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Endocrinology Vol. 143, No. 12 4820-4827
Copyright © 2002 by The Endocrine Society

ARTICLE

In Altering the Release of Glucocorticoids, Ketorolac Exacerbates the Effects of Systemic Immune Stimuli on Expression of Proinflammatory Genes in the Brain

Véronique Blais, Ji Zhang and Serge Rivest

Laboratory of Molecular Endocrinology, Centre Hospitalier de l’Université Laval Research Center and Department of Anatomy and Physiology, Laval University, Québec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Dr. Serge Rivest, Laboratory of Molecular Endocrinology, Centre Hospitalier de l’Université Laval Research Center and Department of Anatomy and Physiology, Laval University, 2705, Boulevard Laurier, Québec, Canada G1V 4G2. E-mail: serge.rivest{at}crchul.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for their antiinflammatory, antipyretic, and analgesic properties. The molecular basis for the therapeutic action of NSAIDs is believed to be in their ability to inhibit cyclooxygenase (COX) activity and thereby blocking the production of prostaglandins. Emerging evidence now suggests that NSAIDs can exert their pharmacological effects through other mechanisms. This study investigated the influence of a nonselective COX-inhibitor ketorolac on IL-1ß- and TNF{alpha}-induced expression of proinflammatory genes in the brain. Systemic injection of both cytokines caused a rapid and transient transcriptional activation of COX-2 gene within the cerebral microvasculature, which was significantly enhanced by ketorolac. Expression of genes encoding the index of nuclear factor {kappa}B activity and the chemokine monocyte chemoattractant protein-1 was also increased by the NSAID. We speculated here that such effect was indirectly mediated via an altered secretion of plasma glucocorticoids because ketorolac is a potent inhibitor of the hypothalamic-pituitary-adrenal axis during systemic inflammation. As expected, pretreatment with the glucocorticoid receptor antagonist RU-486 exacerbated the influence of systemic immune stimuli on proinflammatory signaling. In contrast, exogenous corticosterone abolished the effects of ketorolac on IL-1ß-induced COX-2 and monocyte chemoattractant protein-1 gene expression in the cerebral endothelium. This drug plays therefore a paradoxical role in its ability to inhibit the circulating levels of glucocorticoids that are essential inhibitory feedback on the proinflammatory signal transduction pathways and gene transcription. In altering the production of key prostaglandins that are involved in the control of hypothalamic-pituitary-adrenal axis, ketorolac may have proinflammatory properties in the central nervous system during systemic immune stimuli.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS (PGs) ARE a family of autocrine and paracrine mediators that contribute to many physiological and pathophysiological responses. They regulate vascular homeostasis, kidney function, ovulation, and parturition. They are equally important as mediators of inflam-mation, thrombosis, and pain. Cyclooxygenase (COX) catalyzes the production of PGs from arachidonic acid, generated by phospholipase A2 that causes the release of arachidonic acid from cellular phospholipids, to form PGG2 and subsequently PGH2. The ultimate biologically active PG products are formed in a cell-specific manner. There are two isoforms of the cyclooxygenase enzyme called COX-1 and COX-2. These isoforms are similar in size, substrate specificity, and kinetics but vary in their expression and distribution (1). In general, COX-1 is constitutively expressed, whereas COX-2 is inducible like other immediate-early genes. COX-1 is responsible for the so-called housekeeping functions of PGs, whereas COX-2 plays a key role in inflammatory responses.

Inhibition of COX, with aspirin and nonsteroidal antiinflammatory drugs (NSAIDs), leads to a decrease in the production of all prostaglandins and thromboxane, which accounts for the beneficial antiinflammatory, antipyretic, analgesic, antineostatic, and cardiovascular effects of NSAIDs as well as their gastrointestinal side effects. As shown by numerous clinical trials, the use of aspirin or other NSAIDs can reduce the risk of colon cancer (2, 3), and a reduced risk of developing Alzheimer’s disease was reported in patients on long-term therapy with NSAIDs (4).

NSAIDs have many pharmacological effects other than inhibiting COX activity. Sodium salicylate and aspirin were shown to inhibit the transcription factor nuclear factor {kappa}B (NF{kappa}B) (5). NSAIDs can also activate peroxisome proliferator-activated {gamma} and induce adipocyte differentiation (6). Although quite controversial, recent in vitro studies have demonstrated that NSAIDs have the ability to modulate COX-2 gene expression in several types of cells. They were found to up-regulate the transcriptional activity of COX-2 in cultured microglial cells (7), epithelial cells (8), and osteoblastic MC3T3E1 cells (9) but suppress IL-1ß-induced COX-2 expression in endothelial (10) and smooth muscle (11) cells.

To clarify these seemingly opposing results, we designed an in vivo experiment to study the effects of ketorolac, a water-soluble and nonselective COX inhibitor, on inhibitory factor {kappa}B{alpha} (I{kappa}B{alpha}) and COX-2 expression in rat brain following a single bolus of proinflammatory cytokines. Results from our previous studies demonstrated that ketorolac was very effective in preventing the endogenous release of PGE2 in both vehicle- and IL-1ß-injected rats (12). Surprisingly, ketorolac exacerbated the influence of systemic inflammatory molecules on NF{kappa}B activity and COX-2 gene expression in vascular-associated elements of the brain. Because ketorolac has the ability to modulate the hypothalamic-pituitaryadrenal (HPA) axis in altering the secretion of key PGs, we then investigated whether such properties were dependent on the endogenous secretion of glucocorticoids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Fifty-four adult male Sprague Dawley rats (175–225 g) were acclimated to standard laboratory conditions (14-h light, 10-h dark cycle, lights on at 0600 h and off at 2000 h) with free access to rodent chow and water. Animal breeding and experiments were conducted according to Canadian Council on Animal Care guidelines, as administered by the Laval University Animal Care Committee.

Surgical preparation
Animals receiving iv injection were chronically implanted with sterile cannulas. Rats were anesthetized with an ip injection of 0.25 ml ketamine hydrochloride (91 mg/kg) and xylazine (9.1 mg/kg) mixture and implanted with a catheter into the right jugular vein. Catheters were made from a piece of SILASTIC-brand tubing (SILASTIC medical-grade tubing; inner diameter 0.020 in., outer diameter 0.037 in.; Dow Corning, Inc., Midland, MI) connected to an intramedic polyethylene tubing (PE-50, Caly Adams, Parsippany, NJ). The outlet of cannula was placed at the level of neck, and rats were housed individually in metal cages for a 3- to 5-d recuperating period. On the day of experiment (~0830 h), the outlet portion of each catheter was fixed to a truncated, 2.0-cm, 22-gauge needle, which was attached to a PE-50 tubing. These connectors were then fixed to a 1-ml syringe, and rats were placed individually in a quiet room for at least 2 h before experimentation. This procedure was used to avoid disturbing the rats during injections.

Experimental protocols
Protocol 1.
Intravenous injections of ketorolac and TNF{alpha} or IL-1ß in rats. The water-soluble inhibitor of PG synthesis ketorolac (10 mg/kg body weight, lot no. 0228, Hoffmann-La Roche Ltd., Mississauga, Ontario, Canada) diluted in sterile saline (100 µl) or the vehicle solution was administered through the right jugular vein. Five minutes later rats received either recombinant rat IL-1ß (rrIL-1ß, 1.87 µg/kg body weight; generously provided by Dr. R. P. Hart, Rutgers University, Newark, NJ), recombinant rat TNF{alpha} (rrTNF{alpha}, 2.0 µg/kg body weight, lot no. AGM017071, R&D Systems, Minneapolis, MN) or the vehicle solution (200 µl sterile pyrogen-free saline). The dose of cytokines was selected on the basis of previous experiments, which showed increase in gene expression in both vascular and parenchymal elements of the brain (13, 14, 15). Animals were conscious and freely moving at all times throughout the procedure. At different times after injection (from 1–3 h), rats were deeply anesthetized via an iv injection of a mixture of ketamine hydrochloride and xylazine and then rapidly perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M borax buffer (pH 9.5 at 4 C).

Protocol 2.
Pretreatment with corticosterone, RU-486, and ketorolac in IL-1ß-treated rats. Rats were pretreated with an ip administration of corticosterone [50 mg/kg, C2505, lot no. 119H0779; diluted in dimethylsulfoxide (DMSO), Sigma, St. Louis, MO], RU-486 (50 mg/kg, M8046, lot no. 30K1542, diluted in DMSO, Sigma), or DMSO (100 µl, D2650, lot no. 11K2321, Sigma). Ketorolac or saline solution was administered 1 h later through the right jugular vein, which was followed by a single bolus of recombinant rat IL-1ß (rrIL-1ß, 1.87 µg/kg body weight, lot no. QZ080121, R&D Systems) or saline solution. Animals were killed 1 h after being injected with the cytokine as explained previously.

In situ hybridization histochemistry and double labeling
After transcardiac perfusions, brains were rapidly removed from the skull, postfixed for 1–3 d, and then placed in a solution containing 10% sucrose diluted in 4% paraformaldehyde-borax buffer overnight at 4 C. The frozen brains were mounted on a microtome (Reichert-Jung, Cambridge Instruments Co., Deerfield, IL) and cut in coronal sections (30 µm) from the olfactory bulb to the end of the medulla. The slices were collected in a cold cryoprotectant solution [0.05 M sodium phosphate buffer (pH 7.3) containing 30% ethylene glycol and 20% glycerol] and stored at -20 C. Hybridization histochemical localization of each transcript was carried out on every sixth section of the whole rostrocaudal extent of each brain using 35S-labeled cRNA probes as described previously (15). After being dried under vacuum, the sections were exposed at 4 C to x-ray films for 15–65 h (depending on the messages), defatted in xylene, and dipped in NTB2 nuclear emulsion (diluted 1:1 with distilled water, Kodak, Rochester, NY). Slides were exposed for 7–17 d, developed in D19 developer (Kodak) for 3.5 min at 14–15 C, washed 15 sec in water, and fixed in rapid fixer (Kodak) for 5 min. Thereafter tissues were rinsed in running distilled water for 1–2 h, counterstained with thionin (0.25%), dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with distrene plasticizer xylene mounting medium.

Synthesis of the cRNA probes and combination of immunocytochemistry with in situ hybridization was performed as previously described (12, 13, 16).

Data analysis
The relative intensity of mRNA signals throughout the brain of each animal was assessed on x-ray film images and graded according to the scale of undetectable (-), low (+), moderate (++), strong (+++), or very strong (++++) signal. Dipped emulsion slides were examined under microscopic evaluation to ascertain the subcellular localization of the transcript. Semiquantitative analysis of COX-2 and I{kappa}B{alpha} mRNA hybridization signals was carried out on nuclear emulsion-dipped slides with an Optical System (BX-50, Bmax, Olympus Corp., Melville, NY) coupled to a Macintosh computer (Power PC 7100/66, Apple Computer, Cupertino, CA) and Image software (version 1.61, non-FPU, W. Rasband, NIH). The refraction density in arbitrary units (RDAU) of the hybridization signal was measured under darkfield illumination at a magnification x50. Sections from experimental animals were digitized and subjected to densitometric analysis, yielding measurements of RDAU. For each animal, an average of 5–10 blood vessels in the ventral medulla corresponding to 50–60 endothelial cells was quantified. The RDAU of each specific encircled cell was then corrected for the average background signal, which was determined by sampling cells immediately outside the cell group of interest.

Quantitative analysis of the c-fos hybridization signal was carried out on x-ray films. Transmittance values (referred here as OD) of the hybridization signal was measured under a Northern Light Desktop Illuminator (Imaging Research, Inc., St. Catherines, Ontario, Canada) using a Sony (Tokyo, Japan) camera video system attached to a Micro-Nikon 55-mm Vivitar extension tube set for a lens (Nikon, Melville, NY) and coupled to a Macintosh computer. OD values for each pixel were calculated using a known standard of intensity and distance measurements from a logarithmic specter adapted from Bioimage Visage 110s (Millipore Corp., Ann Arbor, MI). The paraventricular nucleus of the hypothalamus (PVN) from experimental and control animals were digitized and subjected to densitometric analysis, yielding measurements of mean density per area. The OD was then corrected for the average background signal by subtracting OD of area without a positive signal located immediately outside the digitized nucleus. Data are reported as mean values (± SEM) for experimental and control animals. Statistical analysis was performed by a two- or three-way ANOVA, followed by a Bonferroni/Dunn test procedure as post hoc comparisons by means of the Statview program (version 4.01, Macintosh).

Plasma corticosterone levels
Blood samples were taken just before the transcardiac perfusion and collected in ice-cold Vacutainer tubes (Becton Dickinson and Co., Franklin Lakes, NJ) that were centrifuged for 15 min at 3000 g. The plasma samples were frozen at -20 C until the assay. Plasma corticosterone levels of treated and control animals were determined by a RIA kit (Immunochem corticosterone RIA kit for rats, catalog no. RCBK9906A, ICN Biochemical, Costa Mesa, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We initially carried out our experiments to evaluate the inhibitory properties of ketorolac, which were reported in a previous study (12). The endogenous production of the PGs was already high in the liver of vehicle-administered rats, and the inhibitor of COX pathway was very effective in preventing the endogenous release of PGE2 in both vehicle- and IL-1ß-injected groups (1 and 3 h). Ketorolac also prevented TNF{alpha} from releasing PGE2 in the liver (data not shown). Notwithstanding these data, ketorolac enhanced COX-2 gene expression in cerebral microvasculature of immune-challenged animals (see below). COX-2-expressing cells were identified as brain endothelium because they were immunoreactive to von Willebrand Factor (Fig. 1Go and Refs. 13 and 15). On the other hand, the gene encoding I{kappa}B{alpha} was found in both endothelial and microglial cells (Fig. 1Go and Refs. 13 and 15). As depicted by Fig. 1Go, vehicle alone did not provoke notable transcriptional activation of COX-2 gene in vascular-associated elements of the brain. On the other hand, a small number of I{kappa}B{alpha}-expressing cells were detected along few isolated capillaries of saline-injected rats (Fig. 1Go, top right).



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  		Figure 1. Influence of the proinflammatory cytokine IL-1ß (1 h post iv injection, 1.87 µg/kg body weight) and TNF{alpha} (90 min post iv injection, 2.0 µg/kg body weight) on the expression of COX-2 and I{kappa}B{alpha} mRNAs in the blood vessels of rat brain. COX-2- and I{kappa}B{alpha}-expressing cells were also immunoreactive to von Willebrand factor (vWF), used here as a marker of endothelial cells. These double-labeled cells (bottom panel) were taken from rats challenged with a single bolus of TNF{alpha}. Black arrowheads, vWF-ir cells positive for the mRNA encoding COX-2 or I{kappa}B{alpha}. Magnification for darkfield photomicrographs, x50; for brightfield photomicrographs, x100.

 
Effects of ketorolac on cytokine-induced COX-2 gene expression
Intravenous injections of IL-1ß and TNF{alpha} activated COX-2 transcription in the cerebral microvasculature (Fig. 2Go). The hybridized signal peaked at 1 h and declined 3 h after a single iv bolus of IL-1ß. Positive hybridization signal was detected along blood vessels penetrating the brain parenchyma 90 min after iv TNF{alpha} injection. Pretreatment with ketorolac increased the effects of both proinflammatory cytokines because the signal intensity was higher in the central nervous system (CNS) of animals pretreated with the inhibitor of PG synthesis. The RDAU was measured on an average of 5–10 blood vessels containing 50–60 endothelial cells for each rat. As shown in Fig. 3Go, COX-2 mRNA signal in vascular-associated elements was significantly higher in the microvasculature of rats that received both treatments than those injected with either cytokine alone. Quantitative analysis was not possible in the brain of animals challenged with vehicle or ketorolac alone because the hybridization signal in cells lining the blood-brain barrier was comparable with background levels.



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  		Figure 2. Expression of COX-2 in the endothelium of the cerebral capillaries in rats that received either IL-1ß or TNF{alpha} with or without being pretreated with ketorolac. These darkfield photomicrographs show in situ hybridization signals for COX-2 mRNA along the microvasculature after iv of IL-1ß (1 and 3 h post injection, 1.87 µg/kg body weight) or TNF{alpha} (90 min post injection, 2.0 µg/kg body weight), respectively. Note that pretreatment with ketorolac (10 mg/kg body weight, iv injection) significantly enhanced COX-2 mRNA expression in response to systemic injection with the proinflammatory cytokines.

 


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  		Figure 3. Average RDAU of COX-2 hybridization signal within the cerebral endothelium following iv injection of ketorolac and IL-1ß (1.87 µg/kg body weight) or TNF{alpha} (2.0 µg/kg body weight). The analysis of hybridization signals was performed on NTB2 emulsion-dipped slides as described in Materials and Methods. Data are means ± SEM for three rats per group. *, Significantly different (P < 0.05) from their respective saline-treated groups.

 
Effects of ketorolac on I{kappa}B{alpha} gene expression
We also verified the effects of ketorolac on the expression of I{kappa}B{alpha}, because de novo induction of this transcript is a reliable index of NF{kappa}B activity in the CNS. A moderate to strong hybridization signal for the mRNA-encoding I{kappa}B{alpha} was detected along the small and large blood vessels of the leptomeninges, choroid plexus, circumventricular organs, and those penetrating the brain parenchyma following iv injection of IL-1ß (1 h) or TNF{alpha} (90 min). Scattered small cells were also found across the brain parenchyma and these are microglia (15). Pretreatment with ketorolac increased the effects of proinflammatory cytokines on I{kappa}B{alpha} gene expression in vascular-associated structures and parenchymal microglia (Fig. 4Go). The signal intensity that was already strong in rats injected with IL-1 or TNF increased significantly when the cyclooxygenase pathway was inhibited (Fig. 5Go).



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  		Figure 4. Darkfield photomicrographs showing the expression of the gene encoding the inhibitory factor I{kappa}B{alpha} in the brain of rats that received iv injection of ketorolac before the systemic administration of IL-1ß or TNF{alpha}. Rats were killed 1 h or 90 min following iv injection of IL-1ß (1.87 µg/kg body weight) or TNF{alpha} (2.0 µg/kg body weight), respectively. Note the positive hybridization signals for I{kappa}B{alpha} mRNA along the microvasculature after administration of both cytokines, a phenomenon that increased following pretreatment with ketorolac.

 


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  		Figure 5. Effect of ketorolac and cytokines on I{kappa}B{alpha} hybridization signal in vascular-associated elements of the rat brain. Animals were killed 1 h and 90 min after iv injection of IL-1ß and TNF{alpha}, respectively. Average RDAU was measured as described in Materials and Methods, and results represent means ± SEM for two to three rats per group. *, Significantly different (P < 0.01) from the vehicle-treated groups. **, Significantly different (P < 0.05) from all groups of animals pretreated with saline rather than ketorolac.

 
Influence of ketorolac on the HPA axis and proinflammatory signaling
Figure 6Go shows plasma corticosterone and mRNA levels for the immediate-early gene c-fos in the PVN of rats. As previously reported by several studies, systemic IL-1ß caused an increase in circulating levels of glucocorticoids and c-fos expression in the neurons that control this endocrine axis. Pretreatment with ketorolac essentially abolished these effects, and we hypothesized that altered HPA axis was responsible for the proinflammatory properties of the PG inhibitor. Indeed, glucocorticoids are the most potent endogenous immunosuppressors and drugs that alter this axis may have profound consequences on the innate immune response.



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  		Figure 6. Influence of ketorolac on plasma corticosterone (A) and c-fos mRNA levels in the hypothalamic PVN (B) of rats injected with IL-1ß. Plasma corticosterone levels were measured by RIA (top panel), whereas OD (bottom panel) was determined on x-ray films as described in Materials and Methods. Results are means ± SEM for two to four rats per group. *, Significantly different (P < 0.05) from the vehicle-injected rats. See Fig. 7Go for representative examples of c-fos mRNA expression in the hypothalamic PVN.

 
To test this hypothesis, rats received exogenous corticosterone or the glucocorticoid receptor antagonist RU-486 1 h before IL-1ß. Control treatments with RU-486, saline, ketorolac, corticosterone, or combined corticosterone with ketorolac failed to activate expression of the different proinflammatory genes in the CNS (Fig. 7AGo). A weak I{kappa}B{alpha} mRNA signal was nevertheless found in the cerebral capillaries of corticosterone-administered rats. This contrasts with the strong hybridization signal for COX-2, I{kappa}B{alpha}, and macrophage chemoattractant protein-1 (MCP-1) transcripts in the brain of animals that were injected with both IL-1ß and RU-486 (Fig. 7BGo, left column). A robust transcriptional activation occurred for the gene encoding COX-2 in large blood vessels as well as small capillaries. On the other hand, COX-2 transcript was barely detectable in brains of animals treated with corticosterone before IL-1ß and ketorolac. A similar phenomenon was observed for the chemokine MCP-1.



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  		Figure 7. Effects of the glucocorticoid receptor antagonist RU-486 or exogenous corticosterone on IL-1-induced expression of proinflammatory genes and c-fos in the rat brain. These darkfield photomicrographs display positive signal on NTB2-dipped coronal section (30 µm) for COX-2, I{kappa}B{alpha}, and MCP-1 transcripts in vascular-associated elements of the rat brain 1 h after iv IL-1ß injection (B). The cytokine also caused expression of the immediate-early gene c-fos in the hypothalamic PVN (x-ray film, Kodak Biomax), which is prevented by ketorolac. This contrasts with the other transcripts that increased in blood vessels (bv), leptomeninges, and choroid plexus (chp) in response to ketorolac and IL-1ß. Although RU-486 amplified the effect of IL-1ß (B, left column), exogenous corticosterone (CORT) largely abolished the ability of ketorolac to increase COX-2 and MCP-1 gene expression (B, right column). The hybridization signal was comparable with background levels in the microvasculature of all control groups, but a low I{kappa}B{alpha} mRNA signal was found in few blood vessels of CORT-treated animals (A). Magnification for panels depicting COX-2 and MCP-1 gene expression, x25; for I{kappa}B{alpha} mRNA, x10.

 
The gene encoding this chemokine is also under the control of NF{kappa}B and systemic inflammatory stimuli trigger its transcription within vascular-associated elements of the brain (16). As for COX-2, ketorolac and RU-486 treatments exacerbated the effects of iv IL-1ß administration on MCP-1 mRNA levels in the circumventricular organs and choroid plexus (Fig. 7BGo). Once again corticosterone decreased expression of the chemokine transcript in response to a combined treatment of IL-1ß with ketorolac (Table 1Go).


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  		Table 1. Expression of COX-2 and MCP-1 transcripts in the brains of IL-1ß-injected rats pretreated or not with ketorolac, corticosterone, or RU-486

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since the classic work of Vane (17), it is widely accepted that the pharmacological action of NSAIDs is mediated by inhibiting the activity of COX. Although several NSAIDs are also able to directly inhibit NF{kappa}B activation, ketorolac does not interfere with NF{kappa}B-signaling events (18). In the present set of experiments, we found that although ketorolac blocks COX-1 and COX-2 activities, it enhances the effect of systemic immune stimuli on proinflammatory signaling and gene expression in cells of the blood-brain barrier. Activation of the COX-2 pathway leads to the production of key PGs that trigger the activity of the neurons involved in the control of the HPA axis. In altering with this endocrine system, ketorolac exacerbated the effects of IL-1 and TNF on expression of immune-related genes. This proinflammatory action may have a great impact for the innate immune response, which is under the direct control of NF{kappa}B transduction pathway and endogenous glucocorticoids.

Among numerous active products of arachidonate metabolism, the PG of E2 type is likely to have a key role in triggering different populations of neurons that control the HPA axis during systemic inflammation (19). Locally produced PGE2 has the ability to diffuse through the brain parenchyma and target its transmembrane receptors expressed at the surface of the neurons controlling the release of corticotropin-releasing factor from the hypothalamic PVN. As shown here and in numerous other studies, ketorolac abolished the activation of the PVN and the subsequent release of glucocorticoids. Ketorolac tromethamine is among the most water-soluble inhibitors of both COX-1 and COX-2 isoforms that totally prevented the endogenous release of PGE2 for the duration of the experiment. In doing so, it is also able to prevent the critical rise in circulating glucocorticoids and therefore explains the surprising proinflammatory properties of the drug.

The antiinflammatory properties of glucocorticoids are largely mediated by interfering with NF{kappa}B signaling events and the subsequent transcriptional activation of proinflammatory genes, such as COX-2 and MCP-1 (20). Glucocorticoids were shown to suppress NF{kappa}B activity through protein-protein interactions with specific members of NF{kappa}B family and glucocorticoid receptor (21, 22). These protein-protein interactions interfere with initiation of transcription on specific sites of the NF{kappa}B promoter (23). We propose here that enhancement of gene expression by ketorolac is due to the effects of the NSAID on plasma release of glucocorticoids. The data with exogenous corticosterone and GR antagonist support this concept, although we have yet to find how exactly glucocorticoids suppress the innate immune response in the CNS.

There is evidence that NSAIDs up-regulate COX-2 transcriptional activity in cultured microglial cells (7), epithelial cells (8), and osteoblastic cells (9). Flufenamic acid was found to inhibit lipopolysaccharide- and TNF{alpha}-induced COX-2 expression and NF{kappa}B activation in RAW 264.7 and HT-29 cells, respectively (24). In contrast, aspirin suppresses COX-2 mRNA in peritoneal macrophages of lipopolysaccharide-injected mice (10). The discrepancies among studies may be attributable to the different experimental conditions. Our experiments were performed in vivo that are more similar to physiological circumstances in which numerous other factors may interact, such as glucocorticoids that are by far the most potent endogenous inhibitor of most regulated genes participating in the innate immune response. The nature of the stimulus, structure-relationship of different NSAIDs, and use of drugs with different doses may also explain discrepant results.

MCP-1 is one of the principal CC chemokines that plays a primary role in the recruitment of inflammatory cells in different tissues during acute and chronic inflammation. As for COX-2, transcription of the gene encoding MCP-1 is under the direct control of NF{kappa}B and immune ligands that use this signal transduction pathway stimulate the chemokine expression in the CNS (16). Although the pattern and time of induction differed among the immune stimuli and species, vascular-associated elements of the brain exhibited in general a positive signal for MCP-1 transcript in response to a single systemic lipopolysaccharide, TNF{alpha}, or IL-1ß bolus (16). The fact that ketorolac amplified this response reinforces the concept that a lack of appropriate inhibition of NF{kappa}B by glucocorticoids is responsible of the proinflammatory properties of this COX inhibitor. Pretreatment with ketorolac was also associated with profound changes in time and duration of genes encoding several members of complement proteins during endotoxemia (Pescarus, R., S. Nadeau, and S. Rivest, unpublished data). It is tempting to speculate that most genes that are controlled by NF{kappa}B signaling and glucocorticoids will follow the same pattern, although this remains only hypothetical at this time.

In conclusion, our results suggest that other than their inhibitory effects on COX activity, ketorolac may also have different pharmacological effects, such as transcriptional regulation of proinflammatory genes. However, this effect is mediated indirectly through the effects of the NSAID on plasma glucocorticoids that are profound inhibitor of NF{kappa}B activity and gene expression. The dual role of ketorolac on the inhibition of COX activity and subsequent alteration of the HPA axis may yield an as-yet-unrevealed pharmacological properties of the NSAID that would rather have proinflammatory side effects, at least in the cerebral tissue. It will be of great interest to compare these effects with other NSAIDs and within systemic tissues that are likely to exhibit similar patterns of gene expression.


   Acknowledgments
 
The authors thank Dr. Alain Israel (Institut Pasteur, Paris, France) for the gift of the plasmid containing the mouse I{kappa}B{alpha} cDNA; Dr. K. Peri (Ste-Justine Hospital Research Center, Montreal, Canada) for COX-2 cDNA; Dr. I. Verma (Salk Institute, La Jolla, CA) for the c-fos cDNA; and Dr. S. C. Williams (Texas Tech University, Lubbock, TX) for the cDNA containing MCP-1.


   Footnotes
 
This work was supported by the Canadian Institutes in Health Research (CIHR) and CIHR Ph.D. studentships (to V.B. and J.Z.). S.R. is a CIHR Scientist and holds a Canadian Research Chair in Neuroimmunology.

Abbreviations: CNS, Central nervous system; COX, cyclooxygenase; DMSO, dimethylsulfoxide; HPA, hypothalamic-pituitary-adrenal; I{kappa}B{alpha}, inhibitory factor {kappa}B{alpha}; MCP-1, macrophage chemoattractant protein-1; NF{kappa}B, nuclear factor {kappa}B; NSAID, nonsteroidal antiinflammatory drug; PG, prostaglandin; PVN, paraventricular nucleus of the hypothalamus; RDAU, refraction density in arbitrary units.

Received June 7, 2002.

Accepted for publication August 13, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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