Preparation and characterization of the Δ18 COX-2 knock-in mouse. A, preparation of the targeting vector. Experimental details are provided under “Experimental Procedures.” B, diagram comparing wild type (WT) COX-2 and del595–612 PGHS-2 (Δ18) COX-2 mutant alleles. C, PCR analysis of DNA from WT COX-2 (lane 1), WT/Δ18 heterozygous COX-2 (lane 2), and homozygous Δ18 COX-2 (lane 3) mice. The left lane contains Invitrogen markers differing in length by 100 bp. DNA was isolated from tail snips from animals after removal of the LoxP site from the mutated (i.e. Δ18 COX-2) gene and subjected to PCR analysis using primers P3 and P4 as detailed under “Experimental Procedures.” Shown at the top of the panel are intron/exon diagrams illustrating the location of the 18-amino acid (i.e. 54 bp) deletion in Exon 10 (black bar in native allele diagram), the LoxP site in the intron (sidewise arrow in mutant allele diagram), and the expected PCR fragment sizes based on the locations of primers P3 and P4. UTR, untranslated region. D, COX-2 protein expression in dermal fibroblasts from WT, heterozygous, and Δ18 COX-2 mice. Dermal fibroblasts were isolated from the skin of newborn WT (lane 1), WT/Δ18 heterozygous (lane 2), or Δ18 COX-2 (lane 3) mice and grown to about 60% confluence in MEM containing 10% FBS as described under “Experimental Procedures.” The cells were serum-starved for 24 h in MEM containing 0.2% FBS. Serum-starved dermal fibroblasts were then treated with MEM containing 20% FBS for 3 h. The cells were harvested, and cell lysate protein (15 μg/lane) was subjected to Western blotting using antibodies to actin, COX-1, or COX-2. Two different anti-COX-2 anti-peptide antibodies were used directed against either Ser⁵⁹⁸–Lys⁶¹² or Gln⁵⁸³–Asn⁵⁹⁴. E, cyclooxygenase activity of dermal fibroblasts from WT, heterozygous, and Δ18 COX-2 mice. Dermal fibroblasts were cultured and serum-starved for 24 h as described in D and then treated with 1 mm aspirin for 30 min to eliminate COX-1 and residual COX-2 activities. The cells were then washed to remove the aspirin and incubated in MEM containing 20% FBS for 3 h. Finally, the cells were washed with MEM without serum and then incubated with 5 μm [1-¹⁴C]arachidonic acid in MEM for 10 min at room temperature. Lipid products were extracted from the medium and separated by TLC as described under “Experimental Procedures.” Based on scintillation counting, the relative conversion of arachidonic acid to total products was 2.8, 4.3, and 5.8% for cells from WT (lane 1), WT/Δ18 heterozygous (lane 2), and Δ18 COX-2 (lane 3) mice, respectively.

COX-2 protein expression and degradation in tail fibroblasts from WT and Δ18 COX-2 mice. Fibroblasts were isolated from the tails of 1–3 male mice as detailed under “Experimental Procedures” for one mouse. A, WT mice; B, Δ18 COX-2 mice; C, both WT and Δ18 COX-2 mice. The cells were grown and serum-starved as described under “Experimental Procedures” and the legend to Fig. 1. Serum-starved tail fibroblasts were stimulated with DMEM containing 20% FBS for the indicated times in media containing no additions, S-FBP (100 μm), S-FBP plus KIF (25 μm), or KIF alone. C, cells were stimulated with 20% serum containing 100 μm S-FBP for 4 h, and then the medium was changed to DMEM containing 20% serum, 100 μm S-FBP. and 50 μm CHX with or without 25 μm KIF. After a further 1-h incubation, the cells were harvested at the indicated times, and cell lysate protein (15 μg/lane) was subjected to Western blotting using antibodies to actin or COX-2 (αGln⁵⁸³–Asn⁵⁹⁴). The experiments were performed at least twice with similar results.

Prostanoid formation and expression of COX-2 in peritoneal macrophages from WT and Δ18 COX-2 mice. Resident peritoneal macrophages were isolated from 12-week-old adult male mice as described under “Experimental Procedures.” After 24 h, PGE₂ (A) and 6-keto-PGF_1α levels (B) in the media were measured after the following treatments: lane 1, 18 h without LPS; lane 2, 0 h with LPS; lane 3, 2 h with LPS; lane 4, 6 h with LPS; lane 5, 18 h with LPS; lane 6, 18 h with LPS and NS398; lane 7, 2 h with LPS and KIF; lane 8, 6 h with LPS and KIF; lane 9, 18 h with LPS and KIF; lane 10, cells were harvested after 18 h with LPS (as in lane 5), and new medium containing with A23187 was added and the sample incubated for 30 min before measuring PGs; and lane 11, cells were harvested after 18 h with LPS and KIF (as in lane 9), and new medium containing with A23187 was added and the sample incubated for 30 min before measuring PGs. Error bars, ± S.D. *, p < 0.05 for comparison between WT and Δ18 COX-2 mice. Statistical analysis was performed by Student's t test. C, resident peritoneal macrophages were isolated from male and female mice as described under “Experimental Procedures” and treated for 24 h with the following: lane 1, LPS; lane 2, LPS plus S-flurbiprofen; lane 3, LPS plus S-flurbiprofen plus KIF; or lane 4, LPS plus KIF. The cells were homogenized and the extracts subjected to Western transfer blotting for COX-2 and actin as indicated in the legend to Fig. 1.

Urinary PGE₂ metabolites from adult male WT or Δ18 COX-2 mice. Analyses of urinary PGE₂ metabolites (PGE-M) were performed as described in detail under “Experimental Procedures” using urine collected from 12-week-old mice. Data are means ± S.E. * indicates a difference from the value for the male WT; ** indicates a difference from the value for male WT but no difference between the values for the WT and Δ18 female mice (p < 0.05).

Expression of COX-2 in tissues from WT and Δ18 COX-2 mice treated with endotoxin with or without ibuprofen. Microsomal protein was prepared from whole brain, whole kidney, and whole spleen obtained from 12-week-old male WT or Δ18 COX-2 mice. Animals were subjected to an intraperitoneal injection with LPS (10 μg) and were euthanized, and tissues were taken at the indicated times following the LPS injection. Half of the animals received ibuprofen (+IBP) in their drinking water (0.2 mg/ml) beginning 2 days prior to the LPS treatment and also received an intraperitoneal injection of ibuprofen (40 mg/kg) in saline 30 min prior to the LPS injection. C, control WT muCOX-2 (0.05 μg); the adjacent lane contained molecular weight standards (not visible). The same amounts of microsomal protein were applied in each lane. The two Brain panels represent different exposure times for the same Western blot. The columns in each of the panels represent tissue from the same mouse (i.e. one mouse was used for each time point). Anti-COX-2 (Novus Biologicals) antibody was used for Western blotting.

Endotoxin-induced febrile response in adult male WT and Δ18 COX-2 mice. WT and Δ18 COX-2 mice that had had telemeters implanted were treated as detailed under “Experimental Procedures.” A, body temperatures of untreated mice demonstrate normal circadian variability in WT (thin line) and Δ18 COX-2 mice (thick line); B, WT mice were injected with pyrogen-free saline (thin line) or LPS (thick line, 10 μg) at time 0. The horizontal line with asterisk denotes statistically significant differences in mean hourly temperature (p < 0.01). C, Δ18 COX-2 mice were injected with pyrogen-free saline (thin line) or LPS (thick line, 10 μg) at time 0. The horizontal bar with asterisk denotes statistically significant differences in mean hourly temperature (p < 0.01). Body temperature measurements shown in the figure represent mean and S.E. of values obtained from six WT mice and five Δ18 COX-2 mice. D, comparison of the mean hourly difference in body temperature (ΔT_b) between pyrogen-free saline and LPS treatment for WT (thin line) and Δ18 COX-2 mice (thick line). Mean hourly ΔT_b values were calculated as described under “Experimental Procedures.” Horizontal black lines with asterisks denote hour periods during which statistically significant differences (p < 0.05) were revealed between WT and corresponding Δ18 COX-2 mice. Dark bars on the x axis denote the dark period of the light/dark cycle.