Impaired mucosal defense to acute colonic injury in mice lacking cyclooxygenase-1 or cyclooxygenase-2

Abstract

To investigate roles in intestinal inflammation for the 2 cyclooxygenase (COX) isoforms, we determined susceptibility to spontaneous and induced acute colitis in mice lacking either the COX-1 or COX-2 isoform. We treated wild-type, COX-1(-/-), COX-2(-/-), and heterozygous mice with dextran sodium sulfate (DSS) to provoke acute colonic inflammation, and we quantified tissue damage, prostaglandin (PG) E(2), and interleukin-1beta. No spontaneous gastrointestinal inflammation was detected in mice homozygous for either mutation, despite almost undetectable basal intestinal PGE(2) production in COX-1(-/-) mice. Both COX-1(-/-) and COX-2(-/-) mice showed increased susceptibility to a low-dose of DSS that caused mild colonic epithelial injury in wild-type mice. COX-2(-/-) mice were more susceptible than COX-1(-/-) mice, and selective pharmacologic blockade of COX-2 potentiated injury in COX-1(-/-) mice. At a high dose, DSS treatment was fatal to 50% of the animals in each mutant group, but all wild-type mice survived. DSS treatment increased PGE(2) intestinal secretion in all groups except COX-2(-/-) mice. These results demonstrate that COX-1 and COX-2 share a crucial role in the defense of the intestinal mucosa (with inducible COX-2 being perhaps more active during inflammation) and that neither isoform is essential in maintaining mucosal homeostasis in the absence of injurious stimuli.

Figure 1

Basal PGE₂ concentration in unstimulated colonic tissues from WT and COX-1– and COX-2–deficient mice. Tissue PGE₂ concentrations were measured by RIA in snap-frozen tissues homogenized in PBS buffer containing the following antiproteases: 50 μg/mL antipain-dihydrochloride, 2 μg/mL aprotinin, and 0.5 μg/mL leupeptin. Mean ± SEM, n = 7. *P < 0.05 versus WT and COX-2^–/–, n = 7.

Figure 2

Colonic PGE₂ levels in 24-hour culture supernatants from either untreated mice (water controls) or WT, heterozygous, or homozygous mice for COX-1 or COX-2 deletion treated for 5 days with 10% DSS. The colons were removed, opened longitudinally, washed in PBS buffer containing penicillin, streptomycin, and fungizone (P/S/F, 100 U/mL) and kept in cold serum-free media (RPMI 1640 1× with P/S/F). The organs were cut into small pieces in a Petri dish containing fresh media and 100 mg of tissues were incubated at 37°C in 1 mL of fresh media for 24 hours. Culture supernatants were harvested and assayed for PGE₂ secretion by RIA. Mean ± SEM, n = 6. *P < 0.05 versus water controls; ⁺P < 0.05 versus COX-1^+/–.

Figure 3

Body weight loss (a) and clinical score (b) induced by low-dose (2.5%) DSS oral treatment in WT, COX-1^–/–, and COX-2^–/– mice. Mean ± SEM, n = 7. *P < 0.05 versus WT; ⁺P < 0.05 versus COX-1^–/–.

Figure 4

Photomicrographs of the proximal colon of mice treated with low-dose DSS (2.5%) or water only for 5 days, and a DSS-treated COX-1^–/– mouse treated with the selective COX-2 inhibitor NS-398 (1 mg/kg every 8 hours) for 5 days. (a) WT mouse that received water (negative control) showing no mucosal damage. (b) WT mouse that received DSS for 5 days. Focal ulceration is present, but the majority of the mucosa shows no damage. (c) COX-1^–/– mouse that received DSS developed a slightly larger mucosal ulcer. (d) COX-2^–/– mouse developed more extensive mucosal ulceration with inflammation extended into the submucosa. (e) COX-1^–/– mouse that received NS-398 developed extensive colonic ulceration and inflammation. Magnification of all photomicrographs is ×33.

Figure 5

PGE₂ levels (a) and IL-1β levels (b) in 24-hour culture supernatants of colonic tissues from untreated (water controls) or DSS-treated (2.5%) WT and COX-2^–/– mice. The colons were removed, opened longitudinally, washed in PBS buffer containing penicillin, streptomycin, and fungizone (P/S/F, 100 U/mL), and kept in cold serum-free media (RPMI 1640 1× with P/S/F). The organs were cut into small pieces in a Petri dish containing fresh media, and 100 mg of tissues was incubated at 37°C in 1 mL of fresh media for 24 hours. Culture supernatants were harvested and assayed for PGE₂ secretion by RIA and for IL-1β by ELISA (Genzyme Diagnostics). Mean ± SEM, n = 7. *P < 0.05 versus WT; ⁺P < 0.05 versus water controls.

Figure 6

Effect of the selective COX-2 inhibitor NS-398 on the clinical score in 2.5% DSS-treated COX-1^–/– mice. NS-398 (1 mg/kg) was injected intraperitoneally every 8 hours, leading to a cumulative dosage of 3 mg/kg daily. NS-398 was administered starting 2 hours before treatment with 2.5% DSS and for 5 days consecutively until the end of DSS treatment. Control COX-1^–/– mice treated with 2.5% DSS were administered the vehicle for NS-398 under the same conditions. Ten to 15 mice per group were investigated. Mean ± SEM. *P < 0.05 versus vehicle-treated group; **P < 0.01 versus vehicle-treated group.