Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium

Abstract

Introduction: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumour necrosis factor (TNF) family member capable of inducing apoptosis in many cell types.

Methods: Using immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and real-time PCR we investigated the expression of TRAIL, TRAIL receptors and several key molecules of the intracellular apoptotic pathway in human synovial tissues from various types of arthritis and normal controls. Synovial tissues from patients with active rheumatoid arthritis (RA), inactive RA, osteoarthritis (OA) or spondyloarthritis (SpA) and normal individuals were studied.

Results: Significantly higher levels of TRAIL, TRAIL R1, TRAIL R2 and TRAIL R4 were observed in synovial tissues from patients with active RA compared with normal controls (p < 0.05). TRAIL, TRAIL R1 and TRAIL R4 were expressed by many of the cells expressing CD68 (macrophages). Lower levels of TUNEL but higher levels of cleaved caspase-3 staining were detected in tissue from active RA compared with inactive RA patients (p < 0.05). Higher levels of survivin and x-linked inhibitor of apoptosis protein (xIAP) were expressed in active RA synovial tissues compared with inactive RA observed at both the protein and mRNA levels.

Conclusions: This study indicates that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors, survivin and xIAP.

Figure 1

TRAIL, TRAIL R1, TRAIL R2, TRAIL R3 and TRAIL R4 expression pattern. Positive cells are shown in red staining. Staining pattern in active RA synovial tissue demonstrated in the area indicated by arrows. Magnification × 200. Synovial tissues were obtained from normal (1st row), active rheumatoid arthritis (RA) (2nd row), inactive RA (3rd row), osteoarthritis (OA) (4th row) and spondyloarthropathy (SpA) patients (5th row). TRAIL = tumour necrosis factor related apoptosis inducing ligand. Regions of interest in some panels are indicated by the circles and arrows and discussed in the text.

Figure 2

TUNEL, cleaved caspase-3, survivin and xIAP protein expression (red) in five patient groups. Magnification ×200, except for x-linked inhibitor of apoptosis protein (xIAP) × 400. OA = osteoarthritis; RA = rheumatoid arthritis; SpA = spondyloarthropathies; TUNEL = terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling.

Figure 3

Double labelling of active RA tissues for expression of (a) TRAIL (red) and CD68 (blue); (b) TRAIL R1 (red) and CD68 (blue); (c) TRAIL R4 (red) and CD68 (blue); (d) TRAIL (red) and CD3 (blue); (e) TRAIL R1 (red) and CD3 (blue); (f) TRAIL R4 (red) and CD3 (blue). An arrow indicates from where the magnified image at the bottom right corner of panel was obtained in panels a-c. Panel g shows double labelling of cleaved caspase-3 and xIAP. Panel h is a magnified region of the image that is indicated by the arrow and circle in panel g. Magnification of panels is × 200 and magnified areas are approximately × 600.

Figure 4

Expression levels of caspase-3, survivin and xIAP mRNAs in inactive RA, active RA and OA patients. These levels were determined by real-time PCR. The results are normalised to levels of hARP and have been expressed relative to OA control tissue. *p < 0.05, compared with inactive RA. n = 3 (OA), n = 3 (inactive RA), n = 5 (active RA). OA = osteoarthritis; PCR = polymerase chain reaction; RA = rheumatoid arthritis; xIAP = x-linked inhibitor of apoptosis protein.