REPORT
Suppression of fever at near term is associated with reduced COX-2 protein expression in rat hypothalamus

¹ Neuroscience Research Group and ³ Mucosal Inflammation Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Alberta, T2N 4N1 Canada; and ² Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-Ku Kyoto, 606 - 8502, Japan

	ABSTRACT

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The fever response is blunted at near term. As the enzyme cyclooxygenase-2 (COX-2) plays a critical role in fever development, we measured its expression in rat hypothalamus during pregnancy and lactation. Western blot analysis revealed a 72-kDa COX-2-immunoreactive band in non-immune-challenged, pregnant rats at day 15 of pregnancy. In contrast, it was almost undetectable at near term and at lactation day 5. COX-2 was significantly induced at the 15th day of pregnancy and at the 5th lactating day after intraperitoneal lipopolysaccharide (50 µg/kg). However, this COX-2 induction was significantly reduced at near term compared with values before and after term. The protein levels of the EP3 receptor in the hypothalamus, one of the prostaglandin E₂ (PGE₂) receptors suggested to be a key receptor for fever induction, were unaffected throughout the pregnancy and lactation in both non-immune-challenged and lipopolysaccharide-treated rats. These data suggest that suppression of fever at near term is associated with a significantly reduced induction of COX-2 by lipopolysaccharide, resulting in a reduced production of PGE₂. Altered expression of the EP3 receptor does not seem to be involved in this fever refractoriness at near term.

cyclooxygenase-2; parturition; lipopolysaccharide; prostaglandin receptor; EP3 receptor

	INTRODUCTION

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FEVER, A MAJOR PART of host defense, is thought to be of beneficial and adaptive value (15). Thus the inability to develop a fever response to pathogens can be detrimental (16). An absent or reduced fever has been observed in pregnant animals at near term (14) and, in some circumstances, is associated with abortion or mortality (23). This febrile refractoriness has been observed in many species, including guinea pig (55), rabbits (28), sheep (14), and rats (7, 23, 24), and has been observed in response to both peripherally injected pyrogens (14, 23, 55) and, to a lesser extent, to centrally infused prostaglandins (7, 24, 48). The fact that fever suppression is most dramatic in response to systemically administered LPS suggests that several steps in the cascade of responses to peripherally injected LPS may be affected.

In response to bacterial pyrogens such as LPS, immunocompetent cells generate endogenous cytokines, which signal to the central nervous system through either humoral or neuronal pathways (for review, see Ref. 8) to induce expression of cyclooxygenase-2 (COX-2) (6, 22). The activity of this enzyme results in the cyclooxygenation of arachidonic acid and subsequently the production of PGE₂, which acts largely in the anterior hypothalamus/preoptic area (47). COX-2 is present in basal conditions in the brain (3, 41), but in inflammation, it is induced specifically in the endothelium of brain capillaries (18, 25). Levels of COX-2 have been correlated to levels of PGE₂ during LPS-induced fever (54). Because COX-2 plays a critical role in the fever response (22), we explored its expression in this hypothalamic region of pregnant and lactating rats. In this study, we asked: 1) do basal hypothalamic COX-2 protein levels change at near term? and 2) is LPS-induced COX-2 expression in the hypothalamus affected at near term?

PGE₂ actions on hypothalamic neurons are mediated through EP receptors (EP-Rs) (34, 51, 56). Studies in knockout mice suggest that among EP-R subtypes (EP1-R, EP2-R, EP3-R, and EP4-R), the EP3-R might be a candidate receptor to mediate the febrile effect of centrally injected PGE₂ and peripherally administered pyrogens (LPS and IL-1) (51). In this study, therefore, we also explored whether EP3-R protein levels change at near term in non-immune-challenged and LPS-injected rats.

	MATERIALS AND METHODS

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Timed Sprague-Dawley pregnant females from Charles River were individually housed in temperature-controlled quarters under a 12:12-h light-dark cycle (lights on 0700). All experimental protocols were approved by the University of Calgary Animal Care Committee and were carried out in accordance with the Canadian Council of Animal Care guidelines.

Pregnant females and protein extraction. Rats at the 15th day of pregnancy, at near term (within 24 h of labor), and at the 5th day postpartum were divided in two groups. The first group received no injection. The second group received an intraperitoneal injection of LPS (Sigma, St. Louis, MO; E. coli serotype 026:B6) at 50 µg/kg, 3 h before removal of the hypothalami [since 3 h after LPS injection corresponds to the first-phase fever (23)]. In these experiments, all animals tolerated this dose of LPS without any apparent ill effects. The animals were anesthetized with pentobarbital sodium (50-60 mg/kg ip) and transcardially perfused with PBS (pH 7.4) (NaCl: 137 mM; KCl: 2.7 mM; Na₂HPO₄: 10 mM; KH₂PO₄: 1.8 mM) to remove the blood. The basal diencephalon, including the preoptic area and the hypothalamus, was quickly removed and put in lysis buffer composed of PBS, 1% nonionic detergent (IGEPAL, Sigma, I-3021), 0.5% sodium deoxycholate (Sigma, D-6750), and 0.1% SDS (Bio-Rad Laboratories, Cat. #161-0301, Richmond, CA) supplemented with proteinase inhibitors that include 1 mM phenylmethylsulfonyl fluoride (Boehringer Mannheim), 30 µl/ml aprotinin (Sigma, A-6279), 2 mM sodium orthovanadate (Sigma, S-6508), and 10 mM sodium fluoride (Fisher Scientific S-299). After mechanical dissociation of the brain tissue, protein levels were assayed using a bicinchoninic acid protein assay (Pierce Rockford, Reagent A Cat. #23223 and Reagent B Cat. #23224). The proteins were then put in a sample buffer composed of 40 mM Tris · HCl (Sigma, T-1503), 1% SDS, 50 mM dithiothreitol, 7.5% glycerol (Sigma, G-5516), bromophenol blue (Sigma B-5525), boiled for 5 min, and stored at 20°C for Western blot analysis.

Western blot. Hypothalamic protein extracts (30 µg/well) in each series of experiments (i.e., basal or LPS stimulated) were loaded concomitantly into a single gel and were separated on 10% SDS-PAGE using a constant current of 30 mA/gel of 1.2-mm thickness. Molecular weight markers (Bio-Rad, Cat. #161-0372) were used in each individual gel. After separation, the proteins were transferred onto nitrocellulose membrane for 2 h under a constant current (1.2 mA/cm² of gel surface) using a transfer buffer containing 20% methanol, 50 mM Trizma base (Sigma), 40 mM glycine (Sigma), and 0.04% SDS (Bio-Rad). Membranes were then incubated overnight at 4°C with 10% fat-free milk in Tris-buffered saline containing Tween 20 (TBS-T) composed of 20 mM Trizma base (Sigma), 0.15 M sodium chloride (Fisher Scientific), and 0.1% polyoxyethylene-sorbitan monolaurate (Sigma). A well-characterized rabbit COX-2 antibody (Cayman Chemical, Cat. #160126) that does not recognize COX-1 protein (53) was used at 1:2,000. We also measured levels of actin, a housekeeping protein. After detection of the COX-2 band, the membrane was stripped with mercaptoethanol (BDH) and reblotted with rabbit anti-actin antibody (Sigma, Cat. #A2066) at a 1:10,000 dilution. After a 2-h incubation with the primary antibodies at room temperature, the membrane was washed with TBS-T and incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:4,000) (Santa Cruz Biotechnology, sc-2004) for 1 h at room temperature. In some experiments, both COX-2 and actin antibody were used concomitantly. Initial experiments (data not shown) determined that there was no interference between these two antibodies, as the secondary antibody was used at saturating conditions. Both procedures yielded similar results. The specificity of the COX-2 antibody was established by preabsorbing COX-2 antibody with the COX-2 peptide (Cayman Chemical, Cat. #360106) for 1 h at room temperature. This preabsorption eliminated the COX-2 band.

A different series of Western blot experiments was carried out on the same hypothalamic extracts to follow EP3-R expression during pregnancy and lactation. Again, all protein extracts for the immunoblot experiment (basal or LPS treated) were loaded onto the same gel. Rabbit EP3-R antibody was used at a concentration of 2 µg/ml. This antibody has been shown to specifically recognize the EP3-R peptide (30). After a 2-h incubation with the primary antibodies at room temperature, the membrane was washed with TBS-T and incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:4,000) (Santa Cruz Biotechnology, sc-2004) for 1 h at room temperature. After EP3-R detection, the membrane was stripped with mercaptoethanol (BDH) and reblotted with rabbit anti-actin antibody (Sigma, Cat. #A2066) at 1:10,000 dilution. For protein detection, a chemiluminescent substrate was applied to the membrane (ECL Kit, Amersham Pharmacia Biotech) and protein bands were visualized using Kodak X-Omat film (Eastman Kodak).

Data analysis. Proteins were quantified using a densitometer to give COX-2/actin and EP3-R/actin ratios. It is important to note that the actual ratio varies from experiment to experiment as a function of transfer efficiency, film exposure, stripping, etc. Thus ratios between different series of experiments cannot be compared. Data are presented as means ± SE and were subjected to analysis of variance followed by Student-Newman-Keuls post hoc comparisons.

	RESULTS

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In basal conditions, the hypothalamus of pregnant rats expressed a detectable COX-2 band with a molecular mass of ~72 kDa at the 15th day of pregnancy. This COX-2 protein expression decreased at near term and stayed low for at least 5 days after parturition (Fig. 1A, top). In these experiments, actin and COX-2 proteins were simultaneously detected by applying a mixture of COX-2 antibody and actin antibody. Analysis using densitometry of COX-2/actin ratios at each pregnancy and lactation stage shows a significantly (>50%) reduced COX-2 expression at near term and at the 5th day postpartum (P < 0.05, n = 3-5 each data point; Fig. 1A, bottom). Induction of COX-2 following injection of LPS (50 µg/kg ip) was higher in the hypothalamus of both 15-day pregnant and lactating rats at the 5th day postpartum compared with its induced levels at near term (Fig. 1B, top). In these series of experiments, membranes were stripped and actin was subsequently detected. Densitometric analysis at each pregnancy and lactation stage shows COX-2 induction by LPS was significantly reduced (~40%) at near term (P < 0.05, n = 4-5 each data point) compared with LPS-induced levels before and after term (Fig. 1B, bottom).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Expression of cyclooxygenase-2 (COX-2) during different stages of pregnancy and lactation in basal and LPS-injected rats. A: proteins extracted from hypothalami at gestation day 15 (G15), day 22 (G22), and at the 5th day of lactation (L5) were electrophoretically separated, and COX-2 and actin were immunodetected as shown in a representative blot (top). A, bottom: optical density analysis of COX-2/actin ratios (*P < 0.05, n = 3-5 for each data point). B: experiments identical to those in A except that all rats were injected with LPS (50 µg/kg) 3 h before collection of hypothalami. Note that COX-2/actin ratios in A and B are not comparable as they were carried out in independent experiments (*P < 0.05, n = 4-5 for each data point).

We tested the hypothesis that the reduced febrile response in pregnant females at near term was also associated with a reduction of EP3-R expression. In non-LPS-challenged rats, three immunoreactive EP3-R proteins were detected (Fig. 2A, top left). The major band (~50 kDa) and the lower molecular mass band (~39 kDa) correspond to the glycosylated forms of EP3-R previously detected (30). An additional higher molecular mass EP3-R-immunoreactive band (~125 kDa) was also consistently detected. This band may be the result of SDS-resistant dimerized EP3-R protein as has been reported for other metabotropic receptors (50). Preabsorption of EP3-R antibody with the NH₂-terminal part of rat EP3-R protein that was used as an antigen (29) eliminated all three of these EP3-R-immunoreactive bands, thus confirming the specificity of this antibody for this EP3-R sequence (Fig. 2A, top right). The expression levels of these three immunoreactive bands were subsequently analyzed at each of the three representative stages. Their expression levels were found to be similar (P > 0.05, n = 3-5 each data point) in the hypothalamus of females at the 15th day of pregnancy, at near term, and at the 5th day postpartum (Fig. 2A, bottom).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Expression of EP3 receptor (EP3-R) protein during different stages of pregnancy and lactation in basal and LPS-injected rats. A: proteins extracted from hypothalami at G15, G22, and L5 were separated using electrophoresis, and EP3-R and actin were detected (top left). A, top right: immunoblot after preabsorption of EP3-R antibody with EP3-R antigen. A, bottom: optical density analysis of EP3-R/actin ratios. Three EP3-R-immunoreactive bands with low (a), medium (b), and high (c) molecular weights were detected, and their corresponding densitometric values were plotted separately (P > 0.05, n = 3-5 for each data point). B: experiments identical to those in A except that pregnant and lactating rats were injected with LPS (50 µg/kg) 3 h before collection of the hypothalami (P > 0.05, n = 4-5 for each data point). B, top right: representative blot of EP3-R expression in hypothalami of male rats given saline or LPS (50 µg/kg) 3 h earlier.

Intraperitoneal injection of LPS (50 µg/kg) did not affect the relative expression of the three EP3-R-immunoreactive bands throughout pregnancy and lactation (Fig. 2B, top left), and densitometry confirmed this for all three immunoreactive EP3-R bands (P > 0.05, n = 4-5 each data point; Fig. 2B, bottom). Because of the remarkable stability of the EP3-R protein throughout the pregnancy, even after LPS, we carried out additional experiments in male rats (2 rats/group) to determine if its levels would change after LPS. Here also there was no change in EP3-R-immunoreactive bands between LPS-treated and control rats (Fig. 2B, top right).

	DISCUSSION

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This study shows that both the basal and LPS-induced expression of COX-2, the rate-limiting enzyme in the production of PGE₂, are reduced at near term. In contrast, the hypothalamic expression of the PGE₂ receptor EP3-R is unaffected in all pregnancy and lactation stages under both basal and LPS-stimulated states.

Fever response at near term. Since the discovery by Kasting and colleagues (14) of fever suppression at near term in sheep, this phenomenon has been described in several other mammals (23, 28, 55). As the brain is thought to have its own antipyretic system, attention focused on the existence of a possible endogenous antipyretic as the cause of this suppression. A likely candidate was AVP, for which there is persuasive evidence that it acts as an endogenous antipyretic (39). Initial evidence in support of AVP acting to suppress fever at term came from the observation that there is increased immunoreactivity for AVP in the hypothalamus during pregnancy in guinea pigs (27) and in push-pull perfusates of the preoptic area in rats (19). However, as evidence in rats to support a role for AVP as an endogenous antipyretic responsible for the suppression of fever at parturition has been largely negative (7, 10), we turned our attention to a possible alteration in the cascade of events leading to fever. To our best knowledge, this study is the first to show that the basal and LPS-induced expression of hypothalamic inducible COX-2, the key enzyme that generates PGE₂ during fever, was significantly reduced at near term.

COX-2 reduction at near term. The mechanism by which COX-2 expression is reduced at near term is not fully understood. The most likely explanation is that the increased levels of circulating estrogen and progesterone toward the late pregnancy (35) affect COX-2 expression and/or activity in the PGE₂-responsive area of the hypothalamus and thus alter one of the key components of the febrile response. The inhibitory effects of estrogen and progesterone on COX-2 gene and protein expression have been explored in several peripheral tissues under different hormonal conditions, and, in each case, administration of ovarian hormones suppressed both basal and stimulated COX-2 levels and/or prostaglandin production (12, 40). Ovarian hormones might affect basal COX-2 gene expression via an action on its transcription factor sites such as nuclear factor (NF)-IL6, cAMP response element, or activating proteins-1 and -2 (1, 13, 32, 36, 42).

Our data show a dramatically reduced COX-2 induction by LPS at near term that could be partly responsible for fever suppression at this time. A possible mechanism for this might be through the inhibition of the immune-activated transcription factor NF-B by estrogens and progesterone (26, 43). Other possibilities could include altered pituitary-adrenal axis activity (11), increased levels of anti-inflammatory molecules such as the IL-1 receptor antagonist observed at near term (38), reduced levels of proinflammatory cytokines (20), or other antipyretic molecules (21).

We chose a 3-h interval between LPS administration and collection of tissues for COX-2 protein determination, as this allows sufficient time for transcription (18) and translation of the protein (5). However, it has been pointed out (2) that fever onset can sometimes precede the earliest appearance of inducible COX-2. As COX-2 appears obligatory for an LPS fever to develop, it is likely that constitutive COX-2 may contribute to the early phase of fever (46). The reduced levels of COX-2 we identified at term could thus also contribute to the suppression of an early part of the febrile response.

EP3-R at near term. PGE₂ acts locally in the preoptic/anterior area of the hypothalamus (44, 47, 52) where PGE receptor subtypes are expressed: namely EP1-R, EP3-R, and EP4-R (37) that have been implicated in fever. Agonist/antagonist studies concluded that PGE₂ acts mainly, if not solely, through the EP1-R subtype to mediate fever (33). With the use of a colocalization paradigm of in situ detection of mRNA of EP-R subtypes and Fos protein immunoreactivity, others provided evidence that EP4-R and, to a lesser extent, EP1-R- and EP2-R-expressing neurons are activated during LPS-induced fever (34, 56). In contrast, EP3-R would not be expected to activate the c-fos gene (4). However, EP3-R protein and mRNA are also expressed in PGE₂-sensitive regions of the hypothalamus (9, 30, 49) including in neurons thought to be involved in fever generation (31). Specific functional disruption of genes of these four EP-Rs led to the conclusion that only EP3-R knockout mice fail to develop fever in response to centrally injected PGE₂ or peripherally administered IL-1 or LPS (51).

Because we previously observed an attenuated response to intracerebroventricular infusion of PGE₂ at near term (7), we tested the hypothesis that, in addition to alteration in COX-2 expression described above, there is a change in the expression of the EP3-R. In this study, we showed that the febrile refractoriness at near term is not a consequence of a change of the EP3-R levels in the hypothalamus. Indeed, EP3-R levels were similar at all pregnancy and lactation stages tested. Furthermore, LPS also did not affect the amount of EP3-R protein expressed in the hypothalamus of mid-pregnant, near-term, and lactating rats or in male rats. If the central PGE₂ sensitivity changes at near term, our data suggest that the EP3-R may not be the key receptor involved. However, the fact that the levels of EP3-R expression did not change at near term does not exclude a possible change in the binding properties or of the activation of downstream messengers nor does it rule out possible changes in a very small proportion of neurons that could be masked by our sampling of the entire preoptic area and the hypothalamus. Due to the lack of available EP1-R and EP4-R antibodies, we could not test whether this attenuated fever response could also be brought about by the reduced sensitivity of the brain to PGE₂ acting through EP1-R and/or EP4-R.

In summary, COX-2 protein expression in the hypothalamus changes during pregnancy and lactation. The COX-2 level is significantly reduced at near term in basal conditions as well as in response to immune challenge with LPS. Alteration in sensitivity to the COX-2 product (PGE₂) in the hypothalamus does not seem to be mediated through alteration in EP3-R levels, because the expression of this receptor was stable throughout pregnancy and lactation in both LPS-challenged and control rats. Taken together, these data suggest that fever suppression at near term is due, in part, to a reduced expression of COX-2 in the hypothalamus. Future studies must address the possibility that other factors such as alterations in the elaboration of pyrogenic cytokines or synthesis of antipyretic molecules (17, 45) may also play a role.

	ACKNOWLEDGEMENTS

This work was supported by the Canadian Institutes of Health Research. M-S. Clerget-Froidevaux was a Medical Research Council/Hypertension, Foundation del Duca (France) and University of Calgary Fellow, and Q. J. Pittman and J. L. Wallace are Alberta Heritage Foundation for Medical Research Medical Scientists.

	FOOTNOTES

Address for reprint requests and other correspondence: A. Mouihate, Neuroscience Research Group, Dept. of Physiology and Biophysics, Univ. of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada (E-mail: mouihate{at}ucalgary.ca).

10.1152/ajpregu.00258.2002

Received 10 May 2002; accepted in final form 30 May 2002.

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