Skin and synovial biopsy specimens were obtained from patients with chronic plaque psoriasis and PsA (n = 11 synovial biopsies; n = 6 skin biopsies) in a prospective open-label clinical trial, as previously described 4. Clinical characteristics have been previously reported 4. Patients received a weekly dose of alefacept (LFA-3TIP, anti-CD2; Amevive, Biogen, San Diego, CA, USA; 7.5 mg, intravenously) for 12 weeks. In brief, skin biopsies were taken before and after 1 and after 6 weeks, whereas synovial biopsies were taken before and 4 and 12 weeks after treatment. Synovial biopsy specimens from active RA patients (Table 1; n = 10) served as disease control group for IL-20 expression at baseline. Patient's disease activity score (DAS28), visual analogue scale (VAS) for global disease activity (scale, 0 to 100 mm), swollen-joint count (SJC), tender-joint count (TJC), erythrocyte sedimentation rate (ESR), and serum levels of C-reactive protein (CRP) were used to evaluate disease activity. The study was conducted according to International Conference of Harmonization (ICH)/Declaration of Helsinki, approved by the Medical Ethics Committee of Academic Medical Center (AMC)/University of Amsterdam, and all patients gave written informed consent. Psoriasis area and severity index (PASI) was assessed as previously described 28.

All samples were, immediately after collection, embedded en bloc in Tissue Tec OCT (Miles, Elkhart, IN, USA) and subsequently snap frozen. The frozen blocks were stored in liquid nitrogen until processed. Shortly before staining, 5-μm sections were cut and mounted on glass slides (Star Frost; Knittelgläser, Braunschweig, Germany). The glass slides were sealed and stored at -80ºC until immunohistochemical analysis was performed.

In brief, after fixation with acetone, endogenous peroxidase activity was inhibited by using 0.1% sodium azide and 0.3% hydrogen peroxide in phosphate-buffered saline (PBS). The primary antibody against human IL-20 (Zymogenetics, Seattle, WA, USA) was incubated overnight at 4ºC followed by secondary antibody affinity-purified horseradish peroxidase (HRP)-conjugated goat anti-mouse (Dako Cytomation, Glostrup, Denmark) for 30 minutes, followed by subsequent incubation with biotinylated tyramide (Perkin Elmer, Boston, MA, USA) for 30 minutes and HRP-conjugated streptavidin for 30 minutes. HRP activity was detected by using hydrogen peroxide as substrate and amino ethylcarbazole (AEC; SK-4200; Vector Laboratories, Burlingame, CA, USA) as dye. With this procedure, IL-20-positive cells stained red. Slides were counterstained with Mayer hematoxylin (Merck, Darmstadt, Germany) and, after washing with distilled water, mounted in Kayser glycerol gelatin (Merck).

To identify IL-20-expressing cells in PsA synovial tissue, double staining was performed by using anti-IL-20 antibody together with FITC-labeled anti-CD55 (M2192; Sanquin Reagents, Amsterdam, The Netherlands) or FITC-labeled anti-CD68 (clone KP1; Dakocytomation). Fixation and blocking endogenous peroxidase activity was performed as stated earlier. The primary antibody against human IL-20 (Zymogenetics) was incubated overnight at 4ºC followed by secondary antibody affinity-purified horseradish peroxidase (HRP)-conjugated goat anti-mouse (Dako Cytomation) for 30 minutes, and subsequent incubation with biotinylated tyramide (Perkin Elmer) for 30 minutes and HRP-conjugated streptavidin for 30 minutes. After a blocking step of 15 minutes with 10% mouse serum (Sanquin Reagents), FITC-labeled anti-CD55 or FITC-labeled anti-CD68 was added to the sections and incubated for 1 hour at room temperature. Then the sections were incubated for 30 minutes with rabbit anti-FITC followed by alkaline phosphatase-conjugated swine anti-rabbit secondary antibodies (both from Dako Cytomation). HRP activity was detected as stated earlier. Alkaline phosphatase activity was detected by using the Alkaline Phosphatase Substrate III kit (SK-5300; Vector Laboratories). With this procedure, IL-20-positive cells stained red, CD55- or CD68-positive cells stained blue, and double-positive cells stained purple.

Skin biopsy specimens were scored semiquantitatively for IL-20 expression on a 5-point scale by two independent observers who were not aware of the clinical data. The scoring methods were described previously 29. In brief, a score of 0 represented minimal staining, whereas a score of 4 represented widespread expression of IL-20 in this case. Individual readings were identical or differed by only 1 point. Minor differences between the observers were resolved by mutual agreement.

Quantification of IL-20-expressing cells in PsA and RA synovial tissues was performed with computer-assisted image analysis, as previously described 30. The same holds true for CD68 and CD55 quantification. In brief, after immunohistochemical staining, all coded sections (one section per patient per time point) were randomly analyzed (18 high-power fields from different parts of the section were analyzed; for example, the mean of the 18 high-power fields was calculated). The images of the high-power fields were analyzed by using the Qwin analysis system (Leica, Cambridge, UK). Positive staining of CD68 and CD55 was expressed as positive cells/mm². Positive staining for IL-20 was expressed as integrated optical density (IOD/mm²).

SPSS version 15.0 for Windows (SPSS, Chicago, IL, USA) was used for statistical analysis. The Wilcoxon signed ranks test was used for comparison of nonparametric variables in related samples, and the Mann-Whitney U test was used for comparison of nonparametric variables in independent samples. Correlations between synovial IL-20 expression and disease activity score 28 (DAS28), synovial CD4, CD8, CD68, and CD55 were analyzed with Spearman rank correlation. Correlations between skin IL-20 and PASI were analyzed with Spearman rank correlation.

First we analyzed whether IL-20 expression in PsA and RA synovia differ from each other. IL-20 was expressed in the intimal lining layer, in the synovial sublining, and on endothelium in both PsA and RA patients (Figure 1A). Digital image analysis of synovial tissue demonstrated clear IL-20 expression with comparable overall scores in both patient groups (median Integrated Optical Density (IOD) (range)): PsA, 60,064 (1,588 to 567,696); RA, 68,554 (1,171 to 530,218) (Figure 1B). Double staining of inflamed PsA synovium showed that CD68⁺ macrophages and CD55⁺ FLS coexpress IL-20 (Figure 1C).

As previously described, alefacept treatment resulted in clinical improvement and in a reduction of CD4⁺ T cells and CD68⁺ macrophages in the synovial infiltrate 4. Despite the clinical improvement and change in cellular infiltrate 4, no change was found in IL-20 expression in the synovium before and after treatment (Figure 2A and 2B). As CD68⁺ macrophages and CD55⁺ FLSs express IL-20, we investigated whether the levels of synovial IL-20 were associated with the numbers of these cells present in synovial tissue. Although no correlation was present between the levels of IL-20 and the numbers of both total CD68⁺ and sublining CD68⁺ macrophages (data not shown), the levels of IL-20 were positively correlated with the numbers of CD55⁺ FLSs (r = 0.4238; P = 0.0196). In addition, the levels of synovial IL-20 expression were not correlated with disease-activity parameters: DAS28, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) (data not shown).

In contrast to findings in the synovium, IL-20 expression in PsA lesional skin was significantly decreased (P = 0.04) 6 weeks after alefacept treatment (Figure 3A and 3B). IL-20 expression in PsA lesional skin was positively correlated with disease activity (PASI score): r = 0.5062; P = 0.031 (Figure 3C).