Distribution and regulation of cyclooxygenase-2 in carrageenan-induced inflammation

Abstract

1 We characterized the regulation of cyclooxygenase-2 (COX-2) at the mRNA, protein and mediator level in two rat models of acute inflammation, carrageenan-induced paw oedema and mechanical hyperalgesia. 2 Carrageenan was injected in the hind paw of rat at low (paw oedema) and high doses (hyperalgesia). COX-2 and prostaglandin E2 (PGE2) levels were measured by RT-PCR and immunological assays. We also determined the distribution of COX-2 by immunohistochemistry. 3 The injection of carrageenan produced a significant and parallel induction of both COX-2 and PGE2. This induction was significantly higher in hyperalgesia than in paw oedema. This was probably due to the 9 fold higher concentration of carrageenan used to provoke hyperalgesia. 4 Immunohistochemical examination showed COX-2 immunoreactivity in the epidermis, skeletal muscle and inflammatory cells of rats experiencing hyperalgesia. In paw oedema however, only the epidermis showed positive COX-2 immunoreactivity. 5 Pretreatment with indomethacin completely abolished the induction of COX-2 in paw oedema but not in hyperalgesia. 6 These results suggest that multiple mechanisms regulate COX-2 induction especially in the more severe model. In carrageenan-induced paw oedema, prostanoid production have been linked through the expression of the COX-2 gene which suggest the presence of a positive feedback loop mechanism.

Figure 1

Regulation of COX-2 in carrageenan-induced paw ædema and hyperalgesia. (a) COX-2 mRNA levels as determined by RT–PCR (see Methods) under conditions of carrageenan-induced paw ædema and hyperalgesia. Data is expressed as fold of the intensity of COX-2-specific band obtained from ipsilateral (injected) paw over that of contralateral (non-injected) paw. Both sets of data have also been corrected for β-actin. Data are mean±s.e.mean of 12–24 determinations. (b) COX-2 protein levels as determined by ELISA under conditions of carrageenan-induced paw ædema and hyperalgesia. Data is expressed as fold of COX-2 levels in ipsilateral over contralateral paws and are mean±s.e.mean of 6–12 determinations. (c) PGE₂ levels in paws under conditions of carrageenan-induced paw ædema and hyperalgesia. Data is expressed as pg per paw and are mean±s.e.mean of 6–12 determinations. ^*P<0.05 over value obtained at time zero.

Figure 2

Photomicrographs of COX-2 immunoreactivity in rat paws under control conditions (Left column) and 3 h following injection of carrageenan under the paw ædema (centre column) and hyperalgesia protocols (right column). (a–c) Epidermis; (d–f) Loose connective tissue; (g–i) Skeletal muscle. Positive COX-2 immunoreactivity (brown staining) can be detected in the epidermis in paw ædema (b) and in the epidermis (c), loose connective tissue (f) and skeletal muscle (i) in hyperalgesia.

Figure 3

Effect of indomethacin on carrageenan-induced paw ædema and hyperalgesia. (a) paw ædema observed 3 h following the injection of carrageenan in vehicle or indomethacin-pretreated animal (10 mg kg⁻¹ p.o.). Data are expressed as per cent of volume increase observed in control animals. (b) Hyperalgesia response observed 3 h following the injection of carrageenan in vehicle or indomethacin-treated animal (10 mg kg⁻¹ p.o.). Data are expressed as percent of vocalization response observed in control animals. Data are mean±s.e.mean of 8–10 determinations. ^*P<0.05.

Figure 4

Effect of indomethacin on the regulation of COX-2 protein and PGE₂ levels in carrageenan-induced paw ædema (a,c) and hyperalgesia (b,d). Indomethacin (10 mg kg⁻¹ p.o.) or vehicle (methocel) was given 1 h prior (ædema) or 2 h after (hyperalgesia) carrageenan injection. Paws were taken at indicated time and PGE₂ (a,b) and COX-2 protein (c,d) levels were analysed as described in Methods. PGE₂ levels are expressed as pg per paw. COX-2 protein levels are expressed as fold of carrageenan-treated over control paws. Data are mean±s.e.mean of 8–12 determinations. ^*P<0.05 over value obtained at time zero.

Figure 5

Effect of indomethacin pre-treatment on the regulation of COX-2 protein and PGE₂ levels in carrageenan-induced hyperalgesia. Indomethacin (10 mg kg⁻¹ p.o.) or vehicle (methocel) was given 1 h prior to carrageenan injection. Paws were taken at indicated time and COX-2 protein levels were analysed as described in Methods. COX-2 protein levels are expressed as fold of carrageenan-injected over saline-injected paws. Data are mean±s.e.mean of eight determinations. ^*P<0.05 over value obtained at time zero. #P<0.05 between indomethacin-treated and untreated curves.

Figure 6

Effect of indomethacin on the regulation of COX-2 mRNA in carrageenan-induced paw ædema. Indomethacin (10 mg kg⁻¹ p.o.) or vehicle (methocel) was given orally 1 h prior to intraplantar injection of carrageenan. Ipsilateral (injected) and contralateral (non-injected) paws were taken at indicated time, mRNA was isolated and both COX-2 and β-actin mRNA levels analysed by RT–PCR. Data are expressed as fold±s.e.mean of the intensity of the COX-2-specific band over that of β-actin obtained from the injected and non-injected paws of eight animals. ^*P<0.05 between ipsilateral and contralateral curves. #P<0.05 between data obtained at time zero and in naive animals.