Fluvastatin treatment inhibits leucocyte adhesion and extravasation in models of complement-mediated acute inflammation

Abstract

Complement activation plays a relevant role in the development of tissue damage under inflammatory conditions, and clinical and experimental observations emphasize its contribution to inflammatory vasculitides. Statins have recently been shown to reduce cardiovascular morbidity independently of plasma cholesterol lowering and in vitro studies support a direct anti-inflammatory action of these drugs. The aim of this study was to verify the in vivo effect of fluvastatin on complement-mediated acute peritoneal inflammation. The effect of oral treatment with fluvastatin was investigated in normo-cholesterolaemic rats that received intraperitoneal injection of either yeast-activated rat serum (Y-act RS) or lipopolysaccharide to induce peritoneal inflammation monitored by the number of PMN recruited in peritoneal fluid washes. In addition, vascular adherence and extravasation of leucocytes were evaluated by direct videomicroscopy examination on mesentery postcapillary venules topically exposed to Y-act RS. The number of PMN in the peritoneal washes of rats treated with fluvastatin was 38% lower than that of untreated animals (P < 0.05) 12 h after LPS injection, and was even lower (56%) in rats treated with Y-act RS already 8 h after injection (P < 0.02). Firm adhesion to endothelium and extravasation of leucocytes evaluated under direct videomicroscopy observation were significantly inhibited in fluvastatin treated rats (77% and 72%, respectively; P < 0.01), 120 min after treatment with Y-act RS. Our results demonstrate that fluvastatin inhibits in vivo complement-dependent acute peritoneal inflammation and suggest a role for statins in preventing the inflammatory flares usually associated with complement activation in chronic diseases, such as SLE or rheumatoid arthritis.

Fig. 1

Acute peritoneal inflammation induced by yeast activated rat serum. Five groups of 4 rats received an ip injection of one of the following reagents: yeast-activated normal rat serum (Y-act NRS, □), yeast-activated C6 deficient rat serum (Y-act C6def RS, ▪), untreated normal (NRS, Δ) or C6 def rat sera (C6def RS, ▴), and sterile saline. The PMN number was counted in the peritoneal washings obtained at different time intervals after injection. Data are expressed as mean ± SD; *P < 0·05, and **P < 0·01 compared with the other groups at the same time of observation.

Fig. 2

Acute peritoneal inflammation induced by C5a. Four groups of 4 rats received an ip injection of increasing concentrations of C5a or sterile saline and the PMN number was counted in the peritoneal washings obtained at different time intervals after injection. Data are expressed as mean ± SD; *P < 0·05, or **P < 0·01 compared with saline-treated animals (used as controls) at the same time of observation.

Fig. 3

C-mediated acute peritoneal inflammation induced in FLU-treated rats. Six groups of 4 rats were daily treated with either FLU at two different doses of 10 mg/kg (□) or 5 mg/kg (•) body weight, or with vehicle (water plus ethanol alone, ⋄). At the end of treatment each rat received an ip injection of (a) yeast-activated normal rat serum (Y-act RS) or (b) LPS (1 mg/kg). The PMN number was counted in the peritoneal washings obtained at different time intervals after injection. Data are expressed as mean ±SD; *P < 0·05, when compared with the results obtained in the vehicle-treated rats at the same time of observation.

Fig. 4

Intravital videomicroscopy evaluation of C-stimulated leucocyte trafficking in FLU-treated rats. Two groups of 8 rats were treated for 15 days with either 5 mg/kg body weight FLU (□) or vehicle (⋄). On the day of the experiment, all rats received an ip injection of LPS (0·25 mg/kg) followed 4 h later by topical application of yeast-activated normal rat serum (50 µl). An additional group of 8 untreated rats received only a topical application of saline 4 h after the ip treatment with LPS (0·25 mg/kg), and served as controls (•). Data of the rolling flux, adhesion and extravasation of leucocytes are expressed as mean ± SD; *P < 0·05, or **P < 0·01, compared with the vehicle-treated animals at each considered time.

Fig. 5

Videomicrograph images showing the degree of extravasation of fluorescinated leucocytes following exposure to yeast-activated rat serum in the mesentery of rats treated with either (a) vehicle or (b) FLU. (c) Untreated rats exposed to sterile saline served as controls. Magnification, ×10.