Gene expression of selected immune regulatory molecules in neuroimmunological diseases

Background: Multiple sclerosis (MS) is an inflammatory, demyelinating disease that is specific to the central nervous system (CNS). Myasthenia gravis (MG) is a disorder of the neuromuscular junction. Both MS and MG are usually considered autoimmune diseases but how the immune system is involved is poorly understood. Partly contradicting results of cytokines in NIS and MG likely reflect methodological variations such as need for stimulation with antigen in vitro, or a low degree of sensitivity by most methods. The aim of the study was to develop a sensitive, accurate and reliable method to analyze mRNA levels of key immune regulatory molecules in unstimulated cells in MS and MG.

Patients and methods: PBMC were obtained from patients with either MS or MG and healthy volunteers. Total RNA was purified from PBMC without stimulation in vitro and reverse transcribed to cDNA. The mRNA levels of selected genes and reference genes were quantified by a fluorescence based competitive polymerase chain reaction (PCR) procedure. In some studies, protein levels were also detected by Western blot.

Results: Compared with healthy controls, mRNA levels of tumor necrosis factor alpha (TNF-[alpha]) was increased in chronic progressive NIS patients, whereas interleukin-10 (IL-10) was decreased in all NIS patients as well as in clinical subgroups of MS. Interferon gamma (IFN-[gamma]) and IL-4 were without significant differences in NIS patients. CD40 and CD40L were upregulated in NIS patients. Fas ligand (FasL) and TNF-related apoptosis inducing ligand (TRAIL) were similarly upregulated in MS patients. TRAIL receptor 2 was slightly elevated whereas Fas was similar in both patients and controls. In MG patients, TNF-[alpha] was decreased. IL-10 was also decreased, most obviously in non-thymectomized patients. No significant differences of IFN-[gamma] and IL-4 were found in MG patients. Fas and FasL was decreased, which was confined to non-thymectomized patients, especially for patients without immunosuppressive treatment, in which TRAIL-R2 and TRAIL were also decreased. The expression of these genes in thymectomized MG patients were similar as in controls. The protein levels of Fas and FasL detected by Western blot showed a parallel pattern as mRNA in both MS and MG.

Conclusions: With a newly developed method, we are able to quantify selected immune regulatory molecules in PBMC without stimulation in vitro. This method to assess gene expression reflects better the in vivo expression pattern. An increased expression of TNF-[alpha] in chronic progression and a decreased expression of IL-10 in all MS patients suggests an altered cytokine pattern in MS. Increased expression of CD40 and CD40L implies a systemic dysregulation of the immune response in MS. The finding of up-regulation of FasL and TRAIL indicates an up-regulation of apoptosis-associated mechanisms in the periphery in MS. However, further studies are needed to find out whether this procession is involved in the tissue damage. Decreased mRNA levels of TNF-[alpha] and IL-10 suggest an altered cytokine expression pattern in MG. The observation of decreased expression of Fas, TRAIL-R2 and their ligands in MG, especially in non-thymectomized patients, implicate that an altered state of apoptosis in lymphocytes may contribute to MG pathogenesis. The combined observations in MS and MG suggest an intriguing difference between these two immune disorders that deserves analysis in more detail.

List of scientific papers

I. Huang WX, Huang P, Link H, Hillert J (1999). Cytokine analysis in multiple sclerosis by competitive RT - PCR: A decreased expression of IL-10 and an increased expression of TNF-alpha in chronic progression. Mult Scler. 5(5):342-348.
https://pubmed.ncbi.nlm.nih.gov/10516778

II. Huang WX, Huang P, Hillert J (2000). Systemic upregulation of CD40 and CD40 ligand mRNA expression in multiple sclerosis. Mult Scler. 6(2):61-65.
https://pubmed.ncbi.nlm.nih.gov/10773848

III. Huang WX, Huang P, Gomes A, Hillert J (2000). Apoptosis mediators FasL and TRAIL are up-regulated in peripheral blood mononuclear cells in multiple sclerosis. Neurology. 55(7):928-934.
https://pubmed.ncbi.nlm.nih.gov/11061246

IV. Huang WX, Huang P, Fredrikson S, Pirskanen R, Hillert J (2000). Decreased mRNA expression of TNF-alpha and IL-10 in non-stimulated peripheral blood mononuclear cells in myasthenia gravis. Eur J Neurol. 7(2):195-202.
https://pubmed.ncbi.nlm.nih.gov/10809941

V. Huang WX, Huang P, Jin YP, Pirkanen R, Hillert J (2000). Decreased mRNA expression of Fas, TRAIL-R2 and their ligands in peripheral blood mononuclear cells in myasthenia gravis. [Submitted]