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.

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.

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).