The study population consisted of a total of 1180 German individuals. All subjects included in this study were interviewed using a questionnaire to obtain information on lifestyle (including a lifetime history of tobacco use) and occupational history. According to their reported smoking habits, patients were classified into smokers, ex-smokers or never smokers. Individual smoking pack years (PY) were calculated. One PY was defined as smoking 20 cigarettes daily over one year. Written informed consent was obtained from all patients before inclusion in the study.

The control group comprised of 177 unrelated, healthy subjects without known diseases and without any exposure to carcinogenic (or fibrogenic) agents at the work place. Any subject with diseases related to potential tissue fibrosis (e.g. diabetes, chronic lung disease) or with benign or malignant tumors were excluded.

The lung fibrosis patients group contained 605 subjects. Only subjects with confirmed diagnosis of lung fibrosis, according to the WHO criteria (26,27) were included. The lung cancer patient group contained 364 and the malignant mesothelioma group contained 34 patients. Only subjects with histological confirmed diagnosis of primary lung cancer or diffuse malignant mesothelioma, according to the WHO criteria (28), were included.

Diagnosis of pulmonary diseases was based on physical examination, haematological, biochemical and immunological laboratory analyses and pulmonary function tests. All patients underwent diagnostic procedures, including X-ray examination interpreted according to the International Labour Office (ILO) Classification of Radiographs of Pneumoconiosis (29).

To allow further discrimination between different kinds of exposure, several groups of patients suffering from fibrosis or lung cancer were created and compared to a non-exposed control group (Table I). In a second evaluation step, the asbestosis group was further subdivided into groups considering the expansion and severity code of fibrosis. These groups were established using chest X-ray findings according to the ILO Classification of Radiographs of Pneumoconiosis (29).

Chest X-rays were reviewed and graded according to the ILO 2000 classification. According to the ILO standard X-rays, three profusions (1, 2 and 3) and the six size and shapes of the opacities (p, q, r for rounded, s, t, u for irregular, and A, B and C for large opacities) were coded. Asbestos-related abnormalities were classified as asbestosis (parenchymal changes) or as asbestos-induced pleural diseases (Table II) (29). Diffuse pleural thickening without parenchymal bands were only observed in adipose patients and as such were attributed to subpleural fat. The Ethics Committee of the University Hospital, Giessen, Germany, approved the study (AZ:75/06).

Whole blood (3 ml) samples were collected by venipuncture in tubes containing EDTA (Sarstaedt, Nümbrecht, Germany). Genomic DNA was isolated from whole blood using the Versagene™ DNA purification kit (Gentra Systems, Minneapolis, MN, USA). Detection of the polymorphisms was performed by rapid capillary PCR, with melting curve analysis, using fluorescence-labelled hybridisation probes in a LightCycler System (Roche Diagnostics, Mannheim, Germany). The PCR primers as well as the fluorescent-labelled detection probes were synthesized by TIB Molbiol (Berlin, Germany) (Table III). The reaction mixture (20 μl) for IL-1β C+3954T comprised 2 μl of forward primer (10 μM), 1 μl of reverse primer (10 μM) and 0.4 μl of each probe (10 μM), 1.2 μl MgCl₂ (25 mM), 2 μl FastStart DNA Master Hybridization Probes (Roche Diagnostics) and 2 μl DNA. The reaction mixture (20 μl) for IL-6 and IL-10 comprised 0.6 μl of each primer (10 μM) and 0.4 μl of each probe (10 μM), 10 μl ABgene QPCR Capillary Master Mix (Thermo Fisher, Dreieich, Germany) and 2μl DNA. Annealing temperatures were 60°C for IL-1β C+3954T, 57°C for IL-6 G-174C and 63°C for IL-10 G-1082A polymorphism. The melting curves were generated to obtain melting temperatures. PCR contamination was checked by the inclusion of negative control, where cDNA was replaced by water.

Detection of the IL-1β C-511T polymorphisms was performed by restriction fragment length analysis (RFLA). The reaction mixture (22 μl) for the PCR consisted of 1 μl of each primer (TIB Molbiol) (Table III), 1 μl Q-solution, 2 μl 10X buffer, 2 μl MgCl₂, 1 μl HotstarTaq® Plus (all from Qiagen, Hilden, Germany) 0.2 μl dNTPs (Fermentas, St. Leon-Rot, Germany) and 2 μl DNA. Annealing temperatures in 40 cycles was 58°C. As a restriction enzyme AvaI (New England Biolabs, Ipswich, MA, USA) was used. Results were visualised by agarose gel electrophoresis.

The odds ratios (OR) and confidence intervals (CI) assessed the association between genotype distribution and patient status. The OR and 95% CI were used as an estimate of the risk in all cases and were calculated by unconditional logistic regression. Adjustments for age, gender and tobacco smoking (PY) were computed to estimate the association between certain genotypes and diseases. Current smokers at the time of diagnosis were considered smokers. Ex-smokers were all the people who had ever smoked. Information was collected on the usual number of cigarettes smoked per day, the age at which the subject started smoking and, if the person was an ex-smoker, the age at which the subject stopped smoking. PY were calculated for the cumulative cigarette smoking. The smokers were stratified by the PY values. All statistical analyses were performed using the statistical software package, SSPS 17.0 (SPSS Inc., Chicago, IL, USA). Allelic and genotype frequencies were obtained by direct counting. The Hardy-Weinberg equilibrium was assessed by a χ² test with 2 degrees of freedom. Allelic and genotype frequencies in patient and control groups were compared using a 2×2, contingency table and a χ² test or the 2-tailed Fisher’s exact test when the number of expected cases was too low. The level of significance was set at P<0.06.

The frequency of the IL-6 (−174) C allele in the control group as well as in the patients group was 0.45. OR analyses were performed for the genotype G-174G vs. the genotypes carrying at least one C allele (G-174C or C-174C). Data was adjusted for PY, age (years) and gender.

This analysis revealed that patients carrying at least one C allele of the IL-6 −174 polymorphism were at higher risk for fibrotic lung diseases (OR_asbestosis, 1.338; 95% CI, 0.71–2.53; OR_silicosis, 1.226; 95% CI, 0.54–2.81 and OR_{fibrosis other aetiology}, 1.313; 95% CI, 0.58–2.98). Additionally higher risk were revealed for malignant mesothelioma (OR_MM, 1.918; 95% CI, 0.68–5.45) and asbestos-induced lung cancer (OR_{LC asbestos}, 2.112; 95% CI, 0.75–5.92) but not for lung cancer of other aetiology (OR_{LC other aetiology}, 1.00; 95% CI, 0.52–1.93) (Table V).

The frequency of the IL-10 (−1082) G-allele was 0.47 in the control and in the patient group. The OR analyses were performed for the genotype G-1082G vs. the genotypes carrying at least one A allele (G-1082A or A-1082A). Data was adjusted for PY, age (years) and gender. This analysis revealed that patients carrying at least one A allele of the IL-10 −1082 polymorphism were at higher risk for silicosis (OR_silicosis, 2.064; 95% CI, 0.78–5.49) but not for asbestosis (OR_asbestosis, 0.986; 95% CI, 0.48–2.03) or fibrosis of other aetiology (OR_{fibrosis other aetiology}, 0.903; 95% CI, 0.37–2.23). A higher risk was revealed for malignant mesothelioma (OR_MM, 1.726; 95% CI, 0.54–5.51) (Table VI).

The frequency of the less frequent IL-1β (+3954) T allele in the control group was 0.26 and in the patient group 0.25. The frequency of the less frequent IL-1β (−511) T allele was 0.31 in the control as well as in the patient group. The OR analyses were performed for the more frequent genotypes (C+3954C and C−511C) vs. the genotypes carrying at least one of the less frequent (T) allele (C+3954T or T+3954T and C−511T or T−511T). Data was adjusted for PY, age (years) and gender.

Patients carrying at least one less frequent (T) allele of the IL-1β −511 polymorphism showed decreased risk for fibrotic and malignant lung diseases (OR_asbestosis, 0.946; 95% CI, 0.50–1.79; OR_silicosis, 0.67; 95% CI, 0.30–1.51 and OR_LC, 0.597; 95% CI, 0.32–1.13) but not for malignant mesothelioma (OR_MM, 1.28; 95% CI, 0.49–3.33). Rather an increasing risk was seen for severe lung asbestosis (2/1, 2/2 and 2/3 ILO: OR, 1.29; 95% CI, 0.46–3.62; 3/1, 3/2 and 3/+ ILO: OR, 3.583; 95% CI, 0.27–47.47) (Table VII).

This analysis revealed that patients carrying at least one T allele of the IL-1β +3954 polymorphism were at higher risk for moderate lung asbestosis (1/1 and 1/2 ILO: OR, 1.79; 95% CI, 0.69–4.64) as well as for hyaline pleural plaques (OR, 1.44; 95% CI, 0.6–3.5). Additionally, a higher risk was seen in the asbestos-induced lung cancer patients (OR_{LC asbestos}, 1.27; 95% CI, 0.47–3.47) rather than in lung cancer of other aetiology (OR_{LC other aetiology}, 1.055; 95% CI, 0.55–2.01) (Table VIII). Significance could not be gained in the described results.