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© Borgis - Postępy Nauk Medycznych 10/2012, s. 765-770
*Anna Lis-Święty1, Joanna Gola2, Urszula Mazurek2, Ligia Brzezińska-Wcisło1
Aktywność transkrypcyjna genów kodujących czynnik martwicy nowotworów α i jego receptorów u chorych z twardziną układową i objawem Raynauda
Transcriptional activity of genes coding tumour necrosis factor α and its receptors in patients with systemic sclerosis and Raynaud’s phenomenon
1Dermatology Department, Medical University of Silesia, Katowice
Head of Department: prof. Ligia Brzezińska-Wcisło, MD, PhD
2Department of Molecular Biology, Medical University of Silesia, Sosnowiec
Head of Department: prof. Urszula Mazurek, MD, PhD
Wprowadzenie. Znaczenie TNFα w patogenezie twardziny układowej (ang. systemic sclerosis – SSc) budzi nadal kontrowersje. Czynnik ten może odgrywać znaczącą rolę we wczesnym stadium rozwoju SSc, tzn. w zmianach naczyniowych (objaw Raynauda, ang. Raynaud phenomenon – RP) i procesie włóknienia.
Cel pracy. Celem pracy było zbadanie jak się zmienia liczba kopii mRNA genów kodujących TNFα i jego receptory w leukocytach krwi obwodowej chorych z SSc i RP.
Materiał i metody. Badaniem objęto 19 chorych z aktywną limited SSc (lSSc), 11 pacjentek z izolowanym objawem RP i 10 osób zdrowych. Oznaczenie mRNA genów kodujących TNFα, TNFαRI i TNFαRII przeprowadzono techniką ilościowej reakcji amplifikacji z odwrotną transkrypcją w czasie rzeczywistym (ang. Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction, real-time QRT-PCR).
Wyniki. Liczba kopii mRNA dla TNFα była znacząco niższa u chorych z aktywną lSSc w stosunku do grupy kontrolnej. Nie stwierdzono znaczących różnic w aktywności transkrypcyjnej dla TNFα, pomiędzy grupą chorych z aktywną lSSc a pacjentami z RP, jak również pomiędzy chorymi z izolowanym RP a grupą kontrolną. W porównaniu z kontrolą, ekspresja TNFαRI i TNFαRII były znacząco niższe, zarówno u chorych z aktywną lSSc, jak i u pacjentów z izolowanym RP. Stosunek TNFα/TNFαRII mRNA w obu grupach, aktywnej lSSc i izolowanym RP, był znacząco wyższy w porównaniu z grupą kontrolna. Stosunek TNFαRI/TNFαRII mRNA był wyższy u chorych z aktywną lSSc niż w grupie kontrolnej.
Wnioski. Obniżenie aktywności transkrypcyjnej genów TNFα, TNFαRI i TNFαRII może być kluczowe w procesie włóknienia związanym z SSc. Proporcje pomiędzy receptorami mogą być ważne w regulacji aktywności antyfibrogennej TNFα.
Introduction. The role of TNFα in the pathogenesis of systemic sclerosis (SSc) is still controversial. This factor may play a significant role at the early stage of SSc development, i.e. in vascular changes (Raynaud’s phenomenon – RP) and in fibrosis processes.
Aim. The aim of this study was to investigate the changes in the number of mRNA copies of genes coding TNFα and its receptors in peripheral blood leukocytes in patients with SSc and RP.
Material and methods. The research concerned 19 patients with active lSSc, 11 patients with isolated RP and 10 healthy persons. Quantification of TNFα, TNFαRI and TNFαRII genes mRNA was carried out with the use of Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction.
Results. The number of copies of TNFα mRNA in active lSSc patients was significantly lower than in the control group. No statistically significant difference in transcriptional activity of TNFα gene between active lSSc and isolated RP patients as well as isolated RP and the control group was found. Comparing to controls the expressions of TNFαRI and TNFαRII were significantly lower both in active lSSc patients and in isolated RP patients. The TNFα/TNFαRII ratio both in active lSSc and isolated RP patients was significantly higher opposed to the control group. The TNFαRI/TNFαRII mRNA ratio was significantly higher in patients with active lSSc than in the control group.
Conclusions. The decrease in transcriptional activity of TNFα, TNFαRI and TNFαRII genes in SSc may be crucial in fibrosis. Receptors proportion may be important in the regulation of TNFα anti-fibrotic activity.

Systemic sclerosis (SSc) is a systemic connective tissue disease, in which skin, organs and systems, such as digestive tract, lungs, kidneys and heart are undergoing progressive fibrosis. Etiopathogenesis of this disease is still mostly unknown. Usually, first clinical symptom is Raynaud’s phenomenon (RP) – sudden discoloration with subsequent edema of hands – resulting from general vasculopathy of small vessels within the skin and internal organs (1). Destruction and activation of endothelium and fibrosis process are related, among others, with expression of many cytokines. Tumour necrosis factor α (TNFα) belongs to the most important mediators and it stimulates synthesis of many pro-inflammatory cytokines (IL-1, IL-8, IL-6, GMCSF), stimulates proliferation of fibroblasts, decreases matrix metalloproteinase activity, induces expression of ICAM-1, VCAM-1 and E-selectin on endothelial cells, it influences release of: von Willebrand’s factor (vWF), vasoconstriction factors – endotheline-1 (ET-1), many cyclooxygenase products (COX), such as thromboksan A2 (1-4). It also influences expression of endothelial nitric oxide synthetase by shortening its half-life time in endothelial cells and increases both proliferation and apoptosis (by activating caspase-3) in vascular smooth muscle cells, therefore regulating the number of these cells (5, 6). However, the role of TNFα for fibrosis in SSc is controversial. Actually there is good evidence that TNFa has anti-fibrotic effects at least in vitro (7). Moreover, TNFα is rarely detected in sera of the patients with SSc, and its concentration weakly correlates with the clinical status (8-10). This is explained by a short half-life time of this cytokine and the presence of circulating inhibitors, mostly soluble TNFα receptors (10). Soluble TNFαRI and sTNFαRII receptors are present in the circulation of healthy persons in little amounts, but upon activation of the immune system their concentration may significantly increase exceeding by 100 folds concentration of this cytokine (10). We have reported increased TNFαRI levels in sera of patients with SSc and some of the patients with RP evolving to SSc (11). TNFα protein has not been detected in the examined SSc sera in our studies (8).
Therefore, the aim of this work was to asses the changes in the number of copies of mRNA for TNFα and its receptors in leukocytes from the patients with active lSSc and isolated RP.
aim of the study
The study group consisted of 19 patients (18 women and 1 man) with active lSSc and 11 women with isolated RP. The diagnoses of active lSSc and early SSc were based on clinical, laboratory, capillaroscopic and autoantibody findings. Internal organ involvement, namely lung, kidney, heart, gastroitestinal tract or muscle, was also documented by routine investigation in all patients. Antibodies were marked with the indirect immunofluorescence (IIF) method on Hep-2 cells and double immunodiffusion. SSc pattern on NCM was defined as definitely enlarged capillaries and/or capillary loss of grades and/or capillary telangiectases (12).
Active lSSc was diagnosed based on the American College of Rheumatology (13) criteria and activity index in SSc according to European Scleroderma Study Group (14). Skin changes in these patients corresponded with limited SSc – appeared on the skin of the face, upper limbs up to 1/3 of the forearm. Patients with isolated RP had any of the SSc-specific autoantibodies (anti-Scl 70, anti- centromere, anti-RNA polymerase III) and/or an SSc pattern on NCM. RP was defined as a history of at least 2 of 3 phases of color change (white, blue, red), usually induced by cold exposure, and involving at least 1 finger of each hand (14). These patients did not display clinical manifestations of SSc or another CTD, such as sclerodactyly, digital ulcers, or pitting scars, loss of distal finger pad, clinically visible capillary telangiectases, or calcinosis (15).
Presents a clinical characteristic of patients with isolated RP and active lSSc (tab. 1). Patients qualified for the research were not treated earlier with immunosuppressive agents and (or) steroids. Control samples were obtained from 8 healthy women aged 47.6 ± 8.3 years. The Medical University of Silesia Local Research Ethics Committee approved the study and all subjects provided informed consent to participate.
Table 1. Clinical characteristics of patients with isolated RP and patients with lSSc.
 Isolated RP
n = 11
n = 19
Age (years)44.6 ± 14.1*49.5 ± 10.8*
Duration of RP7.1 ± 5.9*10.7 ± 5.9*
Duration of sclerodactyly3.2 ± 1.9*
SSc pattern on NCM719
Immunologic markers
Anti-Scl 70
Anti- RNA polymerase III
Antibody with homogenous pattern of immunofluorescence

Visceral involvement


*Average ± standard deviation
material and Methods
Extraction of total RNA. Total RNA was extracted from the 500 μl whole blood samples with the use of acid guanidinium-thiocyanate phenol-chloroform method /20/. Extracts of total RNA were purified with the use of RNeasy Mini Kit (Qiagen Gmbh, Germany), according to the manufacturer's instructions. The quality of RNA extracts was estimated electrophoretically using 1% agarose gel stained with ethidium bromide. The results were analyzed and recorded with the gel documentation system 1D Bas-Sys (Biotech-Fisher, Perth, Australia). The total RNA concentration was determined by spectrophotometric measurement at 260 nm using a Gene Quant II RNA/DNA Calculator (Pharmacia LKB Biochrom Ltd., Cambridge, UK).
mRNA quantification by Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction. The quantitative analysis was carried out with the use of Sequence Detector ABI PRISM™ 7000 (Applied Biosystems, California, USA). The quantity of PCR products was determined after each round of amplification, using fluorescent dye SYBR Green I (Qiagen Gmbh, Germany) that binds double-stranded DNA. The standard curve was appointed for standards of β-actin cDNA (TaqMan® DNA Template Reagents Kit, Applied Biosystems, California, USA). For this assay positive (β-actin mRNA) and negative (no template) controls were carried out. The nucleotide sequences of the PCR primers used to assay TNF, TNFR1, TNFR2 and β-actin (endogenous control) gene expression, chemical and thermal conditions of amplification were as previously (16, 17).

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otrzymano: 2012-08-22
zaakceptowano do druku: 2012-09-28

Adres do korespondencji:
*Anna Lis-Święty
Dermatology Department Medical University of Silesia
ul. Francuska 20/24, 40-027 Katowice
tel.: +48 602-720-948, fax: +48 (32) 256-11-82
e-mail: annadlis@neostrada.pl

Postępy Nauk Medycznych 10/2012
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