AJP - Regu Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 288: R1774-R1782, 2005. First published February 17, 2005; doi:10.1152/ajpregu.00815.2004
0363-6119/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/6/R1774    most recent
00815.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sapirstein, A.
Right arrow Articles by Bonventre, J. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sapirstein, A.
Right arrow Articles by Bonventre, J. V.

INFLAMMATION AND CYTOKINES

Cytosolic phospholipase A2{alpha} regulates induction of brain cyclooxygenase-2 in a mouse model of inflammation

Adam Sapirstein,1 Hideyuki Saito,2 Sarah J. Texel,1 Tarek A. Samad,3 Eileen O’Leary,4 and Joseph V. Bonventre4,5

1Anesthesiology and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland; 2Department of Pharmacy, Kumamoto University Hospital, Kumamoto, Japan; 3Neural Plasticity Research Group, Department of Anesthesia, Massachusetts General Hospital, Boston; 4Medical Services, Brigham and Women’s Hospital, Boston; and 5Harvard-MIT Division of Health Sciences and Technology, Boston, Massachusetts

Submitted 1 December 2004 ; accepted in final form 4 February 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The products of arachidonic acid metabolism are key mediators of inflammatory responses in the central nervous system, and yet we do not know the mechanisms of their regulation. The phospholipase A2 enzymes are sources of cellular arachidonic acid, and the enzymes cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) are essential for the synthesis of inflammatory PGE2 in the brain. These studies seek to determine the function of cytosolic phospholipase A2{alpha} (cPLA2{alpha}) in inflammatory PGE2 production in the brain. We wondered whether cPLA2{alpha} functions in inflammation to produce arachidonic acid or to modulate levels of COX-2 or mPGES-1. We investigated these questions in the brains of wild-type mice and mice deficient in cPLA2{alpha} (cPLA2{alpha}–/–) after systemic administration of LPS. cPLA2{alpha}–/– mice had significantly less brain COX-2 mRNA and protein expression in response to LPS than wild-type mice. The reduction in COX-2 was most apparent in the cells of the cerebral blood vessels and the leptomeninges. The brain PGE2 concentration of untreated cPLA2{alpha}–/– mice was equal to their wild-type littermates. After LPS treatment, however, the brain concentration of PGE2 was significantly less in cPLA2{alpha}–/– than in cPLA2{alpha}+/+ mice (24.4 ± 3.8 vs. 49.3 ± 11.6 ng/g). In contrast to COX-2, mPGES-1 RNA levels increased equally in both mouse genotypes, and mPGES-1 protein was unaltered 6 h after LPS. We conclude that cPLA2{alpha} regulates COX-2 levels and modulates inflammatory PGE2 levels. These results indicate that cPLA2{alpha} inhibition is a novel anti-inflammatory strategy that modulates, but does not completely prevent, eicosanoid responses.

lipopolysaccharide; prostaglandin E synthase; inflammation; prostaglandin E2

THE INFLAMMATORY PROCESS is important in the propagation of both acute and chronic neurological injuries such as stroke, Alzheimer disease, and multiple sclerosis (10, 30, 44). Inflammatory stimuli generated by cells within the brain and circulating molecules can alter the expression of inflammatory proteins within the brain. The enzyme cyclooxygenase (COX)-2 is among the earliest and best characterized of these molecules. Furthermore, inhibition of COX-2 activity appears to be neuroprotective in a variety of animal models of neurological disease (19, 25).

The COX enzymes convert arachidonic acid to PGH2, the common precursor of all prostaglandins and thromboxanes. Although there are two COX isoforms, the COX-2 enzyme has been characterized as the inducible form that mediates inflammatory prostanoid generation (36). O’Banion demonstrated that COX-2 is induced by IL-1{beta} in cultured glial cells, and studies have demonstrated in vivo induction of COX-2 after inflammatory stimuli (28, 38).

PGE2 is an important mediator of inflammation in the brain, and PGE2 levels correlate directly with levels of inflammation in a model of stroke (9). Three distinct PGE2 synthase (PGES) enzymes convert PGH2 into PGE2: a glutathione-dependent cytosolic form and two membrane-associated PGES forms (mPGES-1 and -2). Analogous to COXs, PGESs appear to have different cellular distributions and expression patterns (45, 47). LPS triggers the release of proinflammatory cytokines, which induce both COX-2 and mPGES-1 in an identical set of vascular endothelial cells of the central nervous system (CNS) (29, 51). The coordinated actions of these enzymes produce high levels of PGE2 within the brain and cerebral spinal fluid of rodents (11, 21). Studies with COX-2 and mPGES-1 knockout mice have proved that increases in brain PGE2 and the febrile response are dependent on COX-2 and mPGES-1 expression (26, 27). Direct cerebral injection of IL-1{beta} increased levels of PGE2, COX-2, and mPGES-1 in mouse brain tissue (31).

It is believed that phospholipase A2 (PLA2) is the major source of the arachidonic acid that is required for PGE2 and other prostanoid synthesis. PLA2 comprises a large enzyme superfamily that produces free arachidonic acid by hydrolyzing the fatty acid at the sn-2 position of membrane glycerophospholipids (reviewed in Ref. 8). The PLA2 family includes secretory, small-molecular weight PLA2 (sPLA2), calcium-independent PLA2, and the intracellular 85-kDa cytosolic PLA2 (cPLA2{alpha}). It has been postulated that cPLA2{alpha} is required for the generation of central arachidonic acid after LPS exposure because cPLA2{alpha} preferentially liberates arachidonic acid and is subject to diverse mechanisms of regulation (7, 17, 51). In addition, peritoneal macrophages derived from cPLA2{alpha}-deficient (cPLA2{alpha}–/–) mice are unable to generate any PGE2 in response to LPS (3), and cytokine treatment of bone marrow-derived mast cells harvested from cPLA2{alpha}–/– mice fail to induce COX-2 mRNA or protein (15).

The roles of the PLA2s in the coordinated regulation of COX and PG production within the CNS are largely unexplored (8). Rosenberger evaluated lipid metabolism in the brains of cPLA2{alpha}–/– mice and found reduced arachidonate turnover (39). Bosetti and Weerasinghe examined cPLA2{alpha}–/– mice and determined that basal levels of COX-2 mRNA and protein were reduced compared with wild-type littermates (4).

Thus cell culture data and previous experiments with the cPLA2{alpha}–/– mice indicate that cPLA2{alpha} has the potential to influence inflammatory PGE2 levels in the brain by three possible mechanisms: 1) cPLA2{alpha} may generate metabolically active arachidonic acid; 2) cPLA2{alpha} may be necessary for COX-2 induction; or 3) cPLA2{alpha} may regulate mPGES-1 induction.

Systemic administration of LPS is a widely used model for the study of COX-2, mPGES-1, and PGE2 in neuroinflammation (51). Here, we studied cPLA2{alpha}–/– and wild-type littermate mice in this model and measured COX-2, mPGES-1, and PGE2 levels to determine the dependence of the CNS inflammatory PGE2 response on cPLA2{alpha}.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. C57BL/6J male and female mice with disruption of the gene encoding for cPLA2{alpha} (cPLA2{alpha}–/–; 10–16 wk old) were used together with their wild-type littermates (3). Body weights were in the 22–30 g range. Escherichia coli LPS was purchased from Sigma (St. Louis, MO). Goat anti-COX-1 and anti-COX-2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cy3-conjugated donkey anti-goat IgG and normal goat serum were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-cPLA2{alpha} antibody was the gift of André Cybulsky.

Mouse treatment. All experiments were conducted with the approval of the Institutional Animal Care and Use Committee and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (20). Unless otherwise specified, mice were injected intraperitoneally with 40 mg/kg LPS or an equivalent volume of pyrogen-free saline (16 ml/kg) using a 27-gauge needle.

Northern blot analysis. Brains were rapidly removed from PBS-perfused mice, frozen in liquid nitrogen, and stored at –80°C until use. Total RNA was extracted using the guanidine isothiocyanate-cesium chloride method (6). RNA blots were hybridized with [{alpha}-32P]dCTP-labeled cDNA probes.

Preparation of cDNA probes. The cDNA probes were created by PCR using primers corresponding to the nucleotide sequences published in GeneBank databases: murine COX-1 (sense, 5'-CGGATCCTACTGGCTCTGGAATTTGTGAATGC-3'; antisense 5'-CGAATTCGGTTATGTTCACGAAGCCAGATCGTG-3'), COX-2 (sense, 5'-CGGATCCATCCTTGCTGTTCCAATCCATGTCAA-3'; antisense, 5'-CGAATTCCCAGGTCCTCGCTTATGATCTGTCTT-3'), murine mPGES (sense, 5'-GGTGTCCCCGAGTTGAAGT-3'; antisense, 5'-GGCATTTTGTGAGGTGAAGG-3'), murine cPLA2{alpha} (sense, 5'-TGGATCCACATGGTACATGTCAACCTTGTACTC-3'; antisense, 5'-CGAATTCGGATATGATGTGTTGAGATTCAAGCC-3'), murine group V PLA2 (sense, 5'-AAGAGGGTTGTAAGTCCAGAGG-3'; antisense, 5'-CAGGGGGCTTGCTAGAACTCAA-3'), and murine GAPDH (sense, 5'-CTTCATTGACCTCAACTACAT-3'; antisense, 5'-CCAAAGTTGTCATGGATGACC-3'). The sense and antisense primers for COX-1, COX-2, mPGES, and cPLA2{alpha} probes had restriction sites for BamHI and EcoRI at their 5' and 3' termini, respectively.

Competitive RT-PCR analysis. Competitive RT-PCR was performed to determine amounts of COX-2 mRNA. The protocol was adopted from a previously published study (37). A deleted DNA construct was generated using the endogenous COX-2 transcript but was missing an internal 132-bp fragment. An amplified COX-2 deletion product (mutCOX-2) of ~430 bp was constructed and subcloned in plasmid Bluescript. RNA for mutCOX-2 was synthesized with T3 RNA polymerase (Promega) and used for subsequent reverse transcription-coupled PCR. Total RNA of mouse brain (1 µg) was reverse transcribed simultaneously with known amounts of mutCOX-2 RNA and then coamplified with the same set of primers. The relative amount of each PCR product was measured with densitometry (NIH Image) of ethidium bromide-stained agarose gels. In Fig. 4B, the logarithm of the ratio of the optical densities of COX-2 to mutCOX-2 was plotted as a function of the logarithm of the amount of mutCOX-2. The best-fit line was determined by linear regression. When the value of the vertical axis is zero, the amount of competitor equals the amount of native COX-2 mRNA.



View larger version (71K):
[in this window]
[in a new window]
  		Fig. 4. LPS increases COX-2 mRNA in cerebral blood vessels and meninges of the cPLA2{alpha}+/+ mouse more than in the cPLA2–/– mouse. Brains of mice injected with saline (A–D) or LPS (E–H) were harvested and flash frozen 6 h after injection. In situ RNA hybridization using an anti-sense probe to COX-2 was performed as described in METHODS. COX-2 mRNA is observed as dark areas (arrows). COX-2 mRNA is highly induced in the penetrating vessels and around the meninges of LPS-treated (E), but not saline-treated (A), cPLA2+/+ mice. COX-2 mRNA increased less in the meninges of LPS-injected cPLA2{alpha}–/– mice (G) compared with saline treatment (C). LPS does not increase COX-2 mRNA over saline treatment in the parenchyma of the brain of either genotype; +/+ saline (B) vs. +/+ LPS (F) and –/– saline (D) vs. –/– LPS (H). A sense probe does not produce a signal (E, inset). Scale bar for all figures is shown in A = 100 µm. Figures are representative of n = 5 independent experiments.

 
In situ hybridization. Digoxigenin-11-UTP (Boehringer-Mannheim, Germany)-labeled RNA probes were prepared from 1 µg of BamHI (for antisense RNA probe)- or EcoRI (for sense RNA probe)-linearized murine COX-2 cDNA using 2 µg/µl T3 or T7 RNA polymerase (Promega). The RNA probe was hydrolyzed in alkaline solution (in mM: 56 NaHCO3, 84 Na2CO3, 7 dithiothreitol, and 3 EDTA) at 60°C for 10 min and ethanol precipitated. RNA concentrations were estimated by comparison to the digoxigenin-labeled control RNA (Boehringer) using an alkaline phosphatase-conjugated antibody (Boehringer).

For in situ hybridization, perfusion-fixed brains were immediately removed and immersed for 30 min in 4% paraformaldehyde in PBS, then in 30% sucrose in PBS at 4°C for 16 h. Coronal brain sections (18 µm thick) were cut with a cryostat (Leica Frigocat, Wetzler, Germany) at –20°C and thaw-mounted on glass slides. The frozen sections were fixed in 4% paraformaldehyde for 10 min, rinsed with PBS, and immersed in 0.2 N HCl for 10 min. After a brief rinse in PBS, the sections were transferred to 2x SSC for 10 min at 70°C, immersed in 0.1 M triethanolamine (pH 8.0) for 5 min, and then acetylated in 0.25% acetic anhydride/0.1 M triethanolamine at room temperature for 10 min. Sections were incubated overnight at 70°C with hybridization solution (50% formamide, 5x SSC, 2.5x Denhardt’s solution, 0.25 mg/ml yeast tRNA, 0.05 mg/ml sonicated denatured herring sperm DNA) containing 400–1,000 ng/ml of either digoxigenin-11-UTP-labeled antisense RNA or sense RNA for control. After washing, 50 µl per section of sheep anti-digoxigenin-alkaline phosphatase conjugate (1:5,000 dilution in buffer containing 1% normal sheep serum) was applied at 4°C for 18 h. The detection of signal was performed by overnight incubation with 150 µl of chromogen solution (20 µl of nitroblue tetrazolium and 15 µl of 5-bromo-4-chloro-3-indolyl-phosphate in 4 ml of buffer) at room temperature. Color development was checked, and the reaction was terminated by immersing the slides in 10 mM Tris·HCl, pH 7.5, 1 mM EDTA. The slides were mounted and examined by light microscope.

Western blot analysis. After death, mouse brains were rapidly removed and a single hemisphere from each mouse was homogenized by 20 strokes of a tight-fitting Dounce homogenizer at 4°C in buffer containing protease inhibitors. The crude homogenate was centrifuged at 10,000 g for 20 min at 4°C, and protein concentration was determined by a modified Bradford assay (Bio-Rad). Proteins were separated on 10–12% acrylamide gels using SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore, Billerica MA).

Immunofluorescence. Three mice of each genotype were treated with saline or 40 mg/kg LPS and killed at 6 h. Brains were perfusion fixed, as described above, and 5-µm coronal sections were cut and thaw-mounted on glass slides, rinsed briefly in PBS, and incubated with 2% BSA in PBS for 20 min. The sections were incubated with the goat anti-COX-2 antibody (1:200). After three washes in PBS, the sections were incubated with the Cy3-conjugated donkey anti-goat IgG (1:500). In some experiments, 4',6-diamino-2-phenylindole (final concentration of 2 µg/ml), a nuclear-specific dye, was added to the solution of secondary antibody. The sections were mounted with Vectashield mounting solution (Vector Laboratories, Burlingame CA).

PGE2 measurement. One hemisphere of each brain was weighed and immediately homogenized in 70% ice-cold methanol. The homogenate was centrifuged at 10,000 g for 25 min at 4°C, and the supernatant was evaporated under a nitrogen stream. The lipids were then resuspended in assay buffer, and PGE2 content was measured by ELISA according to instructions (Amersham, Piscataway, NJ).

Data and statistical analysis. The intensity of Northern blot signals was measured using the National Institutes of Health Image program. The relative signal intensities were normalized to the 18S ribosomal band of the ethidium bromide-stained RNA gel for each lane. Because COX-2 and mPGES mRNAs were barely detected in saline-treated brains of both genotypes, comparisons were made only between LPS-treated mice using paired Student’s t-tests. COX-1 differences were compared using two-factor analysis of variance. The images were analyzed with identical settings and averaged. For in situ RNA hybridization signal measurement, relative threshold densitometry was performed with the Inquiry program (version 3.08; Loats Associates, Westminster, MD). The threshold level was set to eliminate background signal when the sense RNA probe was used. Two mice were analyzed for each condition, and a minimum of three representative images from each mouse were measured. Applicable results are displayed as means ± SE, with P < 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Northern blot analysis. Based on previous cell culture experiments, we postulated that cPLA2{alpha} could effect induced COX-2 expression (15). To evaluate the effects of systemic LPS injection on COX-2 mRNA expression in the cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice, total RNA was extracted from brain, heart, and lung tissue and examined by Northern blot analysis. Figure 1 shows that COX-2 mRNA is scarcely detectable in saline-treated tissues from both cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice. LPS injection markedly increased COX-2 mRNA expression in the mouse brains and lungs. Notably, the LPS-induced upregulation of COX-2 mRNA, corrected relative to 18S intensity, was much less in the brain of cPLA2{alpha}–/– mice (64%, n = 7, P < 0.01).



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 1. Brain cyclooxygenase (COX)-2 mRNA levels are lower in cytosolic phospholipase A2{alpha} (cPLA2{alpha})-deficient (–/–) mice after LPS injection. cPLA2{alpha}-positive (+/+) and cPLA2{alpha}–/– mice were treated with either saline or 40 mg/kg LPS. At 6 h after treatment, the mice were euthanized and the tissues harvested for collection of total RNA. Twenty micrograms of total RNA from brain, heart, and lung were separated on a formaldehyde-agarose gel and transferred to a single nylon membrane. The membrane was sequentially hybridized and stripped with probes recognizing the murine forms of COX-2 (A), COX-1 (B), or cPLA2{alpha} (C). D: ethidium bromide-stained gel is used for standardization. The figure is representative of n = 3 experiments.

 
In contrast, COX-1 mRNA was constitutively expressed at equivalent levels in the brains of wild-type and cPLA2{alpha}–/– mice. Treatment of the cPLA2{alpha}–/– mice with LPS resulted in approximately twofold reduction in brain COX-1 mRNA compared with saline-treated mice (n = 3, P < 0.025).

The band for cPLA2{alpha} in the wild-type mouse tissues shows the expected size for cPLA2{alpha} of 2.8 kbp, as has been reported previously (7). In the saline-treated cPLA2{alpha}+/+ mice, cPLA2{alpha} mRNA was detected in the lung. After LPS injection, cPLA2{alpha} mRNA was increased in the brain, heart, and lung. The increase in levels of brain mRNA is consistent with the finding that seizure activity in rats also induces both COX-2 and cPLA2{alpha} (48). As expected, cPLA2{alpha} mRNA was not seen in the tissues of the cPLA2{alpha}–/– mice. These results demonstrate that induced COX-2 mRNA depends significantly on cPLA2{alpha}.

To determine the time of peak levels of COX-2 mRNA in the brain after LPS injection in our model, we harvested total brain RNA at various times. The upregulation of COX-2 reached a maximum between 3 and 6 h after LPS injection and then decreased to basal levels at 12 h (data not shown). A small but reproducible second phase of increase in COX-2 mRNA levels occurred at 24 h after the LPS injection. We conducted subsequent experiments at the 6-h time point to ensure high levels of COX-2.

Levels of COX-2 and mPGES-1 mRNA and protein have been demonstrated to be coordinately upregulated in the vasculature of the rat brain after LPS injection (51). We inquired whether mPGES-1 regulation was also abnormal in the cPLA2{alpha}–/– mouse. Northern hybridization with a probe specific for murine mPGES-1 revealed four transcripts, as has been demonstrated in murine peritoneal macrophages (Fig. 2) (49). mPGES-1 was not detected in the saline-treated brains of either genotype (Fig. 2B). LPS treatment strongly induced mPGES-1 in both the cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice. The mPGES-1 mRNA in the brains of cPLA2{alpha}–/– mice exhibited a tendency toward increased levels (1.4-fold of wild type, P = not significant, n = 6; Fig. 2B). This is in contrast to the decreased brain levels of COX-2 mRNA in the cPLA2{alpha}–/– mouse after LPS treatment (Fig. 2A) and suggests that cPLA2{alpha}-dependent inflammatory gene regulation is specific.



View larger version (60K):
[in this window]
[in a new window]
  		Fig. 2. Brain COX-2 and microsomal PGE synthase (mPGES) mRNA in saline or LPS-treated mice. cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice were treated with either saline or 40 mg/kg LPS, and brain RNA was harvested after 6 h. Ten micrograms of total RNA were gel separated and applied to a nylon membrane. The same membrane was hybridized with probes recognizing the murine forms of COX-2 (A) and mPGES (B). C: ethidium bromide-stained gel for loading comparison.

 
Competitive PCR analysis. To more precisely determine the magnitude of LPS-induced upregulation of COX-2 message in cPLA2{alpha}+/+ and cPLA2{alpha}–/– mouse brains, we used a highly accurate and specific competitive PCR technique (37). Figure 3A shows representative ethidium bromide-stained gels of competitive PCR products for COX-2 induced in brain of cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice after LPS injection. A higher amount of competitor (mutCOX-2) is needed to compete with the COX-2 products in the cPLA2{alpha}+/+ brain. After LPS injection, the cPLA2{alpha}+/+ brains had 2.3-fold more COX-2 mRNA than did the cPLA2{alpha}–/– mice (cPLA2{alpha}+/+ mice, 177 ± 11 pg/µg of total RNA, n = 4; cPLA2{alpha}–/– mice, 76 ± 7 pg/µg of total RNA, n = 3; P < .05) (Fig. 3C). By this analysis, the LPS-induced brain COX-2 mRNA of the cPLA2{alpha}–/– is only 43% of normal, and this compares favorably to the Northern analysis (Fig. 1).



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 3. Brain COX-2 induction is attenuated by deletion of cPLA2{alpha}. Competitive PCR using a mutated COX-2 probe was performed as described in METHODS. A: representative results of PCR using RNA from the brains of LPS-treated cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice with different amounts of mutant COX-2. B: densitometric analysis of the results from the PCR. C: quantification of COX-2 mRNA concentration in the brains of LPS-treated cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice; n = 3–4 experiments. *P < 0.05.

 
Localization of COX-2 mRNA in brain after LPS injection. We suspected that COX-2 would be induced at specific sites within the brain and performed in situ RNA hybridization 6 h after LPS injection to define the expression sites. COX-2 signals were undetectable in the leptomeninges of the brain of both groups of saline-injected mice (Fig. 4, A and C), whereas signals were detected in neuronal cells of the cerebral cortex (Fig. 4, B and D). At 6 h after LPS injection, there was high expression of COX-2 mRNA in the leptomeninges of the parietal cortex and the blood vessels located on the surface of the cerebral cortex in cPLA2{alpha}+/+ mice (Fig. 4E). In contrast, the intensity of the signal was much less in the corresponding regions of the cPLA2{alpha}–/– brain (Fig. 4G). We performed densitometric analysis and found that COX-2 mRNA in these regions for cPLA2{alpha}–/– mice was ~34% of the amount in wild-type mice. The COX-2 of the parenchymal cells was not influenced markedly by LPS injection in the brains of either cPLA2{alpha}+/+ or cPLA2{alpha}–/– mice (Fig. 4, F and H). No mRNA signal was detectable when the sense probe was used in LPS-treated cPLA2+/+ mice (Fig. 4E, inset). These results demonstrate that cPLA2{alpha}-dependent differences in COX-2 mRNA levels are most pronounced in the meninges and within blood vessels after LPS injection.

COX-2 protein in cPLA2{alpha}+/+ and cPLA2{alpha}–/– brain. We measured cPLA2{alpha}, COX-2, and mPGES-1 protein by the Western blot technique. cPLA2{alpha} was detected in the brains of wild-type, but not cPLA2{alpha}–/– mice (Fig. 5A). The levels of cPLA2{alpha} were not altered 6 h after the treatment with LPS (Fig. 5A). We determined that mPGES-1 protein was expressed equally in the saline-treated wild-type and cPLA2{alpha}–/– brains (Fig. 5C). Interestingly, 6 h after LPS, there was no significant increase in the mPGES-1 protein levels in the brains of either genotype (Fig. 5C). In contrast to these findings, COX-2 expression in the brains of the saline-treated cPLA2{alpha}–/– mice was only 0.31-fold that of the wild-type mice (P = 0.04; Fig. 5B). After 6 h of LPS treatment, COX-2 protein was significantly increased in the brains of both cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice. The levels of COX-2 protein 6 h after LPS treatment were significantly less (0.72-fold, P = 0.03) in the cPLA2{alpha}–/– compared with the LPS-treated wild-type mice.



View larger version (36K):
[in this window]
[in a new window]
  		Fig. 5. Western analysis of cPLA2{alpha}, COX-2, and mPGES-1 proteins in cPLA2{alpha}+/+ and cPLA2{alpha}–/– mouse brains. Brains of mice of each genotype were harvested 6 h after injection with saline or 40 mg/kg LPS. A single hemisphere from each mouse was homogenized, and 25 µg of the 10,000-g supernatant was separated by SDS-PAGE on 10 or 12% acrylamide gel and transferred to polyvinylidene difluoride membrane. The membrane was blotted and stripped sequentially for cPLA2{alpha} (A), COX-2 (B), and actin (not shown). Proteins from the spleens of LPS-treated mice are included as controls. Separate membrane for low-molecular-weight proteins was developed for mPGES-1 (C).

 
We then used immunofluorescence microscopy to compare the expression of COX-2 protein in specific cells of the brains of cPLA2+/+ and cPLA2{alpha}–/– mice. As expected, COX-2 immunoreactivity was rarely detected after saline injection in either genotype (Fig. 6, A–C). In cPLA2{alpha}+/+ mice, COX-2 immunoreactivity was enhanced 6 h after LPS injection in cells of parenchymal, midsize blood vessels (Fig. 6D), blood vessels penetrating cerebral cortex (Figs. 6E), and large blood vessels of the subarachnoid space (Fig. 6G). COX-2 was present in the leptomeninges and the luminal surface of parenchymal blood vessels. As shown by 4',6-diamino-2-phenylindole-staining of nuclei, COX-2 immunoreactivity appears specifically around the nuclear envelope. COX-2 was also detected within cells of the parietal cortex, but the signals were much weaker than those in blood vessels and not affected by LPS injection (data not shown). Substitution of normal goat serum for the primary antibody resulted in images that had essentially no background staining (data not shown).



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6. LPS-induced increases in COX-2 protein expression in the meninges and cerebral blood vessels of cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice. Brains of mice injected with saline (A–C) or 40 mg/kg LPS (D–H) were harvested 6 h after injection and flash frozen. Coronal cryostat sections were stained with a CY-3-conjugated anti-COX-2 antibody and DAPI. COX-2 appears red, whereas nuclei are blue (+, brain boundary; *, vessel lumens). Saline-injected cPLA2{alpha}+/+ (A and B) and cPLA2{alpha}–/– (C) mouse brains express little COX-2. LPS-treated cPLA2+/+ mice express high levels of COX-2 in cells on the luminal side of medium- and large-sized cerebral blood vessels (D–E) and in the leptomeninges (G–H). cPLA2{alpha}–/– cerebral blood vessels (F) and leptomeninges (I) express less COX-2. Images in A, C, G, H, and I are from similar regions of the parietal cortical surface. Images in B, D, E, and F are from similar regions of the cerebral cortex. Scale bar for all figures except E is in A and is = 50 µm; scale bar in E = 20 µm. Images are representative of sections from n = 3 experiments.

 
PGE2 content of cPLA2{alpha}+/+ and cPLA2{alpha}–/– brain. PGE2 caused by inflammation is the final product of the increased activities of PLA2, COX-2, and mPGES-1. We assessed the functional significance of reduced COX-2 induction and the absence of cPLA2{alpha} in the cPLA2{alpha}–/– mouse on PGE2 synthesis by measuring the levels of PGE2 in whole brains (Fig. 7). Saline-injected mice of both genotypes had comparable brain levels of PGE2 (7.7 ± 1.6 vs. 6.8 ± 0.12 ng/g, P = not significant). LPS-induced brain PGE2 was significantly less in the cPLA2{alpha}–/– mice compared with their wild-type littermates (3.6- and 6.4-fold, respectively; P < 0.05). Thus, although basal PGE2 was equivalent in both genotypes, the reduced PGE2 response to LPS correlated very closely with the reduction in induced COX-2 mRNA.



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 7. Brain PGE2 content is higher in cPLA2{alpha}+/+ mice than in cPLA2{alpha}–/– after LPS treatment. cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice were injected with 40 mg/kg LPS ip and killed 6 h later. Brains were recovered, and PGE2 content was measured by ELISA, as described in METHODS. n = 5–6 Experiments. *P < 0.05 compared with LPS-treated cPLA2{alpha}–/– mice; +P < 0.01 compared with saline-treated mice.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
PLA2s, COX-2, and mPGES-1 are important enzyme mediators in the CNS response to inflammation (5, 14, 31). In this study, we examined the relationship of cPLA2{alpha} in the PGE2 response of the CNS to LPS. We found that peak COX-2 mRNA induction after LPS exposure in the brains of cPLA2{alpha}–/– mice was only ~40% of that seen in wild-type mice. Both basal and LPS-stimulated levels of COX-2 protein were significantly decreased in the cPLA2{alpha}–/– mouse brains. We used in situ RNA hybridization and protein immunofluorescence to demonstrate that cPLA2{alpha}–/– brain perivascular and meningeal cells have less COX-2 induction. In contrast, mPGES-1 mRNA responses in the cPLA2{alpha}–/– mouse brain were equal to wild-type responses. PGE2 levels in saline-treated brains were the same in cPLA2{alpha}–/– and wild-type mice. After LPS treatment, however, cPLA2{alpha}–/– mice had significantly less brain PGE2 than wild-type mice. These results show that cPLA2{alpha} plays a significant role in regulating the response of COX-2, but not that of mPGES-1, to inflammatory stimuli in vivo. They also indicate that cPLA2{alpha} does not provide arachidonic acid for basal eicosanoid synthesis in the brain.

Results obtained by comparing genetically altered mice to their wild-type littermates must be interpreted with caution. It is possible that other members of the large PLA2 family are able to compensate for the deficiency of cPLA2{alpha} in the cPLA2{alpha}–/– mouse. Our in vivo results, however, are consistent with studies that used both chemical inhibitors and cells derived from cPLA2{alpha}–/– mice and showed cPLA2{alpha}-dependent COX-2 induction and inflammatory prostaglandin synthesis (3, 15).

It is possible that cPLA2{alpha} has regulatory effects on other eicosanoid synthetic enzymes, such as PGD synthases. We have focused on PGE2 metabolism in this study because of the well-described effects of LPS and other inflammatory agents on PGE2 synthesis (21, 50). Although basal PGD2 levels in the brain are higher than those of PGE2, LPS treatment amplifies PGE2 synthesis to a greater extent than PGD2 (50).

We limited our examination of the PGES enzymes to mPGES-1. Although both mPGES-2 and cytosolic PGES synthesize PGE2, they have been characterized as constitutive enzymes (32, 47). Biochemical analysis has demonstrated enzymatic coupling of COX-2 activity to mPGES-1 (33). Furthermore, in rodents, the rise in central levels of PGE2 after acute systemic injection of LPS appears to be dependent on mPGES-1. Uematsu et al. (49) showed that mPGES-1-deficient mice injected with 1 mg of LPS failed to increase serum levels of PGE2. Engblom et al. (12) found that LPS treatment failed to increase PGE2 in the CSF or PGES activity in the brains of mPGES-1-deficient mice. Moore and colleagues recently demonstrated Il-1{beta}-induced upregulation of cytosolic PGES/p23 in the brain, and it remains to determine whether this PGES contributes to inflammatory PGE2 synthesis in our model (31).

We used a high systemic dose of LPS (40 mg/kg) and examined its acute effects on the cycloxygenase-mPGES-1 axis. This is a well-described model in which both COX-2 and mPGES-1 are induced in vascular endothelial cells (51). We found that COX-2 mRNA levels peak between 3 and 6 h. Other investigators used similar doses of LPS and found that mPGES-1 RNA responses also peak within this time period (49). Given the localization of both the COX-2 mRNA and the immunoreactive protein, the positive cells lining the luminal surface of blood vessels are likely endothelial cells. This finding is consistent with those reported by other groups showing induction of COX-2 in cerebral vascular endothelial cells after LPS treatment (5, 26, 29). It is possible that other cell types, such as astroglia, pericytes, and smooth muscle cells, express COX-2 at the arterioles, a possibility that has important implications for regulation of the neurovascular unit during inflammation (18). The significant reduction of COX-2 responses in the cPLA2{alpha}–/– brain may have been different if we had used an isolated CNS model of inflammation. For example, a 6-day infusion of LPS into the fourth cerebral ventricle of the rat did not result in changes in cPLA2{alpha}, sPLA2, COX-1, or COX-2 protein levels but did increase cPLA2 activity (40).

Other PLA2s or PLA2-independent mechanisms may be the source of arachidonate for PGE2 synthesis in cPLA2{alpha}–/– brain as basal PGE2 concentrations are normal and PGE2 significantly increases after LPS injection (Fig. 7). A model of inflammatory pain in which central COX-2 and PGE2 levels increase dramatically without an increase in central PLA2 activity supports this hypothesis (41). Furthermore, selective overexpression of COX-2 in neurons dramatically increases basal CNS PGE2 levels, indicating that basal arachidonic acid levels are sufficient to produce large increases in PGE2 (50).

It is not surprising that other forms of PLA2 or PLA2-independent mechanisms are responsible for generating metabolically active arachidonate in the brain. The cPLA2{alpha} content of rodent brains is low. In rat brain, calcium-independent PLA2 activity is at least 10-fold greater than sPLA2 activity, and cPLA2{alpha} activity is even less than that of sPLA2s (52). In wild-type mice, the cPLA2{alpha} enzymatic activity of whole brain homogenate was below our ability to detect it using published assays (data not shown) (52). Other laboratories have also been unable to detect differences in total or specific PLA2 activities between cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice (4). Rosenberger and colleagues performed a detailed analysis of brain lipid metabolism in the cPLA2{alpha}–/– mouse and found an increased turnover of arachidonic acid but no difference in free arachidonic acid concentrations (39). They also concluded that other sources of arachidonic acid function in the absence of cPLA2{alpha} (39). Furthermore, it is possible that chronic changes or redundancy of PLA2 activities in the brains of the cPLA2{alpha}–/– mouse compensate for cPLA2{alpha} deficiency. Bosetti and Weerasinghe analyzed in vitro PLA2 activities of brain homogenate and did not find compensation (4). Our findings may also be strain dependent. Group IIA sPLA2 is a major source of PLA2 activity in rat brain (52), but because of a naturally occurring missense mutation, neither the C57BL/6J nor Sv129 mouse strains used to generate the cPLA2{alpha}-mouse express group IIA PLA2 (23). Further studies with application of specific inhibitors and with cPLA2{alpha}–/– mice of different strains clarify these issues.

Our Northern blot analyses did not reveal differences in basal COX-2 RNA levels, but Western blot analysis showed that constitutive COX-2 protein levels were less in the cPLA2{alpha}–/– brain. Bosetti and Weerasinghe (4) found that constitutive levels of both COX-2 protein and RNA are reduced in the cPLA2{alpha}–/– mice. It is possible that the more sensitive PCR-based method of those authors detected smaller differences in basal COX-2 RNA. The basal levels of brain PGE2 in both genotypes were the same in our study, suggesting that small differences in basal COX-2 levels have little impact on unstimulated PGE2 synthesis.

cPLA2{alpha} and other forms of PLA2 regulate the induction of COX-2 by cytokines in cell culture systems. Cytokine induction of COX-2 in murine bone marrow-derived mast cells is dependent on cPLA2{alpha} (15). Balsinde and coworkers (1) used P388D1 murine macrophage-like cells to demonstrate that group V sPLA2 can also modulate COX-2 induction by LPS treatment. Thus it is possible that the ability of the cPLA2{alpha}–/– mice to partially induce COX-2 may be the result of the complementary actions of another form of PLA2, such as group V PLA2.

In contrast to COX-2, the mRNA response of mPGES-1 in the cPLA2{alpha}–/– mice remains intact. In macrophages, mPGES-1 induction is completely dependent on nuclear factor-IL6 and is essential for the PGE2 response to high-dose LPS (49). The transcriptional responses of COX-2 and mPGES-1 to inflammatory agents share many features. However, in orbital fibroblasts, the increase in mPGES-1 is a purely inductive process, whereas levels of COX-2 mRNA are partially dependent on mRNA stabilization (16). Our results showing no difference between basal mPGES-1 expression are in agreement with those of Bosetti and Weerasinghe (4). Given the apparent increase in mPGES-1 mRNA 6 h after LPS, we were surprised that protein levels did not also increase. In a model of cerebral ventricle injection of IL-1{beta}, Moore and colleagues (31) found that, although brain COX-2 protein levels appear to peak by 6 h, the mPGES-1 levels continued to rise up to 24 h after injection. It is possible that, in our model of systemic LPS injection, the mPGES-1 protein levels respond more slowly than COX-2. A more detailed temporal analysis might resolve this question.

What actions of cPLA2{alpha} enhance COX-2 levels? cPLA2{alpha} might alter the cytokine milieu and thus transcription of COX-2. We found that cPLA2{alpha}–/– peritoneal macrophages treated with LPS secreted significantly more TNF-{alpha} than did wild-type macrophages (data not shown). In a mouse multiple sclerosis model, a cPLA2{alpha} inhibitor prevented COX-2 and altered cytokine expression (22). The arachidonic acid or other lipid byproducts of cPLA2{alpha} might activate intracellular signaling pathways. For example, Serou et al. (43) demonstrated that IL-1{beta}-stimulated induction of COX-2 in cultured rat hippocampal neurons is dependent on platelet-activating factor. Finally, products of cPLA2{alpha} activity, such as PGE2, may stabilize COX-2 mRNA (13). After injection of IL-1{beta}, however, COX-2 inhibition does not affect COX-2 levels but does prevent increases in mPGES-1 (31). We postulate that the cytokine response to LPS induces both COX-2 and mPGES-1 but that a cPLA2{alpha}-dependent eicosanoid or platelet-activating factor is required for maximal COX-2 response. This is an attractive hypothesis because cPLA2{alpha} has a preference for phosphatidylcholine-containing phospholipids that are the precursors of platelet-activating factor (7).

In the future, experiments with specific PLA2 inhibitors will help determine the mechanisms of cPLA2{alpha}-dependent COX-2 regulation. There is also a need to further dissect the cPLA2{alpha} signaling cascade within the CNS and determine the importance of cPLA2{alpha} in other models of neuroinflammation using both in vivo and cell culture techniques. The availability of mice deficient in other forms of PLA2, such as group V or calcium-independent PLA2, will further this work (2, 42). Investigation of such knockouts can provide new insights into the production of inflammatory eicosanoids.

The coordinated regulation of COX-2 by cPLA2{alpha} has important implications for potential therapies. Inhibition and gene knockout of cPLA2{alpha} have already demonstrated profound improvement in neurological outcomes of mice after inflammatory, oxidative, and immunologic injuries (3, 22, 24, 46). It remains to be determined whether the benefit of eliminating cPLA2{alpha} activity in the CNS goes beyond downregulation of the COX-2 response. In the lung, cPLA2{alpha} propagates injury by COX-2-independent mechanisms of injury propagation (34, 35). If such effects are also found in the CNS, a specific cPLA2{alpha} inhibitor may provide benefits beyond those found in traditional nonsteroidal or selective COX-2 inhibition.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-39773, DK-38452, NS-10828 (to J. Bonventre), DK-02493 (to A. Sapirstein), and American Heart Association Grant 0150504N (to A. Sapirstein). H. Saito received a Fellowship from the Ministry of Education, Science, Sports, and Culture of Japan for Research Abroad.


   ACKNOWLEDGMENTS
 
We thank Drs. David Borsook and David Linden for helpful comments. We thank Drs. Paolo Ciceri and Peter C. Isakson for advice on PGE2 measurement, Kathleen Blizzard for technical assistance, and Tzipora Sofare for editorial assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Sapirstein, Anesthesiology and Critical Care Medicine, Johns Hopkins School of Medicine, 600 N. Wolfe St./Meyer 297-A, Baltimore, MD 21287-7294 (E-mail: Asapirs1{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Balsinde J, Shinohara H, Lefkowitz LJ, Johnson CA, Balboa MA, and Dennis EA. Group V phospholipase A2-dependent induction of cyclooxygenase-2 in macrophages. J Biol Chem 274: 25967–25970, 1999.[Abstract/Free Full Text]
  2. Bao S, Miller DJ, Ma Z, Wohltmann M, Eng G, Ramanadham S, Moley K, and Turk J. Male mice that do not express group VIA phospholipase A2 produce spermatozoa with impaired motility and have greatly reduced fertility. J Biol Chem 279: 38194–38200, 2004.[Abstract/Free Full Text]
  3. Bonventre JV, Huang Z, Taheri MR, O’Leary E, Li E, Moskowitz MA, and Sapirstein A. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 390: 622–625, 1997.[CrossRef][Medline]
  4. Bosetti F and Weerasinghe GR. The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse. J Neurochem 87: 1471–1477, 2003.[ISI][Medline]
  5. Cao C, Matsumura K, Ozaki M, and Watanabe Y. Lipopolysaccharide injected into the cerebral ventricle evokes fever through induction of cyclooxygenase-2 in brain endothelial cells. J Neurosci 19: 716–725, 1999.[Abstract/Free Full Text]
  6. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[ISI][Medline]
  7. Clark JD, Milona N, and Knopf JL. Purification of a 110-kilodalton cytosolic phospholipase A2 from the human monocytic cell line U937. Proc Natl Acad Sci USA 87: 7708–7712, 1990.[Abstract/Free Full Text]
  8. Diaz BL and Arm JP. Phospholipase A2. Prostaglandins Leukot Essent Fatty Acids 69: 87–97, 2003.[CrossRef][ISI][Medline]
  9. Dore S, Otsuka T, Mito T, Sugo N, Hand T, Wu L, Hurn PD, Traystman RJ, and Andreasson K. Neuronal overexpression of cyclooxygenase-2 increases cerebral infarction. Ann Neurol 54: 155–162, 2003.[CrossRef][ISI][Medline]
  10. Eikelenboom P, Bate C, Van Gool WA, Hoozemans JJ, Rozemuller JM, Veerhuis R, and Williams A. Neuroinflammation in Alzheimer’s disease and prion disease. Glia 40: 232–239, 2002.[CrossRef][ISI][Medline]
  11. Ek M, Engblom D, Saha S, Blomqvist A, Jakobsson PJ, and Ericsson-Dahlstrand A. Inflammatory response: pathway across the blood-brain barrier. Nature 410: 430–431, 2001.[Medline]
  12. Engblom D, Saha S, Engstrom L, Westman M, Audoly LP, Jakobsson PJ, and Blomqvist A. Microsomal prostaglandin E synthase-1 is the central switch during immune-induced pyresis. Nat Neurosci 6: 1137–1138, 2003.[CrossRef][ISI][Medline]
  13. Faour WH, He Y, He QW, de Ladurantaye M, Quintero M, Mancini A, and Di Battista JA. Prostaglandin E2 regulates the level and stability of cyclooxygenase-2 mRNA through activation of p38 mitogen-activated protein kinase in interleukin-1 beta-treated human synovial fibroblasts. J Biol Chem 276: 31720–31731, 2001.[Abstract/Free Full Text]
  14. Farooqui AA, Litsky ML, Farooqui T, and Horrocks LA. Inhibitors of intracellular phospholipase A2 activity: their neurochemical effects and therapeutical importance for neurological disorders. Brain Res Bull 49: 139–153, 1999.[CrossRef][ISI][Medline]
  15. Fujishima H, Sanchez Mejia RO, Bingham CO, 3rd Lam BK, Sapirstein A, Bonventre JV, Austen KF, and Arm JP. Cytosolic phospholipase A2 is essential for both the immediate and the delayed phases of eicosanoid generation in mouse bone marrow-derived mast cells. Proc Natl Acad Sci USA 96: 4803–4807, 1999.[Abstract/Free Full Text]
  16. Han R, Tsui S, and Smith TJ. Up-regulation of prostaglandin E2 synthesis by interleukin-1beta in human orbital fibroblasts involves coordinate induction of prostaglandin-endoperoxide H synthase-2 and glutathione-dependent prostaglandin E2 synthase expression. J Biol Chem 277: 16355–16364, 2002.[Abstract/Free Full Text]
  17. Hefner Y, Borsch-Haubold AG, Murakami M, Wilde JI, Pasquet S, Schieltz D, Ghomashchi F, Yates JR, 3rd Armstrong CG, Paterson A, Cohen P, Fukunaga R, Hunter T, Kudo I, Watson SP, and Gelb MH. Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases. J Biol Chem 275: 37542–37551, 2000.[Abstract/Free Full Text]
  18. Iadecola C. Neurovascular regulation in the normal brain and in Alzheimer’s disease. Nat Rev Neurosci 5: 347–360, 2004.[CrossRef][ISI][Medline]
  19. Iadecola C, Niwa K, Nogawa S, Zhao X, Nagayama M, Araki E, Morham S, and Ross ME. Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice. Proc Natl Acad Sci USA 98: 1294–1299, 2001.[Abstract/Free Full Text]
  20. ILAR. Guide for the Care and Use of Laboratroy Animals. Washington, DC: National Academy Press, 1996.
  21. Inoue W, Matsumura K, Yamagata K, Takemiya T, Shiraki T, and Kobayashi S. Brain-specific endothelial induction of prostaglandin E2 synthesis enzymes and its temporal relation to fever. Neurosci Res 44: 51–61, 2002.[CrossRef][ISI][Medline]
  22. Kalyvas A and David S. Cytosolic phospholipase A2 plays a key role in the pathogenesis of multiple sclerosis-like disease. Neuron 41: 323–335, 2004.[CrossRef][ISI][Medline]
  23. Kennedy BP, Payette P, Mudgett J, Vadas P, Pruzanski W, Kwan M, Tang C, Rancourt DE, and Cromlish WA. A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains. J Biol Chem 270: 22378–22385, 1995.[Abstract/Free Full Text]
  24. Klivenyi P, Beal MF, Ferrante RJ, Andreassen OA, Wermer M, Chin MR, and Bonventre JV. Mice deficient in group IV cytosolic phospholipase A2 are resistant to MPTP neurotoxicity. J Neurochem 71: 2634–2637, 1998.[ISI][Medline]
  25. Klivenyi P, Kiaei M, Gardian G, Calingasan NY, and Beal MF. Additive neuroprotective effects of creatine and cyclooxygenase 2 inhibitors in a transgenic mouse model of amyotrophic lateral sclerosis. J Neurochem 88: 576–582, 2004.[CrossRef][ISI][Medline]
  26. Li S, Ballou LR, Morham SG, and Blatteis CM. Cyclooxygenase-2 mediates the febrile response of mice to interleukin-1{beta}. Brain Res 910: 163–173, 2001.[CrossRef][ISI][Medline]
  27. Li S, Wang Y, Matsumura K, Ballou LR, Morham SG, and Blatteis CM. The febrile response to lipopolysaccharide is blocked in cyclooxygenase-2(–/–), but not in cyclooxygenase-1(–/–) mice. Brain Res 825: 86–94, 1999.[CrossRef][ISI][Medline]
  28. Marcheselli VL and Bazan NG. Sustained induction of prostaglandin endoperoxide synthase-2 by seizures in hippocampus. Inhibition by a platelet-activating factor antagonist. J Biol Chem 271: 24794–24799, 1996.[Abstract/Free Full Text]
  29. Matsumura K, Cao C, Ozaki M, Morii H, Nakadate K, and Watanabe Y. Brain endothelial cells express cyclooxygenase-2 during lipopolysaccharide-induced fever: light and electron microscopic immunocytochemical studies. J Neurosci 18: 6279–6289, 1998.[Abstract/Free Full Text]
  30. McCullough L, Wu L, Haughey N, Liang X, Hand T, Wang Q, Breyer RM, and Andreasson K. Neuroprotective function of the PGE2 EP2 receptor in cerebral ischemia. J Neurosci 24: 257–268, 2004.[Abstract/Free Full Text]
  31. Moore AH, Olschowka JA, and O’Banion MK. Intraparenchymal administration of interleukin-1{beta} induces cyclooxygenase-2-mediated expression of membrane- and cytosolic-associated prostaglandin E synthases in mouse brain. J Neuroimmunol 148: 32–40, 2004.[CrossRef][ISI][Medline]
  32. Murakami M, Nakashima K, Kamei D, Masuda S, Ishikawa Y, Ishii T, Ohmiya Y, Watanabe K, and Kudo I. Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2. J Biol Chem 278: 37937–37947, 2003.[Abstract/Free Full Text]
  33. Murakami M, Naraba H, Tanioka T, Semmyo N, Nakatani Y, Kojima F, Ikeda T, Fueki M, Ueno A, Oh S, and Kudo I. Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2. J Biol Chem 275: 32783–32792, 2000.[Abstract/Free Full Text]
  34. Nagase T, Uozumi N, Ishii S, Kita Y, Yamamoto H, Ohga E, Ouchi Y, and Shimizu T. A pivotal role of cytosolic phospholipase A2 in bleomycin-induced pulmonary fibrosis. Nat Med 8: 480–484, 2002.[CrossRef][ISI][Medline]
  35. Nagase T, Uozumi N, Ishii S, Kume K, Izumi T, Ouchi Y, and Shimizu T. Acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase A2. Nat Immun 1: 42–46, 2000.[CrossRef][ISI][Medline]
  36. Needleman P and Isakson PC. The discovery and function of COX-2. J Rheumatol 24, Suppl 49: 6–8, 1997.[Medline]
  37. Nogawa S, Zhang F, Ross ME, and Iadecola C. Cyclo-oxygenase-2 gene expression in neurons contributes to ischemic brain damage. J Neurosci 17: 2746–2755, 1997.[Abstract/Free Full Text]
  38. O’Banion MK, Miller JC, Chang JW, Kaplan MD, and Coleman PD. Interleukin-1{beta} induces prostaglandin G/H synthase-2 (cyclooxygenase-2) in primary murine astrocyte cultures. J Neurochem 66: 2532–2540, 1996.[ISI][Medline]
  39. Rosenberger TA, Villacreses NE, Contreras MA, Bonventre JV, and Rapoport SI. Brain lipid metabolism in the cPLA2 knockout mouse. J Lipid Res 44: 109–117, 2003.[Abstract/Free Full Text]
  40. Rosenberger TA, Villacreses NE, Hovda JT, Bosetti F, Weerasinghe G, Wine RN, Harry GJ, and Rapoport SI. Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide. J Neurochem 88: 1168–1178, 2004.[CrossRef][ISI][Medline]
  41. Samad TA, Moore KA, Sapirstein A, Billet S, Allchorne A, Poole S, Bonventre JV, and Woolf CJ. Interleukin-1{beta}-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity. Nature 410: 471–475, 2001.[CrossRef][Medline]
  42. Satake Y, Diaz BL, Balestrieri B, Lam BK, Kanaoka Y, Grusby MJ, and Arm JP. Role of group V phospholipase A2 in zymosan-induced eicosanoid generation and vascular permeability revealed by targeted gene disruption. J Biol Chem 279: 16488–16494, 2004.[Abstract/Free Full Text]
  43. Serou MJ, DeCoster MA, and Bazan NG. Interleukin-1{beta} activates expression of cyclooxygenase-2 and inducible nitric oxide synthase in primary hippocampal neuronal culture: platelet-activating factor as a preferential mediator of cyclooxygenase-2 expression. J Neurosci Res 58: 593–598, 1999.[CrossRef][ISI][Medline]
  44. Steinman L. Medicine: collateral damage repaired. Nature 422: 671–672, 2003.[CrossRef][Medline]
  45. Stichtenoth DO, Thoren S, Bian H, Peters-Golden M, Jakobsson PJ, and Crofford LJ. Microsomal prostaglandin E synthase is regulated by proinflammatory cytokines and glucocorticoids in primary rheumatoid synovial cells. J Immunol 167: 469–474, 2001.[Abstract/Free Full Text]
  46. Tabuchi S, Uozumi N, Ishii S, Shimizu Y, Watanabe T, and Shimizu T. Mice deficient in cytosolic phospholipase A2 are less susceptible to cerebral ischemia/reperfusion injury. Acta Neurochir Suppl (Wien) 86: 169–172, 2003.
  47. Tanioka T, Nakatani Y, Semmyo N, Murakami M, and Kudo I. Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis. J Biol Chem 275: 32775–32782, 2000.[Abstract/Free Full Text]
  48. Tu B and Bazan NG. Hippocampal kindling epileptogenesis upregulates neuronal cyclooxygenase-2 expression in neocortex. Exp Neurol 179: 167–175, 2003.[CrossRef][ISI][Medline]
  49. Uematsu S, Matsumoto M, Takeda K, and Akira S. Lipopolysaccharide-dependent prostaglandin E2 production is regulated by the glutathione-dependent prostaglandin E2 synthase gene induced by the Toll-like receptor 4/MyD88/NF-IL6 pathway. J Immunol 168: 5811–5816, 2002.[Abstract/Free Full Text]
  50. Vidensky S, Zhang Y, hand T, Goellner J, Shaffer A, Isakson P, and Andreasson K. Neuronal overexpression of COX-2 results in dominant production of PGE2 and altered fever response. Neuromolecular Med 3: 15–28, 2003.[CrossRef][ISI][Medline]
  51. Yamagata K, Matsumura K, Inoue W, Shiraki T, Suzuki K, Yasuda S, Sugiura H, Cao C, Watanabe Y, and Kobayashi S. Coexpression of microsomal-type prostaglandin E synthase with cyclooxygenase-2 in brain endothelial cells of rats during endotoxin-induced fever. J Neurosci 21: 2669–2677, 2001.[Abstract/Free Full Text]
  52. Yang HC, Mosior M, Johnson CA, Chen Y, and Dennis EA. Group-specific assays that distinguish between the four major types of mammalian phospholipase A2. Anal Biochem 269: 278–288, 1999.[CrossRef][ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/6/R1774    most recent
00815.2004v1
Right arrow