D. Matusevicius

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.

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.

Tumor necrosis factor-α, 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.

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‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α) and lymphotoxin (LT) that may make MS worse, and anti‐inflammatory cytokines like IL‐4, IL‐10 and transforming growth factor‐β (TGF‐β) that may act beneficially. Substances that down‐regulate cytokines such as TNF‐α or promote IL‐10 or TGF‐β can be anticipated to affect MS beneficially. Material and methods ‐ In situ hybridization to detect and enumerate IFN‐γ, TNF‐α, LT, IL‐4, IL‐10 and TGF‐β 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‐γ secreting blood MNCLinomide was used. Results ‐ Here we report that Linomide, a synthetic immunomodulator, at concentrations effective in vivo reduces the number of MBP‐reactive TNF‐a and increases MBP‐reactive IL‐10 and TGF‐β 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‐β 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.

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. Perform mRNA expressing CSF MNC were not detected in any of the patients lacking active lesions on brain MRI. The expression of tumor necrosis factor‐α, 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.

Autoantigen-induced IL-13 mRNA expression is increased in blood mononuclear cells in myasthenia gravis and multiple sclerosis

Evidence has been presented for the involvement of immune mechanisms in the pathogenesis of myasthenia gravis (MG) and multiple sclerosis (MS). The production of autoantibodies in both diseases is regulated by T‐cells by means of cytokines. Interleukin‐13 (IL‐13) is mainly produced by T‐helper type 2 cells and induces B‐cell proliferation and antibody class switch. The role of IL‐13 in MG and MS is not known. We employed in situ hybridization with synthetic radiolabelled oligonucleotide probes to detect and enumerate blood and cerebrospinal fluid (CSF) mononuclear cells (MNC) expressing IL‐13 mRNA from patients with MG, MS, optic neuritis (ON), other inflammatory neurological diseases (OIND) and healthy controls. MG is associated with elevated levels of acetylcholine receptor (AChR) reactive IL‐13 mRNA expressing blood MNC compared to control patients. In MS, numbers of MBP‐reactive IL‐13 mRNA expressing MNC were higher compared to cultures without antigen stimulation. The levels of MBP‐reactive IL‐13 mRNA positive MNC were higher in MS compared to MG, but not other controls. There were no differences in spontaneous IL‐13 mRNA expressing blood MNC numbers between MG, MS, ON and control patients. The data suggest the involvement of IL‐13 in both MG and MS.

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 ± 106) 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.

Experimental Myasthenia Gravis in Mice: The role of costimulatory molecules

Activation of T-cells requires two signals. One signal is provided by the cognate TCR/MHC-peptide interaction. The second costimulatory signal is mediated through ligation of CD28 by B7 [1]. The CD28 molecule is a 44-kD glycoprotein, which is expressed on all murine and human T cells, while its counter receptor pair B7-1 (CD80) and B7-2 (CD86) are expressed mainly on antigen presenting cells (APCs) [1]. In addition to the conventional signals 1 and 2, a direct signal delivered to T-cells via triggering or cross-linking of the CD40 ligand (CD40L) is also required for full activation of T cells to perform effector functions and to produce cytokines [2]. T-helper cells activate resting B-cells through TCR recognition of MHC class II-peptide complexes and costimulation through CD40L/ CD40 interactions [2]. CD40L is a 33-kDa membrane protein that is preferentially expressed on activated CD4+ T cells [2], while the counter-receptor for CD40L is CD40, expressed on APCs [2]. The mechanisms for CD28 and CD40L in the regulation of the immune system at the cellular and molecular levels are just beginning to be elucidated. Interactions of CD28-B7 and CD40-CD40L have been implicated in T cell-mediated autoimmune diseases such as experimental allergic encephalomyelitis (EAE) and insulin-dependent diabetes mellitus [3-6].

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.