Cyclooxygenase Isoform Exchange Blocks Brain-Mediated Inflammatory Symptoms

Abstract

Cyclooxygenase-2 (COX-2) is the main source of inducible prostaglandin E2 production and mediates inflammatory symptoms including fever, loss of appetite and hyperalgesia. COX-1 is dispensable for fever, anorexia and hyperalgesia but is important for several other functions both under basal conditions and during inflammation. The differential functionality of the COX isoforms could be due to differences in the regulatory regions of the genes, leading to different expression patterns, or to differences in the coding sequence, resulting in distinct functional properties of the proteins. To study the molecular underpinnings of the functional differences between the two isoforms in the context of inflammatory symptoms, we used mice in which the coding sequence of COX-2 was replaced by the corresponding sequence of COX-1. In these mice, COX-1 mRNA was induced by inflammation but COX-1 protein expression did not fully mimic inflammation-induced COX-2 expression. Just like mice globally lacking COX-2, these mice showed a complete lack of fever and inflammation-induced anorexia as well as an impaired response to inflammatory pain. However, as previously reported, they displayed close to normal survival rates, which contrasts to the high fetal mortality in COX-2 knockout mice. This shows that the COX activity generated from the hybrid gene was strong enough to allow survival but not strong enough to mediate the inflammatory symptoms studied, making the line an interesting alternative to COX-2 knockouts for the study of inflammation. Our results also show that the functional differences between COX-1 and COX-2 in the context of inflammatory symptoms are not only dependent on the features of the promoter regions. Instead they indicate that there are fundamental differences between the isoforms at translational or posttranslational levels.

Fig 1. Attenuated febrile response in COX-1>COX-2 animals after administration of LPS.

A. Telemetric recordings in freely moving animals showing the deep body temperature of COX-1>COX-2 animals or WT littermates after the administration of LPS (100 μg/kg) or vehicle intraperitoneally. B Average body temperature after LPS administration is completely normalized in COX-1>COX-2 animals compared to WT animals for the duration of the febrile response (2.5-8h post LPS). ***p<0,001

Fig 2. Cumulative food intake in COX-1>COX-2 and WT littermates during the first 3 hours after LPS injection (10μg/kg).

The COX-1>COX-2 animals are significantly protected from the acute, anorexigenic effects of LPS (***p<0.001).

Fig 3. Quantification of the nociceptive response (flinching) to formalin-induced pain.

(A) Nociceptive score was calculated for each 5 minutes during a total 60 minutes after subcutaneous injection of 2,5% formalin in the right hind paw. (B) The second phase of the nociceptive response was clearly attenuated in COX-1>COX-2 animals (**p<0.01).

Fig 4

Quantification of COX-1 (A) and COX-2 (B) mRNA levels in hypothalami of COX-1>COX-2 animals and WT littermates 3 h after LPS injection (100μg/kg). A significant up-regulation of COX-1 is seen in COX-1>COX-2 animals (A). As expected, COX-2 was strongly induced in WT but not COX-1>COX-2 mice. The COX-1 expression signal in COX-1>COX-2 animals after LPS is at least as strong as the combined expression signal of COX-1 and COX-2 in WT animals after LPS, indicating that the induction of COX-1 at least corresponds to the increase in COX-2 mRNA in absolute terms (C). COX-1>COX-2 heterozygous (HZ) animals were included as comparison. (*p<0.05; **p<0.01)

Fig 5. Photomicrographs showing the COX-1/COX-2 expression pattern in the brain 3 hours after intraperitoneal injection of NaCl or LPS (100 μg/kg).

Basal COX-2 expression in WT animals was prominent in parts of the cerebral cortex (a) whereas, in WT mice treated with LPS, strong COX-2 expression was seen also in cells with endothelial morphology lining cerebral vessels (b). No COX-2 could be detected in brains from COX-1>COX-2 mice treated with LPS (c). Strong COX-1 labeling was found in cells with microglial morphology in both WT and COX-1>COX-2 mice (d, e; cerebral cortex). Brain sections from mice lacking COX-1 displayed no COX-1 labeling (f). Neuronal COX-2 expression was seen in the cerebral cortex of WT mice (g) injected with LPS, but no corresponding expression pattern of COX-1 was seen in COX-1>COX-2 mice (h). Instead, only cells with microglial morphology expressed COX-1 in the corresponding region of the brain in COX-1>COX-2 mice (h) in a pattern identical to COX-1 in WT mice (i). Vascular COX-2 expression in LPS-treated WT mice (j, k) was compared to COX-1 expression in COX-1>COX-2 mice (l, m) and COX-1 expression in WT animals (n, o). Neither in the hypothalamus (j, l, n) nor elsewhere (k, m, o; striatum), the COX-1 expression in COX-1>COX-2 mice matched that of COX-2 in WT mice. Dual labeling analysis and confocal microscopy showed that activated endothelial cells, identified by detection of Lcn2 expression, expressed COX-2 in WT mice (p) but that no corresponding COX-1 expression could be seen in activated endothelial cells in COX-1>COX-2 mice (q, r). Scale bar (same for all figures within a given row) in a = 200 μm, d, g, j and p = 50 μm.