Dynamics of early synovial cytokine expression in rodent collagen-induced arthritis : a therapeutic study using a macrophage-deactivating compound

Abstract

This study was performed to elucidate pathophysiological events before and during the course of collagen-induced arthritis in Dark Agouti rats, a model for rheumatoid arthritis. Kinetic studies of local cytokine responses were determined using immunohistochemical techniques, quantified by computer-assisted image analysis. We recently reported that the macrophage-pacifying agent CNI-1493 successfully ameliorated collagen-induced arthritis. In the present trial, we investigated the potential of CNI-1493 to down-regulate pro-inflammatory cytokines. Synovial cryosections were analyzed at various time points for the presence of interleukin (IL)-1beta, tumor necrosis factor (TNF), and transforming growth factor (TGF)-beta. Unexpectedly, an early simultaneous TNF and IL-1beta expression was detected in resident cells in the lining layer, preceding disease onset and inflammatory cell infiltration by >1 week. The predominant cytokine synthesis by synovial (ED1+) macrophages coincided with clinical disease. TNF production greatly exceeded that of IL-1beta. CNI-1493 treatment did not affect the early disease-preceding TNF and IL-1beta synthesis in the lining layer. However, after disease onset, CNI-1493 intervention resulted in a pronounced reduced IL-1beta and in particular TNF expression. Furthermore, CNI-1493 significantly up-regulated synthesis of the anti-inflammatory cytokine TGF-beta and thereby shifted the balance of pro-inflammatory and anti-inflammatory cytokines in the arthritic joint in a beneficial way.

Figure 1.

Clinical expression of CIA in CNI-1493-treated animals and controls. Three untreated and three animals treated with CNI-1493 were sacrificed at the time points indicated. Each individual animal is represented with a bar, which shows the arthritis index at time point of sacrifice. CNI-1493 treatment was withdrawn on day 27 after immunization (arrow), and three animals from each group were monitored without therapy until day 38 after immunization, when the trial was terminated.

Figure 2.

Quantified expression of cytokines and phenotype markers during the course of CIA. Synovial specimens from knee joints were analyzed by immunohistochemistry in animals sacrificed at the time points indicated on the x axis. The expression of the cytokines TNF (A), IL-1β (B), and TGF-β (C), and the cell surface markers MHC class II (OX-6) (D), surface marker for rat macrophages and monocytes (ED1) (E), and α/β T cell receptor (R73) (F), respectively, were studied and quantified at an original magnification of ×250 using a computerized image analysis system. Each bar represents an individual animal and expresses the total positive area for the given cytokine within 1.2 mm above and below the patella bone. A significant difference (P < 0.05) between treatment groups was noted at the time points indicated (asterisk).

Figure 3.

Representative micrographs illustrating brown (diaminobenzidine) immunoperoxidase staining of cryopreserved synovial tissue for expression of TNF (A) and IL-1β (B) at an early time point, 3 days after immunization, ie, preceding expected disease onset by 10 days. A thin, nonproliferative synovia is evident at this time point. The TNF- and IL-1β-producing cells are particularly located in the synovial lining layer but also within the blood vessel endothelium and occasionally as isolated cells. C: A sequential section is stained with irrelevant control antibody. Bone is characterized by dark blue staining in all figures. Original magnification, ×125.

Figure 4.

Representative micrographs illustrating brown (diaminobenzidine) immunoperoxidase staining of cryopreserved synovial tissue from arthritic animals. Untreated animal analyzed at the time point of maximal arthritis (day 21 after immunization) with sequential sections stained for expression of TNF (A), IL-1β (B), ED-1 (C), and an irrelevant isotype-matched control (D). CNI-1493-treated animal with clinical arthritis day 27 after immunization stained for TNF (E) and ED1 (F). Note the marked cell infiltration and area of ED1⁺ cells far exceeding that of TNF-producing cells in sections from the CNI-1493-treated animal. Synovitis, articular cartilage, and bone (dark blue staining) are evident in all figures. Original magnification, ×100.

Figure 5.

Representative micrographs illustrating brown (diaminobenzidine) immunoperoxidase staining of cryopreserved synovial tissue day 38 after immunization in a post-CNI-1493-treated animal with CIA (treatment withdrawn day 27 after immunization), stained for TGF-β (A) and a sequential section stained with irrelevant control antibody (B). Synovitis, articular cartilage, and bone (dark blue staining) are evident in all figures. Original magnification, ×100.