Wen-Xin Huang

CTLA-4 gene expression is influenced by promoter and exon 1 polymorphisms.

CTLA-4, expressed mainly on activated T cells, helps maintain, through its inhibitory function, immune-system homeostasis. Polymorphisms in the CTLA-4 gene (CTLA4) are known to be important in several autoimmune diseases, including multiple sclerosis (MS). Here, we have performed genotyping for CTLA4 polymorphisms, and investigated expression by peripheral blood mononuclear cells of CTLA-4 mRNA and protein, in patients with MS and myasthenia gravis and in healthy controls. Expression levels for mRNA and protein were similar in the patient and control groups; however, there was a clear relationship between genotype and CTLA-4 expression. Specifically, individuals carrying thymine at position −318 of the CTLA4 promoter (T−318) and homozygous for adenine at position 49 in exon 1 showed significantly increased expression both of cell-surface CTLA-4 after cellular stimulation and of CTLA-4 mRNA in non-stimulated cells. The association was seen most clearly for unsorted CD3+ cells and was absent in the CD8+ subset. The T−318 allele has been shown to be negatively associated with susceptibility to MS in an earlier study by our group. Thus, we propose that the susceptibility-influencing role of CTLA4 in MS may be related to genotypically conditioned promoter function, whereby high gene expression may decrease the risk of disease.

Apoptosis mediators fasL and TRAIL are upregulated in peripheral blood mononuclear cells in MS.

Objective: To investigate the expression of apoptosis-inducing ligand and receptor molecules in patients with MS. Background: Dysregulation of apoptosis may induce autoimmune conditions, possibly through inadequate termination of immune responses, and could be of importance for pathogenesis of MS. Methods: Messenger RNA (mRNA) levels of two apoptosis-related members of the tumor necrosis factor (TNF) receptor family, Fas and TNF-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2), and their ligands, Fas ligand (FasL) and TRAIL, were quantified by competitive reverse transcription PCR in unstimulated peripheral blood mononuclear cells in 47 untreated patients with MS and 46 control subjects. Results: The expression of FasL was increased in patients with MS compared with healthy control subjects. Analysis of clinical subgroups revealed that the increase was marked in relapsing-remitting MS, being especially high in remission (p = 0.0002), but less so in chronic progressive MS (p = 0.14). Compared with healthy control subjects, TRAIL mRNA levels were also upregulated in patients with MS (p = 0.0001) but did not differ between clinical subgroups. The expression of TRAIL-R2 was slightly elevated in patients with MS (p = 0.02) whereas the expression of Fas was similar in patients and control subjects. The ratio of expression levels for two isoforms of TRAIL-R2, TRICK2a and TRICK2b, in patients with MS differed from healthy control subjects (p = 0.04). Conclusions: There was increased expression of both FasL and TRAIL in peripheral blood lymphocytes. It remains to be determined whether this increased expression represents a disease-promoting autoimmune process or is merely the effect of a secondary compensatory mechanism that downregulates the inflammatory response.

Increased expression of caspase-1 and interleukin-18 in peripheral blood mononuclear cells in patients with multiple sclerosis

Multiple sclerosis (MS) is supposedly a T-cell mediated autoimmune disorder of the central nervous system. Cytokines and other molecules involved in the regulation of apoptosis are thought to be of importance for the pathogenesis of MS. In this study, the mRNA levels of interleukin 18 (IL-18), IL-1b and their processing enzyme caspase-1 were quantified by a competitive RT-PCR method in unstimulated peripheral blood mononuclear cells (PBMCs) in MS patients never treated with disease modifying drugs. Western blot was used to support the expression pattern at the protein level. We found that the expression of caspase-1 and IL-18 was significantly increased in MS patients compared with healthy controls. Analysis of clinical subgroups revealed that caspase-1 was increased in all subgroups, whereas IL-18 was upregulated in chronic progression (P-0.001) and relapsing MS patients in remission (P-0.002) but not significantly during relapses (P-0.12). mRNA levels of IL-1b were not significantly altered in MS except for a possible decrease in chronic progression (P-0.03). An increased IL-18 expression, potentially augmented at the mature protein level, may indicate a pathway worth considering in future therapeutic strategies in MS.

Cytokine analysis in multiple sclerosis by competitive RT - PCR: A decreased expression of IL-10 and an increased expression of TNF-α in chronic progression

Multiple sclerosis (MS) is an inflammatory, demyelinating disease that is specific to the central nervous system. Cytokines are thought to be key mediators of the autoimmune attack against central nervous system myelin in MS. To investigate the involvement of cytokines in MS, the mRNA levels of interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), interleukin-4 (IL-4) and interleukin-10 (IL-10) in peripheral blood mononuclear cells without stimulation in vitro were quantified by a competitive reverse transcription polymerase chain reaction technique. The level of IL-10 specific mRNA was significantly decreased in 47 MS patients compared with 42 healthy controls (P50.0001). TNF-a was significantly increased in MS patients compared with healthy controls (P=0.014), especially in the patients with chronic progressive MS (P=0.0003). Thus we conclude that there are significant in vivo alterations in cytokine gene expression in the periphery in MS.

Dendritic cells derived from patients with multiple sclerosis show high CD1a and low CD86 expression

Dendritic cells (DC) are important antigen presenting cells (APC) and play a major role in initiating and orchestrating immune responses by priming T cells. Little is known about involvement of DC in multiple sclerosis (MS), where auto-aggressive T cells against myelin autoantigens are considered to contribute to inflammation and demyelination in the central nervous system. In this study, we compared phenotype and cytokine secretion of DC from patients with MS, other neurological diseases (OND) and healthy subjects. DC were generated from blood adherent mononuclear cells (MNC) by culture for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The yield and morphology of DC were similar in MS patients and controls. In both, the DC phenotype was that of immature myeloid lineage, comprising CD1a+ and CD11c+. The proportion of CD1a+ DC, being important for presentation of lipid antigens to T cells, was higher in MS patients compared to controls. The proportion of CD86+ DC, a co-stimulatory molecule that is assumed to promote Th2 differentiation, was low in MS. Low proportions of CD86+ DC were only observed in untreated MS patients but not in patients treated with IFN-b. Production of IL-10 and IL-12 p40 by DC did not differ in MS patients and controls. These findings indicate that alterations of functionally important surface molecules on DC are associated with MS.

An angiotensin‐converting enzyme gene polymorphism suggests a genetic distinction between ischaemic stroke and carotid stenosis

Ischaemic cerebrovascular disease (ICVD) is a heterogeneous syndrome to which different genetic factors may contribute. We have investigated the distribution of alleles of the angiotensin‐converting enzyme (ACE) gene, which has been suggested to be of possible importance in ischaemic stroke or cardiovascular disease, in groups of patients with ischaemic stroke and carotid artery stenosis (CS).

Th1-like cell responses to peripheral nerve myelin components over the course of experimental allergic neuritis in Lewis rats

Experimental allergic neuritis (EAN) is a T cell‐mediated animal model of Guillain‐Barré syndrome characterized by inflammation and demyelination of peripheral nerves. EAN can be induced by immunization of rats with bovine peripheral nerve myelin (BPM) or the myelin proteins P2 or PO, but the extent of T cell responses over the course of EAN is incompletely defined. We studied the T cell responses to these proteins and the glycolipid GM1 by enumerating T helper type 1 (Th1)‐like cells secreting interferon‐γ (IFN‐γ) after short‐term culture of mononuclear cells (MNC) in presence of antigen. Already 7 days post immunization (p.i.) with BPM and before onset of clinical EAN, lymph nodes contained elevated levels of P2 responsive T cells. At the height of EAN on day 14 p.i. and during recovery, T cell levels responding to BPM, PO and GM1 were also elevated. The same temporal profiles and specificities were registered for antigen reactive spleen MNC. The results implicate that Th1‐like cells with multiple specificities including the glycolipid GM1 occur at increased levels in lymphoid organs in EAN rats, and that IFN‐γ may be an important effector molecule in the induction of nerve damage.

Lipoprotein lipase gene polymorphisms in ischaemic stroke and carotid stenosis

Ischaemic stroke is pathogenetically heterogeneous, but there is strong evidence that genetic as well as environment factors contribute to the risk of the individual. Here we report the similar distribution of polymorphic markers of the lipoprotein lipase (LPL) gene in 128 patients with ischaemic stroke, 56 patients with carotid artery stenosis and 95 healthy control subjects, in spite of a significant influence of the Asn291→ Ser mutation on serum levels of triglycerides. We conclude that these LPL polymorphisms do not contribute greatly to the overall risk of ischaemic stroke in the general population.

Systemic upregulation of CD40 and CD40 ligand mRNA expression in multiple sclerosis.

It is increasingly clear that the CD40 and CD40 ligand (CD40L) receptor-ligand pair mediates a crucial activation signal in both cell-mediated and humoral immune responses. Here, we detected mRNA levels of CD40 and CD40L in non-stimulated peripheral blood mononuclear cells in 46 patients with multiple sclerosis (MS) and 46 healthy controls by a competitive RT-PCR procedure allowing quantification without previous culture or antigenic stimulation. The levels of CD40 and CD40L mRNA were markedly increased in MS patients (P <0.0001) compared with healthy controls. There was no difference between clinical MS subgroups or stage of disease. Our findings indicate that, although MS is an organ specific disorder an increased signaling via the CD40 and CD40L pathway may be present at the systemic level. The nature of this upregulation, whether primary or secondary to the organ-specific autoimmune response, is yet to be determined. Since interference with CD40/CD40L is an effective way to interfere with autoimmune model diseases such as experimental autoimmune encephalomyelitis, it may be relevant to investigate further the role of these molecules in the pathogenesis of MS.

T-Cell Immunity to Acetylcholine Receptor and its Subunits in Lewis Rats over the Course of Experimental Autoimmune Myasthenia Gravis

Lymph nodes, spleen and thymus obtained from Lewis rats were examined over the course of experimental autoimmune myasthenia gravis (EAMG) for the distribution and the number of antigen‐reactive CD4+ T helper cells which, upon recognition of Torpedo acetylcholine receptor (AChR) or the α, β, γ or δ subunits of Torpedo AChR, responded by secretion of interferon‐gamma (IFN‐γ). T cells with these specificities were detected in these three immune organs. Numbers were highest in lymph nodes. In spleen and thymus, numbers of antigen‐reactive T cells did not differ. T cells reacting against the intact AChR were more frequent than T cells recognizing any of the subunits. The immunogenicity between the four subunits did not differ, with the exception that the α subunit induced a slightly higher T‐cell response. No restriction of the T‐cell repertoire to the four subunits was detected during early compared to late phases of EAMG. The AChR and subunit‐reactive T cells could—via secretion of effector molecules including IFN‐γ—play an important role in the initiation and perpetuation of EAMG. and consequently also of human myasthenia gravis. T cells with the same specificities were also detected in control animals injected with adjuvant only, but at much lower numbers which were within the range of T cells recognizing the control antigen myelin basic protein. They could represent naturally occurring autoimmune T cells.