Introduction

Rheumatoid arthritis (RA) is a systemic inflammatory auto-immune disease characterized by chronic polyarthritis of unknown etiologies (1). Although it is manifested principally by involvement of joints, extra-articular tissues, such as the skin, heart, lungs, eyes, and the nervous system may also be affected. The number and severity of extra-articular symptoms may vary depending on the duration and severity of the disease (2,3). Pulmonary involvement is a well-known extra-articular involvement form of RA and it is the most common second factor causing death, following infection (4). Incidence rate of pulmonary involvement in RA is known to range between 16 and 81 percent (5). Recently, specific antibodies against epidermal filagrin (filamentaggregating protein) were determined in 40-60% of patients with RA. Citrulline is a rare amino acid found in filagrin molecule. In recent studies, it was shown that citrulline found in filagrin is a structural component of antigenic epitope. Antibodies against cyclic peptide containing citrulline (anti-CCP; anti-cyclic citrullinated peptide) are reported as a new parameter, which is far more specific than rheumatoid factor (RF). Anti-CCP antibodies are mostly in IgG class, and their specificity rate for RA is 97 percent. Those antibodies can be easily detected using ELISA (Enzyme-Linked Immunosorbent Assay) due to development of synthetic peptides containing citrulline (6,7).

The aim of this studywas to determine the relationship between serum anti-CCP antibodiesand disease activity score-28 (DAS28), using the Stanford HealthAssessment Questionnaire (HAQ) in patients with RA, whose pulmonary involvement was detected by computed tomography (CT).

Methods

Totally 49 patients with RA diagnosis fulfilling 1987 the American Rheumatism Association (ARA) classification criteria, who had referred to outpatient clinic of ImmunologyRheumatology Division, Internal Medicine Department, were enrolled into the present study. Considering inclusion of patients, no age-and sex-related limitations were applied. Patients were divided into two groups: patients with and without pulmonary involvement according to the results of high-resolution computed tomography examinations (HRCT). The HRCT images were acquired using single-detector CT in the supine position and at maximum inspiration. The scans covered the whole lung with a slice thickness of 1 mm and interslice gaps of 10 mm. Images were reconstructed using an edge-enhancing algorithm and a matrix of 512-512 pixels. One and the same HRCT scanner was used in all patients. One observer (radiologist) documented the following features on HRCT images.

Disease Activity was quantified using the DAS28 and the Stanford Health Assessment Questionnaire (HAQ). According to the results of disease activity assessment performed using DAS28, two shoulders, 2 elbows, 2 wrists, 10 metacarpaphalangeal joints, 10 proximal interphalangeal joints, 2 knee joints were examined for tenderness and swelling. Tender joint count (TJC) and swollen joint count (SJC) were recorded. Erythrocyte sedimentation rate (ESR) and general health status (GHS) were also required for calculation of the DAS28 using the formula given below:

DAS 28= (0,56x√TJC) + (0,28x√SJC) + (0,70 log ESR) + (0,014xGHS)

It is considered that a DAS-score below 2.6 indicates remission, a score between 2.6 and 3.2 indicates low clinical activity, a score between 3.2 and 5.1 indicates intermediate clinical activity, and a score over 5.1 indicates high clinical activity. For assessment of the GHS, the patients were asked to rate their GHS on a self-administered scale ranging between 0 and 100.. 0= no pain, 100= intolerable pain. The point marked by the patient was used as GHS.

Using the HAQ, the patients were questioned about dressing, raising, eating, walking, hygiene, reaching, griping and common daily activities over the past week. Scores included 0=without any difficulty, 1=with some difficulty, 2= with much difficulty, 3=unable to do, and total of scores were recorded. They were also asked about special instruments used for performing those activities and need for another person.

Anti-CCP antibodies were studied by ELISA IgG kit following the below-mentioned steps. Serum samples were diluted 1:101 with sample buffer. Calibrators, control and diluted serum samples, all in volume of 100 μl, were placed in ELIZA wells and were incubated at room temperature for 60 minutes. After rinsing the wells three times using rinsing solution, 100 μl of the enzyme conjugate (peroxidaselabeled anti-human IgG) was added and the mixture was incubated at room temperature for 30 minutes. 100 μl of stop solution was added to each well and the reading was performed using a spectrophotometer at 450 nm. Samples with value >5 RU/ml were considered positive.

Statistics

All analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 13. All data were expressed as mean±standard deviation (SD) or as a percentage, as appropriate. Qualitative parameters were analyzed with the Chi-Square test and Fisher’s exact test. Nonparametric variables were analyzed using the MannWhitney U test.

Results

A total of 49 patients with the diagnosis of RA-42 females (85.7%) and 7 males (14,3%) aged between 23 and 96 years (mean: 55.86±13.31 years) were examined. The duration of the disease was 0-2 years in 17 patients (34.7%) and over 2 years in 32 patients (65.3%) (Table 1).

The mean value of DAS 28 in all cases was 3.52±1.55, but it was 2.99±1.41 in 24 patients with pulmonary involvement and 4.03±1.52 in 25 patients without pulmonary involvement. When the difference in the values of DAS 28 between the groups with and without pulmonary involvement was regarded, it was found that the mean DAS 28 was significantly higher in the group with pulmonary involvement (p= 0.017).

The mean score of the HAQ in all cases was 10.10±12.59, but it was 6.75±9.41 in 24 patients with pulmonary involvement and 13.32±14.50 in 25 patients without pulmonary involvement. No significant difference was found between the mean HAQ scores of groups with and without pulmonary involvement (p= 0.067) (Table 2).

When the disease activity was examined using DAS 28 and the HAQ, although DAS28 activity was found higher in the patients with positive anti-CCP, no statistically significant difference could be observed (p=0.119; 0.787) (Table 3).

19 patients were anti-CCP-positive (38.8%) and 30 were negative (61.2%). Anti-CCP was positive in 10 out of 24 patients with pulmonary involvement (41.6%) and negative in the remaining 14 patients (58.4%), whereas it was positive in 9 out of 25 patients without pulmonary involvement (36%) and negative in the remaining 16 patients (64%). No significant difference could be found between pulmonary involvement and positivity and negativity of anti-CCP test (p=0.773) (Table 4).

HRCT findings in RA patients with pulmonary involvement included pulmonary nodule in 17 cases (70.8%), bronchiectasia in 14 (58.3%), interlobular septal thickening in 13 (54.2%), peribronchial thickening in 7 (29.2%), ground glass opacity in 5 (20.8%), reticular infiltration in 5 (20.8 %), pleural thickening in 4 (16.7%), emphysema in 4 (16.7%), atelectasia in 3 (12.5%), air trapping in 3 (12.5%), fibrous band in 2 (8.3%), pleural nodule in 2 (8.3%), honeycomb formation in 2 (8.3%) and bullae in 2 cases (8.3%) (Table 5).

Discussion

RA is a disease that particularly affects the joints, however, it may involve all the organ systems (3). In approximately 50 percent of RA cases, occurrence of extra-articular manifestations including serositis, pneumonitis, myocarditis, renal involvement, myositis, arteritis, peripheral neuritis, involvement of the central nervous system and hematological changes have been demonstrated. Extra-articular involvement may occur at any age following onset of the diseases. Although the true prevalence of pulmonary involvement is unknown, it has been reported to be 1-40 % of all RA patients (8,9). Middle age, male sex, serious destructive arthritis, high RF titrations, presence of subcutaneous nodule and other extra-articular involvement findings are known predisposing factors for pulmonary involvement (10). Respiratory involvement (9.9%) is the third most common cause of death in patients with RA, following infections (23.5%) and cardiovascular diseases (17,3 percent) (11). Particularly, development of pulmonary fibrosis may adversely affect the clinical course. Following diagnosis of pulmonary fibrosis, five-year survival was reported to be 39 percent (12). Since pulmonary involvement increases mortality and morbidity, the diagnosis should be made and treatment should be started at an early stage.

While anti-CCP antibodies are highly specific to RA, the relationship of these antibodies with clinical activity of the disease is not clear. It was shown that patients with antiCCP positive RA have relatively higher disease activity and DAS than patients with anti-CCP negative RA (13). In the present study, disease activity determined using DAS 28 was found higher in anti-CCP(+) group, although it was not significant. No association could be found between disease activity determined using the HAQ and anti-CCP positivitynegativity.

It has been claimed that anti-CCP antibodies have prognostic value and are dominantly present in individuals with erosive disease (1,14). Erre et el. stated in their study that anti-CCP antibody, among all anti-filagrin antibodies, is the most definitive antibody for diagnosis of RA and, moreover, it has determinant role in the early phase of arthritis in terms of persistency of the disease and development of radiologically evidenced damage (15). Meyer et al. had prospectively observed 191 patients with recently diagnosed RA for 5 years and found using the Sharp-van der Heijde method that radiological damage, erosions and narrowing in joint space had been three times more prevalent in anti-CCP positive patients (16). Similarly, this relationship had also been demonstrated in studies conducted by Vencovsky et al. and Lee et al. Nevertheless, in the study conducted by Dündar et al., no significant difference could be found between modified Larsen scores of right and left hand in anti-CCP positive and negative patients (17). In the present study, using the Sharp-van der Heijde method, no significant difference could be found in terms of joint erosion and narrowing on hand images of anti-CCP positive and negative patients using Sharp-van der Heijde method.

In pulmonary involvement, one of extra-articular findings in patients with RA, known predisposing factors include middle age, male sex, serious destructive arthritis, high RF titrations and presence of subcutaneous nodules (18). However, we could not find any study in the literature examining the relationship between anti-CCP antibodies and pulmonary involvement. In the present study, we could find no significant relations between pulmonary involvement and anti-CCP positivity-negativity.

In conclusion, as pulmonary involvement significantly influences morbidity and mortality in patients with RA, it is important to determine pulmonary involvement at an early phase and to plan treatment modality. Recently, significance of anti-CCP antibodies in early diagnosis of RA and their relationship with extra-articular symptoms of RA has been demonstrated. In the present study, it was observed that pulmonary involvement in RA is not related with levels of anti-CCP antibodies, which have high specificity for early diagnosis of RA and that disease activity is decreased as patients with pulmonary involvement are more aggressively treated. However, we decided that future studies in larger patient series are required.