Etanercept suppresses arteritis in a murine model of kawasaki disease: a comparative study involving different biological agents

Abstract

Coronary arteritis, a complication of Kawasaki disease (KD), can be refractory to immunoglobulin (IVIG) treatment. To determine the most effective alternative therapy, we compared the efficacy of different agents in a mouse model of KD. Vasculitis was induced by injection of Candida albicans water-soluble fractions (CAWS) into a DBA/2 mouse, followed by administration of IVIG, etanercept, methylprednisolone (MP), and cyclosporine-A (CsA). At 2 and 4 weeks, the mice were sacrificed, and plasma cytokines and chemokines were measured. CAWS injection induced active inflammation in the aortic root and coronary arteries. At 2 weeks, the vasculitis was reduced only by etanercept, and this effect persisted for the subsequent 2 weeks. At 4 weeks, IVIG and CsA also attenuated the inflammation, but the effect of etanercept was more significant. MP exerted no apparent effect at 2 or 4 weeks. The suppressive effect exerted by etanercept on cytokines, such as interleukin- (IL-)6, IL-12, IL-13, and tumor necrosis factor- α (TNF- α ), was more evident than that of others. The extent of arteritis correlated with the plasma TNF- α levels, suggesting a pivotal role of TNF- α in KD. In conclusion, etanercept was most effective in suppressing CAWS-induced vasculitis and can be a new therapeutic intervention for KD.

Figure 1

Induction of vasculitis by CAWS administration. Arterial inflammation around the aortic root in control mice (a, b) and the CAWS group (no treatment) at 2 weeks (c, d) and at 4 weeks (e, f). (a) and (b): no significant histological abnormalities are evident in the aortic root and coronary arteries in the negative control group. (c) and (d): at 2 weeks, the active inflammatory infiltrates, composed of numerous neutrophils and lymphocytes, segmentally or partially involved the aortic root ((c), arrowheads). The elastic lamillae are focally disrupted ((d), arrowheads). Exudation of fibrin was seen in the subendothelial region (arrow). (e) and (f): at 4 weeks, the inflammation circumferentially involved the aortic root extending from the adventitia throughout the surrounding connective tissue and heart ventricles ((e), arrowheads). The disruption of the vascular walls due to loss of internal and external elastic lamina was more extensive compared to that at 2 weeks ((f), arrowheads). Chronic changes, represented by accumulation of collagen fibers, progressed. Hematoxylin and eosin (H&E) stain ((a), (c), (e)), Elastica-Masson Goldner (EMG) stain ((b), (d), (f)). (a)–(d) Image at original magnification of ×100; (e) and (f) images at original magnification of ×40. Scale bars: 500 μm (a)–(d) and 1 mm (e, f).

Figure 2

Effects of different biological agents on the development of vasculitis. (a) Incidence of panvasculitis (%), (b) number of segments with arteritis, (c) severity score, and (d) total area of inflammation (mm²). Data at 2 weeks are indicated by white bars and those at 4 weeks by black bars. ^# P < 0.001 versus control, *P < 0.05 versus CAWS, **P < 0.001 versus CAWS. Abbreviations: control (Con), CAWS with no drug treatment (CAWS), immunoglobulin (IVIG), etanercept (EC), methylprednisolone (MP), and cyclosporine-A (CsA).

Figure 3

Histological observations on the development of vasculitis treated with different biological agents. Arterial inflammation around the aortic root at 2 weeks (a)–(d) and at 4 weeks (e)–(l) treated with IVIG ((a), (e), (i)), etanercept ((b), (f), (j)), methylprednisolone ((c), (g), (k)), and cyclosporine A ((d), (h), (l)). (a) Segmental inflammatory changes were observed in IVIG-treated mice (arrowheads). (b) Rare significant inflammatory changes were observed yet in etanercept-treated mice. (c, d) In methylprednisolone and cyclosporine-A groups, the inflammatory changes were still segmental (arrowheads), and appeared comparable with each other. (e, i) In IVIG-treated mice at 4 weeks, the inflammatory cell infiltration was more pronounced with loss of elastic lamina (arrowheads) extending into the surrounding tissue. (f, j) In the etanercept group at 4 weeks, vascular architecture was almost preserved except for a small segmental inflammatory area (arrowheads). (g, k) In methylprednisolone-treated mice at 4 weeks, the vascular architecture was diffusely distorted or misshapen due to dense inflammatory cell infiltration that expanded into the hear ventricles and connective tissues (arrowheads). (h, l) In cyclosporine-A-treated mice at 4 weeks, the inflammatory cell infiltration was segmental with focal loss of elastic lamina (arrowheads). H&E stain (a)–(h), EMG stain (i)–(l). All images at original magnification of ×40. All scale bars: 1 mm.

Figure 4

Correlation between histological changes and plasma cytokine levels. Correlation between the total area of inflammation and cytokine levels at 4 weeks. Relationship between the degree of vasculitis, indicated by the total area of inflammation (mm²), and plasma levels (pg/mL) of TNF-α (a), IL-13 (b), and IL-6 (c).