Systemic sclerosis (SSc) is a disease of the connective tissue characterized by vascular changes and immunological dysfunctions which lead to progressive skin and internal organ fibrosis. First clinical manifestation is usually Raynaud’s phenomenon (RP) (paroxysmal blanching with subsequent cyanosis and swelling of hands) connected with a generalized vasculopathy of minor vessels in the skin and internal organ areas (1). A consequence of rupturing the endothelium is a migration of mononuclear peripheral blood cells to the extravascular space and creation of inflammatory infiltrations characteristic for SSc (2, 3). T-cells and monocytes are dominant in this infiltration. Whilst producing a series of cytokines and growth factors these cells are able to initiate a series of intercellular interactions leading to vessel changes as well as disregulation of synthesis and degradation of extracellular matrix components. The main role in the fibrosis processes in SSc could be played by transforming growth factor β1 (TGFβ1) produced in excess by peripheral blood mononuclear cells (PBMC) (4). It has been proved that this cytokine can stimulate gene transcription of collagen by stimulation of synthesis or activation of specific transactive DNA binding factors (5, 6). In patients with limited SSc (lSSc) and diffuse SSc (dSSc) treated with pamidronate (aminobisphosphonate) a approx. 30% decrease in TGFβ1 production by the PBMC was noticed which could explain a positive therapeutic effect (7). Hasegawa et al. (8) demonstrated an increase in TGFβ1 production by PBMC in patients with SSc in comparison with a healthy persons control group. However, this data was not confirmed in other papers. In research of Giacomelli et al. (9) the TGFβ1 concentration in the serum and supernatants of the PBMC culture from SSc patients in spontaneous conditions as well as after phytohemagglutinin (PHA) stimulation was not different from the control group. The concentration of TGFβ1 in serums of patients with SSc can remain unchanged, reduced in relation to the control group or be below the lower limit of detection, however TGFβ1 can be present in large amounts in the tissue (10-14). Reasons for this are faintly sensitive methods or inhibitors appearing in the serum. In physiologic states TGFβ1 binds with proteins (latency-associated peptide, α2-macroglobulin), which can largely conceal its presence in the blood and are responsible for a non-linear diagnosed sample dilution curve line and a divergence in relation to the standard curve in the ELISA method (15, 16).

An analysis of gene expression in the earlier stage of this process, that is the transcription level, not only doesn’t possess such limitations but also allows for the detection of molecular changes preceding changes at protein level. Therefore the main aim of the study was to evaluate the number of mRNA copies of genes coding TGFB1 and its receptors in peripheral blood leukocytes changes in patients with SSc and isolated RP in which capillaroscopy and (or) immunological markers presence suggested a risk of SSc development.

The study group consisted of 27 patients (26 women and 1 man) with RP, aged 18 to 65, average 48.1 ± 11.6 years hospitalized in Medical University of Silesia – Dermatology Department in Katowice with a suspicion or diagnosed SSc. The RP lasted for 0.3 to 25 years, average 8.2 ± 5.9 years. A capillaroscopy examination of the nailfold was carried out for each patient, antinuclear antibodies were marked with the indirect immunofluorescence (IIF) method on Hep-2 cells and detailed diagnostic research was conducted allowing for an assessment of the internal organs affected by pathological changes. Changes in the esophagus were diagnosed based on confirmed peristaltic dysfunction and/or smoothing out the mucous membrane folds in a radiological examination of the esophagus. Influence on the lungs was attested to bilateral fibrosis changes in chest X-ray. Cardiologic changes with characteristics of arrhythmia, conductivity disorders in ECG examination or during transesophageal electrostimulation and syndromes of right ventricle failure, prior to lung hypertension were diagnosed as heart muscle involvement in course of SSc. Influence on the kidney by the disease process was diagnosed based on a persistent proteinuria and coexisting arterial hypertension. Myositis type muscle changes aside from clinical symptoms: muscle weakness and pain were diagnosed based on increased activation of muscle enzymes (creatine phosphokinase and aldolase) as well as aberrations in electromyography and histopathology examinations. Apart from this, routine laboratory tests were carried out: ESR, blood morphology, general urine test. Other tests were: Waaler-Rose reaction, latex-R, electrophoresis of serum protein division and assessment of kidney functions.

In 19 patients SSc was diagnosed based on the American College of Rheumatology (17) criteria, remaining 8 were female patients with an isolated RP. Skin changes in patients with SSc corresponded with lSSc – appeared on the skin of the face, upper limbs up to 1/3 of the forearm. Table 1 presents a clinical characteristic of patients with RP without clinical symptomes of connective tissue diseases and patients with lSSc. Patients qualified for the research were not treated earlier with immunosuppressive agents and (or) steroids. Control samples were obtained from 8 healthy volunteers. The Medical University of Silesia Local Research Ethics Committee approved the study and all subjects provided informed consent to participate.

Total RNA was isolated from the 500 μl whole blood samples using acid guanidinium-thiocyanate phenol-chloroform method (18). Extracts of total RNA were purified with the use of RNeasy Mini Kit (Qiagen Gmbh, Hilden, Germany), in accordance with manufacturer protocol. The quality of RNA was estimated by electrophoresis on a 1% agarose gel stained with ethidium bromide. The RNA concentration was determined by absorbance at 260 nm using a Gene Quant II spectrophotometer (Pharmacia LKB Biochrom Ltd., Cambridge, UK).

The quantitative analysis was carried out with the use of Sequence Detector ABI PRISM™ 7000 (Applied Biosystems, Kalifornia, USA). The quantity of PCR products was determined after each round of amplification, using fluorescent dye SYBR Green I (Qiagen Gmbh, Hilden, Germany) that binds double-stranded DNA. The standard curve was appointed for standards of β-actin (Applied Biosystems, Kalifornia, 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 gene TGFβ1, TGFβR1, TGFβR2, TGFβR3 and β-actin (endogenous control) expression, chemical and thermal conditions of amplification were as previously (19-21).

Sequence specificity of amplimers was proved by analysis with ABI PRISM™ 377 DNA Sequencer (Applied Biosystems, Kalifornia, USA). Melting temperatures of amplimers were assessed by SYBR Green I Dissociation assay (Dissociation Curve Software – Applied Biosystems, Kalifornia, USA). The PCR products and molecular weight marker pBR 322/Hae III (Fermentas International Inc., Ontario, Canada) were separated on 8% polyacrylamide gel and visualized using silver staining (LKB-Pharmacia). The length of amplified fragments was assessed by analysis with GelScan v.1.45 software (Kucharczyk TE, Warsaw, Poland).

The values were expressed as median and range. Quantitative data were compared by a nonparametric Mann-Whitney U test. Correlations were evaluated using the Spearman rank correlation coefficient test. P < 0.05 was considered significant. All calculations were performed with Statistica Version 6.0 software (StatSoft Inc., Oklahoma, USA). The expression of the TGFβ1, TGFβR1, TGFβR2, TGFβR3 and β-actin genes was expressed as a ratio of the mRNA copy number to the 1 μg of total RNA in samples studied.

In all samples analyzed mRNA of β-actin gene was demonstrated, thus indicating the integrity of the RNA extracts.