Fig. 1. Leukocytes from wild-type (A_2AR^+/+) mice express more COX-2 in response to LPS than do knockout (A_2AR^–/–) mice. (A) Dorsal air pouches from wild-type (A_2AR^+/+) CD1 mice were injected with PBS alone or containing 500 ng LPS for 4 hours; migrated leukocytes were collected and processed for the determination of indicated mRNA expression by real-time PCR, as described in Materials and Methods. Typical real-time PCR results from one mouse are shown. (Inset) Enumeration of leukocytes which migrated into the air pouch of wild-type (+/+) or homozygote knockout mice (–/–). Results are expressed as the mean±s.e.m., with values between brackets being the number (n) of animals in each condition. *Statistically different from PBS-injected mice. (B) Comparative GAPDH, COX-1 and COX-2 mRNA expression in migrated leukocytes between LPS-injected A_2AR^+/+ and A_2AR^–/– mice. Results show the mean±s.e.m. from at least four animals. *Statistically different from GAPDH and COX-1. **Statistically different from A_2AR^+/+. (Inset) Comparative mRNA expression in resident cells (mean±s.e.m., n=4). (C) Air pouches were injected with LPS for the indicated times. Migrated leukocytes were collected, enumerated, and samples, equalized by cell numbers, were processed for the determination of COX-2 by western immunoblotting, as described in Materials and Methods. (D) Air pouches were injected with LPS for 60 minutes. Migrated leukocytes were collected, enumerated, and samples were processed for the determination of COX-2 by western immunoblotting. *Statistically different from A_2AR^+/+. In each panel, immunoblots from one experiment typical of at least three separate experiments are shown. Bar graph depicts the expression of COX-2 protein levels in leukocytes from LPS-injected mice, relative to that observed in A_2AR^+/+ mice, as determined by densitometric analysis of the bands in three independent experiments (mean±s.e.m.; n=3).