared with patients examined during the relapsing-remitting phase. Levels of IL-15 mRNA-positive blood MNC were similar in patients with AM, myasthenia gravis, non-inflammatory neurological diseases and healthy controls. Taken together these data indicate that IL-15 mRNA expression is up-regulated in MS, further suggesting a role for proinflammatory cytokines in the pathogenesis of MS.

Ischemic stroke is associated with a systemic increase of blood mononuclear cells expressing interleukin-8 mRNA.

BACKGROUND AND PURPOSE: Ischemic brain injury secondary to an arterial occlusion is characterized by acute local inflammation. Blood polymorphonuclear leukocytes (PMNL), primarily neutrophils, adhere to endothelial cells and rapidly invade the injured brain after the arterial occlusion. This neutrophilic invasion might correlate with the production of certain chemoattractants by blood mononuclear cells (MNC). We evaluated mRNA expression of the CXC chemokine interleukin (IL)-8, and the CC chemokines monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta in blood MNC from patients with ischemic stroke. METHODS: Peripheral blood was obtained at 8 AM on days 1 to 7 (mean, day 3) after onset of symptoms. In situ hybridization with radiolabeled synthetic oligonucleotide probes for the chemokines was adopted to measure chemokine mRNA expression in MNC. An enzyme-linked immunosorbent assay for IL-8 was used to measure IL-8 levels in plasma. RESULTS: Most patients with ischemic stroke had high numbers of IL-8 mRNA expressing blood MNC, regardless of the time interval between onset of clinical symptoms and examination. There was a marked difference between patients with ischemic stroke and healthy subjects (median, 6228 versus 885 positive cells per 10(5) MNC; P<.0001). IL-8 levels in plasma correlated positively to IL-8 mRNA expression in examined patients (n=7) with ischemic stroke (r=.78, P<.05). In contrast, mRNA expression for the CC chemokines showed no significant difference between patients with ischemic stroke and healthy control subjects. CONCLUSIONS: This study demonstrated a systemic increase of IL-8 mRNA expressing MNC and IL-8 levels in plasma from patients with ischemic stroke, suggesting that IL-8 could be involved in recruiting blood PMNL to the sites of cerebral ischemia.

Optic neuritis and cytokines: no relation to MRI abnormalities and oligoclonal bands.

Acute unilateral monosymptomatic optic neuritis (ON) is a common first manifestation of MS if associated with multiple MS-like lesions on brain MRI and oligoclonal IgG bands (OB) in the CSF, whereas ON patients lacking these laboratory abnormalities are considered to have a good prognosis regarding future MS development. Several cytokines involved in immune regulation are upregulated in blood and even more noticeable in CSF in MS. To study a possible relation between cytokine profiles and presence versus absence of MS-like brain MRI lesions and CSF OB, we used in situ hybridization to examine mRNA expression of the proinflammatory interleukin-12 (IL-12), interferon-gamma, and tumor necrosis factor-alpha and the immune response downregulating IL-10, transforming growth factor-beta, and IL-4 in blood and CSF mononuclear cells (MNC) from 59 patients with untreated ON. There were no differences in numbers of MNC in blood or CSF expressing any of the cytokines under study, upon subgrouping the ON patients regarding presence (n = 31) versus absence (n = 28) of MRI lesions, presence (n = 45) versus absence (n = 14) of OB, or duration after onset of ON (<1 month, n = 30, versus >1 month, n = 29). Similarly, no differences were observed for numbers of myelin basic protein-reactive blood MNC expressing any of these cytokines after subgrouping according to these variables. Our findings suggest that the cytokine profile, as examined in this study, is less useful to determine the risk of future development of clinically definite MS in ON patients or as indicator for therapeutic interventions in ON. An upregulation of both pro- and anti-inflammatory cytokines in ON patients seems to be more related to the CNS disease per se, whether limited to the optic nerve or not, than to the inflammatory process characteristic for MS.

Influence of IFN-beta1b (Betaferon) on cytokine mRNA profiles in blood mononuclear cells and plasma levels of soluble VCAM-1 in multiple sclerosis.

Inflammatory cell infiltration within the central nervous system (CNS) and upregulation of both pro- and anti-inflammatory cytokines are characteristic for multiple sclerosis (MS). Treatment with interferon-beta 1b (IFN-beta1b) reduces the number and severity of MS relapses. To examine whether treatment with IFN-beta1b affects levels of cytokine mRNA expressing blood mononuclear cells (MNC) we employed in-situ hybridization with synthetic oligonucleotide probes to detect and enumerate IFN-gamma, TNF-alpha, IL-10, TGF-beta and perforin mRNA expressing cells in MS patients before treatment with IFN-beta1b and during treatment for 3-6 weeks and for 3-6 months. Numbers of blood MNC spontaneously expressing TNF-alpha and IL-10 mRNA were lower after 3-6 months of treatment, while numbers of IFN-gamma, TGF-beta and perforin mRNA expressing MNC were not affected by treatment. IFN-beta1b had no influence on levels of MBP-reactive IFN-gamma, TNF-alpha, TGF-beta, IL-10 or perforin mRNA expressing blood MNC determined after 3-6 weeks or 3-6 months of treatment. Parallel measurements of plasma concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1) revealed elevated levels after 3-6 weeks of treatment and these levels remained higher after 3-6 months of treatment. The results suggest that IFN-beta1b treatment upregulates plasma levels of sVCAM-1, but has little effects on numbers of blood MNC expressing mRNA of the pro- and anti-inflammatory cytokines under study.Copyright Lippincott-Raven Publishers

Neutralising and binding anti-interferon-beta-I b (IFN-beta-I b) antibodies during IFN-beta-I b treatment of multiple sclerosis.

Interferon-beta-I b (IFN-beta-I b) is an immunomodulatory therapy of multiple sclerosis (MS), reducing the numbers and severity of exacerbations and the total lesion load measured by magnetic resonance imaging of the brain. The benefits of IFN-beta-I b could be hampered by the development of neutralising antibodies against the compound. Our results confirmed earlier studies, showing that 42% of MS patients treated with IFN-beta-I b for more than 3 months had developed neutralising antibodies. The occurrence of binding anti-IFN-beta-I b antibodies, presently not believed to impede the clinical efficacy of IFN-beta-I b, were demonstrated by an immunoassay in some patients after 1 month of treatment and in 78% after 3 months. The development of binding antibodies seemed to be an early phenomenon, preceding the appearance of neutralising antibodies. Antibodies crossreacting with IFN-beta-I a and natural IFN-beta were also found in a majority of IFN-beta-I b treated patients with high titres of binding antibodies. Employing a solid-phase enzyme-linked immunospot (ELISPOT) assay, 68% of MS patients treated with IFN-beta-I b for 1-23 months had elevated numbers of anti-IFN-beta-I b-antibody secreting cells in blood, compared to 18% of untreated MS patients and 20% among patients with other neurological diseases. Thus, our findings confirm that IFN-beta-I b is immunogenic in MS patients. High levels of anti-IFN-beta-I b antibody secreting cells were, however, also found in two untreated control patients with inflammatory diseases, suggesting that anti-IFN-beta-I b antibodies might also occur spontaneously.

Soluble CD30 levels in plasma and cerebrospinal fluid in multiple sclerosis, HIV infection and other nervous system diseases.

The CD30 molecule, a member of tumor necrosis factor superfamily, has been suggested to be preferentially expressed and released in soluble form by activated T cells that produce T helper 2 type (Th2) cytokines. To evaluate whether determination of soluble CD30 (sCD30) levels could have a diagnostic value in diseases associated with Th1 and Th2 cytokine involvement, we investigated sCD30 in plasma and cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS), HIV infection and other nervous system diseases. There was no statistically significant difference for plasma sCD30 levels in these clinical groups. However, patients with HIV infection had higher levels of sCD30 in CSF than MS patients. The mean sCD30 values were 3 to 6 folds higher in plasma than in CSF in all patient groups. No relationships were found between sCD30 levels and different clinical variables of MS and HIV infection, except that higher plasma sCD30 levels in HIV-infected patients were found in those with higher CD4+ T cell counts (> 200 x 10(6)) compared to the group with lower cell counts. The findings indicate that determinations of plasma and CSF sCD30 levels in MS and HIV infection have limited or no value as diagnostic or prognostic indicator.

Tumor necrosis factor-alpha, lymphotoxin, interleukin (IL)-6, IL-10, IL-12 and perforin mRNA expression in mononuclear cells in response to acetylcholine receptor is augmented in myasthenia gravis.

Myasthenia gravis (MG) is a neuromuscular disorder mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) on the postsynaptic membrane of the neuromuscular junction. The production of antibodies is regulated by T cells by means of immunoregulatory cytokines. To investigate the involvement of TNF-alpha, lymphotoxin (LT), IL-6, IL-10, IL-12 and perforin in MG, numbers of cytokine mRNA expressing blood mononuclear cells (MNC) were examined in patients with MG and controls. LT belongs to the Th1 cell related cytokines, IL-6 and IL-10 to the Th2 type, TNF-alpha is produced by both Th1 and Th2 cells, IL-12 induces T cell switch towards the Th1 type and perforin is an effector molecule inducing cell lysis. Short term culture of MNC with AChR revealed augmented levels of AChR-reactive TNF-alpha, LT, IL-6, IL-10, IL-12 and perforin mRNA expressing cells in MG compared to levels obtained without AChR or in presence of control antigen. AChR-reactive TNF-alpha, IL-6, IL-10, IL-12 and perforin mRNA expressing cells were higher in MG than controls. These findings suggest that the cytokines TNF-alpha, LT, IL-6, IL-10 and IL-12, and the cytolytic effector molecule perforin are also involved in MG.

Multiple sclerosis: the proinflammatory cytokines lymphotoxin-alpha and tumour necrosis factor-alpha are upregulated in cerebrospinal fluid mononuclear cells.

Lymphotoxin-alpha (LT-alpha) and tumour necrosis factor-alpha (TNF-alpha) promote inflammation in autoimmune diseases and have been detected in the multiple sclerosis (MS) brain lesions and blood, suggesting these cytokines are also present in the cerebrospinal fluid (CSF). To study this, mononuclear cells (MNC) were examined for transcripts of LT-alpha and TNF-alpha, using in situ hybridization (ISH) with synthetic oligonucleotide probes. Most patients with MS had LT-alpha and TNF-alpha mRNA-expressing MNC in their CSF at mean frequencies of about 1/2800 cells for both cytokines. Numbers were dramatically higher than in the paired blood specimens. Control patients with other inflammatory neurological diseases (OIND) also had LT-alpha and TNF-alpha mRNA-expressing cells in CSF but at mean frequencies of only 1/36,000 and 1/18,000 cells, respectively. In blood, levels were similar in OIND and MS. To elucidate the influence of myelin antigen stimulation on LT-alpha and TNF-alpha expression, MNC were cultivated with or without myelin basic protein. Strongly elevated levels of MBP-reactive TNF-alpha mRNA-expressing cells were detected in the MS patients' CSF, in particular when examined during clinical exacerbations, as well as MBP-reactive LT-alpha mRNA-expressing MNC. No such patterns were observed in the OIND controls. The strong accumulation of LT-alpha- and TNF-alpha-producing cells and of MBP-reactive LT-alpha and TNF-alpha mRNA-positive cells in the immediate vicinity of the demyelinating process in MS patients implicates a role of these cytokines in the development of MS.

Increased interleukin-6 mRNA expression in blood and cerebrospinal fluid mononuclear cells in multiple sclerosis.

The increased intrathecal production of immunoglobulins within the cerebrospinal fluid (CSF) compartment commonly observed in multiple sclerosis (MS) implicates participation of B cell activating factors. One effect of the cytokine interleukin (IL)-6 is induction of immunoglobulin production by activated B cells. Employing in situ hybridization (ISH) with synthetic oligonucleotide probes, we measured numbers of IL-6 mRNA-expressing mononuclear cells (MNC) in blood and CSF from patients with MS, aseptic meningo-encephalitis (AM), and in blood from patients with other neurological diseases (OND) and healthy subjects. Numbers of IL-6 mRNA-expressing MNC were elevated in blood (mean frequency 1 per 33,000 MNC) and even further enriched in the CSF (1 per 10,000 MNC) of MS patients, and to a similar extent in AM patients' blood. Cultivation in the presence of myelin basic protein and proteolipid protein revealed strong augmentation of IL-6 mRNA-positive cells in MS but not in OND. The results suggest that IL-6 is one of several cytokines which are upregulated in MS, in particular locally in the CSF. A role of IL-6 in MS, whether disease- promoting or protective, remains unclear.