Effect of regulated expression of human cyclooxygenase isoforms on eicosanoid and isoeicosanoid production in inflammation

Abstract

To examine the role of cyclooxygenase (COX) isozymes in prostaglandin formation and oxidant stress in inflammation, we administered to volunteer subjects placebo or bolus injections of lipopolysaccharide (LPS), which caused a dose-dependent increase in temperature, heart rate, and plasma cortisol. LPS caused also dose-dependent elevations in urinary excretion of 2,3-dinor 6-keto PGF(1alpha) (PGI-M) and 11-dehydro thromboxane B(2) (Tx-M). Platelet COX-1 inhibition by chronic administration of low-dose aspirin before LPS did not alter the symptomatic and febrile responses to LPS, but the increment in urinary PGI-M and Tx-M were both partially depressed. Pretreatment with ibuprofen, a nonspecific COX inhibitor, attenuated the febrile and systemic response to LPS and inhibited prostanoid biosynthesis. Both celecoxib, a selective COX-2 inhibitor, and ibuprofen attenuated the pyrexial, but not the chronotropic, response to LPS. Experimental endotoxemia caused differential expression of the COX isozymes in monocytes and polymorphonuclear leucocytes ex vivo. LPS also increased urinary iPF(2alpha)-III, iPF(2alpha)-VI, and 8,12-iso-iPF(2alpha)-VI, isoprostane (iP) indices of lipid peroxidation, and none of the drugs blunted this response. These studies indicate that (a) although COX-2 predominates, both COX isozymes are induced and contribute to the prostaglandin response to LPS in humans; (b) COX activation contributes undetectably to lipid peroxidation induced by LPS; and (c) COX-2, but not COX-1, contributes to the constitutional response to LPS in humans.

Figure 1

(a) The effect of chronic administration of low-dose aspirin (ASA; 81 mg for 10 days) and acute administration of the nonselective NSAID ibuprofen (800 mg) on the pyrexial response to 4 ng/kg LPS. Ibuprofen, but not aspirin, significantly attenuated the pyrexial response to endotoxin (P < 0.01). (b) The effect of LPS on heart rate is not altered by either aspirin or ibuprofen (^AP = NS). (c) Pretreatment with either celecoxib (800 mg) or ibuprofen (800 mg) significantly (P < 0.01) depressed the pyrexial response to LPS (4 ng/kg). Body temperature did not differ significantly between subjects receiving celecoxib or ibuprofen before LPS. NS, not significant.

Figure 2

(a) LPS induces an increase in excretion of Tx-M (P < 0.001). A dose-response effect is evident with peak excretion at 3–6 hours. No change in Tx-M excretion was seen with placebo. (b) LPS causes a dose-dependent increase in excretion of PGI-M. The maximal effect at 3–6 hours coincided with the maximal constitutional effects of the endotoxin (P < 0.001). ^AP < 0.05; ^BP < 0.001.

Figure 3

(a) The effect of LPS on Tx-M excretion with selective COX-1 and nonselective COX inhibition. Inhibition of platelet COX-1 with low-dose aspirin partially inhibits excretion of TX-M evoked by 4 ng/kg LPS. By contrast, ibuprofen completely inhibits the increase in thromboxane biosynthesis induced by the endotoxin (P < 0.01). (b) The effect of LPS on PGI-M excretion after selective and nonselective COX inhibition. The increment in PGI-M excretion evoked by 4 ng/kg LPS is partially inhibited by low-dose aspirin. Nonselective inhibition of both COX isoforms completely inhibits PGI-M excretion. (c) The effect of LPS on PGI-M excretion after selective and nonselective COX inhibition. Although celecoxib substantially depressed the increment in PGI-M, it is depressed further by ibuprofen. ^AP < 0.05; ^BP < 0.001.

Figure 4

(a) Low-dose aspirin and ibuprofen fail to depress LPS-evoked urinary iPF_2α-III excretion. (b) Celecoxib or ibuprofen fail to depress LPS-evoked urinary 8,12-iso-iPF_2α-VI excretion. (P = NS).

Figure 5

(a) Western blot of monocyte COX-1. LPS increases the expression of COX-1 in peripheral blood monocytes. This is maximal in cells that have been isolated from blood drawn at 30 minutes after LPS injection (4 ng/kg). The time from blood withdrawal to detection lasted 2–2.5 hours (see the text). All samples are drawn from a single individual. This result is representative of three separate experiments. (b) Immunoprecipitation of monocyte COX-2. LPS does not alter the expression of COX-2 in monocytes ex vivo in blood drawn at the time points shown or in monocytes harvested at 1.5, 3, 4, 6, and 12 hours after LPS administration (data not shown). As before, the time for blood withdrawal to detection lasted 2–2.5 hours (see the text). All samples were drawn from a single individual administered 4 ng/kg LPS, and this study is representative of three separate experiments. –C denotes a negative control, unstimulated monocytes in vitro. +C is a positive control and denotes monocytes stimulated with LPS (10 μg/mL) in vitro. (c) Immunoprecipitation of neutrophil COX-2. Increased expression of COX-2 was observed in peripheral blood neutrophils isolated from blood drawn at 90 and 180 minutes after LPS injection (4 ng/kg). Time from harvesting to detection was 1 hour (see the text). No alteration in expression was observed after placebo administration. All samples were drawn from a single individual. This result is representative of three separate experiments.