Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of NFkappaB

Abstract

The antiinflammatory action of aspirin generally has been attributed to direct inhibition of cyclooxygenases (COX-1 and COX-2), but additional mechanisms are likely at work. These include aspirin's inhibition of NFkappaB translocation to the nucleus as well as the capacity of salicylates to uncouple oxidative phosphorylation (i.e., deplete ATP). At clinically relevant doses, salicylates cause cells to release micromolar concentrations of adenosine, which serves as an endogenous ligand for at least four different types of well-characterized receptors. Previously, we have shown that adenosine mediates the antiinflammatory effects of other potent and widely used antiinflammatory agents, methotrexate and sulfasalazine, both in vitro and in vivo. To determine in vivo whether clinically relevant levels of salicylate act via adenosine, via NFkappaB, or via the "inflammatory" cyclooxygenase COX-2, we studied acute inflammation in the generic murine air-pouch model by using wild-type mice and mice rendered deficient in either COX-2 or p105, the precursor of p50, one of the components of the multimeric transcription factor NFkappaB. Here, we show that the antiinflammatory effects of aspirin and sodium salicylate, but not glucocorticoids, are largely mediated by the antiinflammatory autacoid adenosine independently of inhibition of prostaglandin synthesis by COX-1 or COX-2 or of the presence of p105. Indeed, both inflammation and the antiinflammatory effects of aspirin and sodium salicylate were independent of the levels of prostaglandins at the inflammatory site. These experiments also provide in vivo confirmation that the antiinflammatory effects of glucocorticoids depend, in part, on the p105 component of NFkappaB.

Figure 1

Aspirin and sodium salicylate, but not indomethacin, are antiinflammatory in the murine air-pouch model of inflammation. Air pouches were formed on mice treated with saline, aspirin (200 mg/kg), sodium salicylate (200 mg/kg), or indomethacin 10 mg/kg), as described in Methods. Inflammation was induced by injection of 1 ml of a suspension of 2% (wt/vol) carrageenan in PBS into the air pouch, as we have previously described (14, 15). After 4 h, the mice were killed; the pouches were flushed with 2 ml of PBS; and the exudates were harvested. In some animals, DMPX (10 μM) or ADA (0.125 units/ml) was injected into the air pouch with the inflammatory stimulus. ADA and DMPX significantly diminished the inhibition of leukocyte accumulation mediated by treatment with either aspirin or sodium salicylate (P < 0.008 for all; n = 7–10 mice per group, studied on two separate occasions). We have previously established that the effects of ADA and DMPX in the murine air-pouch model are specific for adenosine A2 receptors, because neither agent reverses the antiinflammatory effects of dexamethasone and because the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine does not reverse the antiinflammatory effect (14). The data are expressed as the mean percentages ± SEM of inhibition of leukocyte accumulation. Mean leukocyte accumulation in animals treated with saline was (2.6 ± 0.2) × 10⁶ cells per ml.

Figure 2

Aspirin is antiinflammatory in animals rendered deficient in COX-2 activity [COX-2 knockouts (KO)]. Air pouches were formed on female COX-2 knockout mice (C57BL/6; 129–Ptgs2^fm1Jed; The Jackson Laboratory) or female wild-type mice (C57BL/6; 129), and the mice were treated with aspirin or saline, as described in Methods. There were significantly fewer leukocytes present in the air pouches of COX-2 knockout mice treated with aspirin (P < 0.01; n = 5) than in those of controls (n = 4). Similarly there were significantly fewer leukocytes present in the air pouches of wild-type mice treated with aspirin (P < 0.05; n = 5) than in those of controls (P < 0.05). The leukocyte counts are shown as means ± SEM.

Figure 3

Aspirin, sodium salicylate, and sulfasalazine, unlike dexamethasone, are no less antiinflammatory in p105 (NFκB) knockout mice than in wild-type mice. Breeding pairs of NFκB (p105; −/−) knockout mice (B6; 129–Nfkb1^fm1Bal) and wild-type controls (+/+; C57BL/6; 129) were obtained from The Jackson Laboratory and bred in the New York University Medical Center animal facility. Air pouches were formed, and the animals were treated with aspirin or sodium salicylate before induction of inflammation and quantitation of leukocytes, as described in Methods and Results. Animals treated with dexamethasone were given a single i.p. dose of dexamethasone (1.5 mg/kg) 1 h before induction of inflammation in the air pouch, and animals treated with sulfasalazine were given sulfasalazine (100 mg/kg) by gastric gavage for 3 days before and on the day of the induction of inflammation. The inhibition of leukocyte accumulation differed significantly between aspirin-treated and dexamethasone-treated knockout and wild-type mice (P < 0.02 for both; Student’s t test). The data are presented as the mean percentages ± SEM of inhibition of leukocyte accumulation in 6–10 mice per group, studied on at least two separate occasions.