Prostaglandin Levels in Stimulated Macrophages Are Controlled by Phospholipase A2-activating Protein and by Activation of Phospholipase C and D*

Deborah A. RibardoDagger, Sheila E. Crowe§, Kristine R. Kuhl, Johnny W. Peterson, and Ashok K. Chopra

From the Department of Microbiology and Immunology and § Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1070

Received for publication, July 26, 2000, and in revised form, November 9, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prostaglandins (PG), which are responsible for a large array of biological functions in eukaryotic cells, are produced from arachidonic acid by phospholipases and cyclooxygenase enzymes COX-1 and COX-2. We demonstrated that PG levels in cells were partly controlled by a regulatory protein, phospholipase A2 (PLA2)-activating protein (PLAA). Treatment of murine macrophages with lipopolysaccharide, interleukin-1beta , and tumor necrosis factor-alpha increased PLAA levels at early time points (2-30 min), which correlated with an up-regulation in cytosolic PLA2 and PGE2 levels. Both COX-2 and secretory PLA2 were also increased in lipopolysaccharide-stimulated macrophages, however, at later time points of 4-24 h. The role of PLAA in eicosanoid formation in macrophages was confirmed by the use of an antisense plaa oligonucleotide. Within amino acid residues 503-538, PLAA exhibited homology with melittin, and increased PGE2 production was noted in macrophages stimulated with melittin. In addition to PLA2, we demonstrated that activation of phospholipase C and D significantly controlled PGE2 production. Finally, increased antigen levels of PLAA, COX-2, and phospholipases were demonstrated in biopsy specimens from patients with varying amounts of intestinal mucosal inflammation, which corresponded to increased levels of phospholipase activity. These results could provide a basis for the development of new therapeutic tools to control inflammation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inflammation is a hallmark of many diseases (e.g. peritonitis, carditis, and arthritis) and is characterized by edema and tissue injury due to the release of numerous chemotactic and pro-inflammatory cytokines (e.g. IL-1beta ,1 TNFalpha , and IL-8), phospholipase A2 (PLA2), and eicosanoids (e.g. prostaglandins (PGE2) and leukotriene B4) (1-7). Phospholipase and eicosanoid production are inextricably linked through a pathway originating from arachidonic acid (AA), a 20-carbon, unsaturated fatty acid that is hydrolyzed from membrane phospholipids by secretory PLA2 (sPLA2) and cytosolic PLA2 (cPLA2) (8). Among these PLA2s, a 14-kDa type IIa sPLA2, which hydrolyzes phospholipids from the sn-2 position, has been extensively studied. The sPLA2 requires millimolar amounts of calcium for activity and has been linked to many other inflammatory diseases, notably adult respiratory distress syndrome (5), rheumatoid arthritis (1), and asthma (2, 9, 10). Recently, cPLA2 (85-110 kDa) has been characterized and found to preferentially use glycerophospholipids as a substrate and requires submicromolar amounts of calcium for activity (11). A third PLA2 is calcium-independent PLA2 (iPLA2), and, although not as extensively characterized as the others, iPLA2 is thought to be responsible for the late phase production of prostaglandins (PGs) when intracellular calcium levels in cells have been depleted (12-14). Several groups (15-20) have reported that sPLA2 is the primary phospholipase involved in PG production in target cells. Conversely, other investigators have documented that cPLA2 is the predominant phospholipase involved in PG formation (13, 21-27). The contribution of the other phospholipases (PLC and PLD) in PG production has not been as extensively studied (28). It is plausible that various phospholipases may be involved in PG production, and the induction of a particular phospholipase is dependent on the cell type involved, the stimulus given, and the activation state of the cell before induction (11, 29-32).

Once AA is liberated from cell membranes, it becomes the substrate for cyclooxygenases or lipoxygenases to form prostaglandins/thromboxanes or leukotrienes, respectively. There are two isoforms of cyclooxygenase, constitutive COX-1 and inducible COX-2. COX-2-induced PG production has been implicated in inflammation, as elevated levels of this protein have been found in synovial cells treated with inflammatory mediators or from patients with rheumatoid arthritis (33-37). The expression of the cox-2 gene appears to be highly regulated by a number of transcription factors, in particular by NF-kappa B (35, 37-40).

Prostaglandin PGE2 is particularly important, since it is a bronchodilator and vasodilator in the lungs and stimulates adenylate cyclase with the formation of cAMP, which stimulates intestinal chloride secretion during noninflammatory and inflammatory diarrheas (7, 8). PGE2 has been shown to increase cPLA2 and COX-2 levels and thereby amplify its own production, as well as to increase synthesis of some inflammatory cytokines resulting in a perpetuation of the immune response (23, 41, 42).

Several drugs have been developed to interrupt PG formation and, therefore, the severity of the inflammatory response. Two central targets have been COX-2 and PLA2 (43). Recently, a phospholipase A2-activating protein (PLAA) cDNA has been cloned from a human monocyte cell line (U937) by our research group (44). The cDNA-encoding PLAA was originally cloned from murine BC3H1 cells using antibodies to melittin, a 26-amino acid (aa)-long peptide and a major component in bee venom (45). At the aa level, murine, rat, and human PLAA exhibit 93-97% homology (44). When murine PLAA was injected into the synovium of rabbits, an increased amount of inflammation occurred that correlated with an increase in sPLA2 activity (46). Likewise, sPLA2 and PLAA have been implicated in a number of other cell functions, including Golgi tubulation and retrograde trafficking, peroxisomal motility in Chinese hamster ovary cells, and calcium oscillations/amylase secretion in rat pancreatic acini (47-49).

Inflammation is a prominent characteristic of inflammatory bowel diseases (IBD), which include Crohn's disease (CD) and ulcerative colitis (UC) (50); however, the etiology and the pathogenesis of these diseases are not yet fully understood (6). It is widely accepted that there are two central components to IBD (8). The first is the initiation of an aberrant immune response, which may result from absorption of foreign antigens of microbial origin, possibly from resident flora in the intestinal lumen. The second is the amplification and perpetuation of this aberrant immune response (7, 8), which is responsible for tissue injury due to the involvement of cytokines, eicosanoids, reactive oxygen species, and complement (6). Examination of biopsy specimens from patients with UC and CD has revealed a marked increase in all of these factors and of PLA2 and PGE2 (4, 51). We initiated studies to assess the involvement of PLAA in PLA2-associated eicosanoid production leading to inflammation in patients with UC and CD. Our studies using immunohistochemistry detected PLAA in inflammatory cells from patients with UC and CD and in mice chemically treated with dextran sodium sulfate, which induced colitis (52). The tissues from normal patients and untreated mice were devoid of any staining for PLAA.

The objectives of this study were as follows: 1) to establish whether PLAA was induced in inflammatory cells (e.g. macrophages) by LPS, IL-1beta , and TNFalpha ; 2) whether an antisense oligonucleotide to plaa cDNA would inhibit AA release from stimulated cells, which could lead to the development of potentially important therapeutic PLAA peptides controlling inflammation; 3) whether other phospholipases (e.g. PLC and PLD) contribute to PGE2 production; and 4) whether levels of PLAA, phospholipases, and COX-2 could be correlated to the extent of inflammation in patients with IBD.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Polyclonal antibodies to a synthetic peptide representing aa residues 185-199 (NSKDFVTTSEDRSLR) of murine PLAA were generated in rabbits (52). This region was highly conserved in murine, rat, and human PLAA (44). Polyclonal antibodies to cPLA2, COX-2, and all of the secondary antibodies used in enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal antibodies to cPLA2 and COX-2 were to regions conserved between mouse, rat, and human. Monoclonal anti-human sPLA2 type II antibodies were from Roche Molecular Biochemicals. Polyclonal antibody to phosphorylated proteins (serine, threonine, and tyrosine) was purchased from Zymed Laboratories Inc. (South San Francisco, CA). Bacterial lipopolysaccharide (LPS) from Escherichia coli, interleukin-1beta (IL-1beta ), and protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-benzoyl-L-tyrosine ethyl ester (BTEE) were from Sigma. Tumor necrosis factor-alpha (TNFalpha ) was obtained from PeproTech, Inc. (Rocky Hill, NJ). LY311727 was a gift from Lilly. Phospholipase C inhibitor D609 and phospholipase D inhibitor propranolol were purchased from Biomol (Plymouth Meeting, PA). Prostaglandin E2 enzyme immunoassay kit and protein G-Sepharose were purchased from Amersham Pharmacia Biotech. Gel shift assay kits were from Promega (Madison, WI). The probe for cox-2 cDNA was from Cayman Chemical Co. (Ann Arbor, MI). The probe for TNFalpha -encoding cDNA was generated by reverse transcription-polymerase chain reaction using specific TNFalpha reverse transcription-polymerase chain reaction amplimers (CLONTECH, Palo Alto, CA) and total RNA from macrophage cell line RAW264.7. A 354-base pair cDNA fragment was cloned into TA cloning vector pCR2.1 (Invitrogen, Carlsbad CA). The recombinant plasmid was digested with the EcoRI restriction enzyme to obtain the fragment. The probe for glyceraldehyde-3-phosphate dehydrogenase cDNA was purchased from CLONTECH. Synthetic melittin, a 26-aa-long peptide (53), was synthesized and purified by high pressure liquid chromatography at the M.D. Anderson Cancer Center (Houston, TX).

LPS/IL-1beta /TNFalpha /Melittin Stimulation of Macrophages and Western Blot Analysis-- RAW264.7 cells (ATCC, Manassas, VA), a murine macrophage-like cell line (1 × 106 cells/ml), were cultured in Dulbecco's modified Eagle's medium and 10% fetal bovine serum with 50 µg/ml gentamicin and 100 units/ml penicillin/streptomycin at 37 °C, 5% CO2. The cells were treated either with 10 ng to 10 µg/ml LPS, 10 ng/ml IL-1beta , 100-500 ng/ml TNFalpha , and 1 µg/ml synthetic melittin for selected times of 2, 10, 30, and 60 min and 2, 4, 8, and 24 h. After incubation with LPS, IL-1beta , TNFalpha , or melittin, cell supernatants were collected and stored at -70 °C until further analysis. The cell monolayer was rinsed with phosphate-buffered saline (PBS) at room temperature and then lysed in RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors (100 µg/ml phenylmethylsulfonyl fluoride, aprotinin at 30 µl/ml, and 100 mM sodium orthovanadate at 10 µl/ml) as described by the manufacturer (Santa Cruz Biotechnology). The cell lysate was passed through a 21-gauge needle to shear the DNA and centrifuged at 15,000 × g for 20 min to remove cellular debris. The protein concentration of the resulting supernatant was determined using a BCA protein assay kit according to the manufacturer's instructions (Pierce). Equal amounts of protein were loaded onto an SDS-12% polyacrylamide gel and electrophoresed at 200 V for 45 min followed by transfer to a nitrocellulose membrane. The blots were briefly rinsed in 1× Tris-buffered saline (TBS) and then incubated overnight in 3% gelatin to block nonspecific binding of antibodies to the membrane. The blots were then washed twice for 10 min with 1× TBS containing 0.5% Tween 20 (TTBS) and incubated with the appropriate primary antibody (1 µg/ml) for 2 h followed by washing twice for 10 min each with 1× TTBS. The blots were then incubated with the appropriate secondary antibody (1:25,000) for 1 h. Finally, blots were washed three times with 1× TTBS and once with 1× TBS for 10 min each followed by development using an enhanced chemiluminescence (ECL) kit (Pierce) (54). The blots were scanned using a gel documentation system (Bio-Rad) to calculate fold increase in the antigen level.

Immunoprecipitation of Phosphorylated Proteins-- RAW264.7 cells previously treated with LPS (10 ng/ml) and analyzed for PLAA expression via Western blot analysis were subjected to immunoprecipitation studies. An aliquot of a protein sample (20 µg) from untreated and LPS-stimulated RAW cells was incubated with 10 µg of an antibody against three phosphorylated proteins (serine, threonine, and tyrosine) for 1 h at 37 °C. Following incubation, 100 µl of protein G-Sepharose was added, and the slurry was mixed at room temperature for 1 h. The slurry was centrifuged at 13,000 × g for 5 min, and the resulting supernatant was transferred to microcentrifuge tubes. The pellet was washed twice with PBS. Both the pellet and the supernatant were boiled for 5 min in 200 µl of SDS sample buffer and loaded onto an SDS-12% polyacrylamide gel as described previously. Western blot was probed with anti-peptide antibodies to PLAA.

Treatment of Macrophages with Various Inhibitors and Northern Blot Analysis-- RAW264.7 cells (1 × 106/ml) were treated with various phospholipase/protease inhibitors concurrently with LPS (10 ng/ml) or with melittin (1 µg/ml). Cells were treated with 25 µM LY311727 (sPLA2 inhibitor), 10 µM BTEE, and TPCK (chymotrypsin-like serine protease inhibitors), 50 µM D609 (PLC inhibitor) or 70 µM propranolol (PLD inhibitor) for periods of 0, 10, 30 min and 2, 4, 8, 24 h. Supernatants were collected and stored at -70 °C for further analysis. RNA was isolated using TriZol reagent according to protocols outlined by Life Technologies, Inc. Briefly, 1 ml of TriZol reagent was added to each 35-mm dish and allowed to shake for 5 min. Cell lysate was transferred to a microcentrifuge tube with subsequent addition of 200 µl of chloroform and shaken for 15 s, followed by centrifugation at 12,000 × g for 15 min. The aqueous phase was transferred to a new microcentrifuge tube, and the RNA was precipitated using 0.5 ml of isopropyl alcohol. RNA was allowed to precipitate at room temperature for 10 min and centrifuged at 12,000 × g for 10 min. The supernatant was removed, and the RNA pellet was washed with 1 ml of 80% ethanol followed by centrifugation at 7,500 × g for 5 min. The RNA pellet was air-dried for 5 min and resuspended in diethyl pyrocarbonate-treated water. RNA was quantitated using a spectrophotometer (Amersham Pharmacia Biotech). Subsequently, RNA was separated on a 3% formaldehyde/agarose gel and visualized by staining with ethidium bromide (55). RNA was transferred to a nylon membrane and baked at 80 °C for 2 h. Membranes were prehybridized for 15 min at 68 °C with Quikhyb (Stratagene, La Jolla CA), followed by the addition of an appropriately labeled [alpha -32P]dCTP probe (1 × 106 cpm/ml) (ICN, Costa Mesa CA) and 10 mg/ml salmon sperm DNA (Stratagene) in the hybridization solution as described (54). Hybridization was performed for 2 h at 68 °C followed by 4 washes for 20 min each with 2× SSC (1× SSC is 0.15 M sodium chloride plus 0.015 sodium citrate, pH 7.4) + 0.1% SDS (twice) and 1× SSC + 0.1% SDS (twice) at 68 °C. Membranes were allowed to air dry for 5 min before being exposed to film overnight at -70 °C. Diethyl pyrocarbonate-treated water was used to prepare all the reagents used for the RNA work.

Gel Shift Assay-- Gel shift assays were performed according to the manufacturer's recommendations (Promega, Madison, WI). Briefly, 1 × 106 cells/ml were washed twice with 1× PBS, followed by the addition of 1 ml of PBS, and the cells were scraped into a cold Eppendorf tube. Cells were spun down at 3,000 rpm for 30 s, and the resulting supernatant was removed. Solution A (50 mM HEPES, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml aprotinin, and 0.5% Nonidet P-40) was added to the pellet in a 2:1 ratio (v/v) and allowed to incubate on ice for 10 min. The cells were pelleted at 6,000 × g for 30 s, and the supernatant was removed. Solution B (Solution A + 1.7 M glucose) was added to the pellet in a 1:1 ratio (v/v) and centrifuged at 12,000 × g for 10 min followed by removal of the supernatant. Solution C (solution A + 10% glycerol and 400 mM KCl) was added to the pellet in a 2:1 ratio (v/v) and vortexed on ice for 20 min. The cells were centrifuged at 12,000 × g for 20 min, and the resulting nuclear extract supernatant was collected in a chilled Eppendorf tube. Protein concentrations of nuclear extracts were quantitated using a BCA assay kit (Pierce). Consensus oligonucleotides were end-labeled using T4 polynucleotide kinase and [gamma -32P]dATP (ICN) for 10 min at 37 °C and the unincorporated 32P removed (54). Gel shift reactions were assembled as follows where the term sample denotes nuclear extracts from treated RAW cells: negative control (7 µl of water and 2 µl of gel shift binding buffer), positive control/sample (5 µl of water, 2 µl of gel shift binding buffer, 2 µl of HeLa nuclear extract/sample (10 µg/ml), specific competitor of a transcription factor (4 µl of water, 2 µl of gel shift binding buffer, 2 µl of HeLa nuclear extract/sample (10 µg/ml), 1 µl of unlabeled competitor oligonucleotide), supershift reaction (3 µl of water, 2 µl of gel shift binding buffer, 2 µl of HeLa nuclear extract/sample (10 µg/ml), 2 µl of specific supershift antibody (e.g. p50/p65)) (Santa Cruz Biotechnology). Reactions were assembled and allowed to incubate at room temperature for 10 min followed by the addition of 1 µl (50,000-200,000 cpm) of 32P-labeled oligonucleotide and another 20 min of incubation at room temperature. Subsequently 1 µl of gel loading buffer was added to each reaction and loaded onto a 4% nondenaturing gel and electrophoresed until the dye was three-fourths of the way down the gel. The gel was dried at 80 °C for 2 h and exposed to film overnight at -70 °C.

PLA2 Activity and PGE2 Assay from Stimulated Macrophages-- Cell culture supernatants and whole cell lysates with the original culture medium were assayed for PLA2 activity according to protocols described previously (54). Briefly, 10 µl of supernatant or cell lysate from stimulated RAW cells was incubated with 10 µl of [3H]AA E. coli cells, 30 µl of high pressure liquid chromatography grade water, and 10 µl of buffer (500 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM CaCl2, 5 mg/ml bovine serum albumin, 50% glycerol) for 15 min at 37 °C. After incubation, 150 µl of ice-cold PBS was added, and the solution was centrifuged at 13,000 × g for 10 min. An aliquot (50 µl) of the supernatant was transferred to scintillation vials and counted for the release of radioactivity. Prostaglandin levels were determined using an EIA kit according to the manufacturer's instructions (Amersham Pharmacia Biotech). Briefly, 50 µl of standard/sample was pipetted into a precoated goat anti-mouse 96-well plate. Aliquots (50 µl) of a mouse polyclonal PGE2 antibody and PGE2 conjugated to alkaline phosphatase were added to each well and allowed to incubate at room temperature for 1 h. After incubation, wells were washed six times with 400 µl of 1× PBS + 0.5% Tween 20, followed by the addition of 150 µl of 3,3',5,5'-tetramethylbenzidine substrate (Sigma). Wells were read at 670 nm with an ELISA reader (Molecular Dynamics, Sunnyvale, CA) 30 min after addition of substrate.

Pretreatment of Macrophages with Antisense plaa Oligonucleotide/cPLA2 Antisense Oligonucleotide Prior to Stimulation with LPS-- RAW264.7 cells at a concentration of 1 × 106 cells/ml were incubated with 3H-labeled AA overnight (1 µCi/ml) and washed three times with Dulbecco's modified Eagle's medium to remove unincorporated label. Cells were pretreated for 4 h with 25 µg/ml antisense plaa oligonucleotide (5'-TGGAGTGGCCGCTCATACAGTGCAT-3') or a random oligonucleotide of similar base composition and length (44). Following pretreatment, cells were stimulated with 10 ng/ml LPS for 24 h, and aliquots of the supernatants were collected and counted to determine AA release. Similarly a synthetic oligonucleotide to antisense cPLA2 was purchased from Cayman Chemical Co. (Ann Arbor, MI) and applied to cells (10 µg/ml) using Clonfectin (CLONTECH) for 4 h prior to stimulation with LPS. Cell supernatants were collected and assayed for PGE2 levels. The cell lysates were analyzed for cPLA2 and COX-2 antigen levels by Western blot analysis.

Biopsy Specimens-- Over 200 biopsy specimens were collected under an Institutional Review Board-approved protocol from individuals undergoing endoscopic procedures for diagnostic purposes, cancer surveillance, colon cancer screening, and assessment of diarrhea and of responses to therapy. In addition to these specimens, which were categorized as either normal control samples or other types of intestinal inflammation based on pathology reports, we collected specimens from patients diagnosed with IBD (UC and CD), and these samples consisted of both macroscopically inflamed and uninflamed areas.

Statistical Analysis-- Data were analyzed by a multiple group comparison test (Scheffé), which allowed all groups to be compared with each other, and hence is the most stringent statistical method available. In addition, the Student's t test was performed on a number of samples for statistical significance between two variables. A p value of <=  0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LPS Stimulates PLAA Production in Macrophages-- To ascertain whether PLAA was increased by inflammatory mediators, we stimulated RAW264.7 cells with LPS (10 ng to 10 µg/ml) followed by Western analysis for PLAA using anti-PLAA peptide antibodies. As seen in Fig. 1A, 10 ng/ml LPS resulted in increased levels of a 72-kDa polypeptide, which corresponded to the size of full-length PLAA. PLAA levels became maximal within 10-30 min (3-fold increase) followed by a reduction at 4 h, which continued to decrease through the last time point tested of 24 h. Significant increases in PLAA levels were observed at higher concentrations of LPS (10 µg/ml, data not shown). We selected a more physiological relevant concentration (10 ng/ml) of LPS in all of our subsequent experiments. The PGE2 levels in the culture supernatants of these cells were similarly elevated with significant increases within 30 min, becoming maximal at 24 h (Fig. 2). The PLA2 activity, as measured by AA release, also revealed a significant increase within 30 min in culture supernatants and cell lysates. The PLA2 activity decreased at 2 h followed by an increase up to 8 h (Fig. 2). Other enzymes utilized for prostaglandin formation were also elevated upon stimulation of macrophages with LPS. Cytosolic PLA2 (cPLA2) showed an increase within 30 min and continued to increase for 8 h, resulting in a 5-fold increase in protein levels at 8 h based on Western blot analysis (Fig. 1B). Interestingly, neither COX-2 nor secretory PLA2 (sPLA2) protein levels increased until 4 h after LPS stimulation of macrophages and continued to increase until the last time point tested of 24 h with 50- and 5-fold increases in protein levels, respectively, based on Western blot analysis (Fig. 1, C and D).



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Fig. 1.   Western blot analysis on proteins from LPS (10 ng/ml)-treated murine macrophage RAW264.7 cell line. Fifty micrograms of total proteins were loaded into each lane of an SDS-12% PAGE, and the blots were probed with polyclonal anti-peptide antibodies to PLAA (A), polyclonal antibodies to cPLA2 (B), polyclonal antibodies to COX-2 (C), or monoclonal antibodies to sPLA2 (D). Cell lysates (A-C) were prepared in RIPA buffer with appropriate protease inhibitors as described under "Experimental Procedures." Cell supernatants (D) were directly loaded on to the gel. Appropriate secondary antibodies conjugated with horseradish peroxidase were used, followed by an enhanced chemiluminescence substrate kit (Pierce) to develop the blots. The blots were scanned to calculate fold increase in the antigen level. Three independent experiments were performed, and a representative blot is shown.



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Fig. 2.   LPS increases PLA2 activity and PGE2 levels in macrophages. PLA2 and PGE2 levels were measured in culture supernatants or cell lysates from LPS (10 ng/ml)-treated murine macrophage RAW264.7 cell line. The PLA2 activity was measured using oleic acid-labeled E. coli as a substrate, whereas PGE2 was measured by a radioimmunoassay kit (for details see "Experimental Procedures"). Three independent experiments were performed, and an average mean with standard errors was plotted. Asterisks denote statistically significant values at p <=  0.05

IL-1beta and TNFalpha Stimulates PLAA Production in Macrophages-- In addition to LPS, we also examined other inflammatory mediators such as pro-inflammatory cytokines for their effects on PLAA and PLA2 production. Stimulation of RAW264.7 cells with 10 ng/ml IL-1beta revealed an increase in PLAA antigen levels within 2 min as determined by Western blot analyses (Fig. 3A). PLAA levels continued to increase for up to 30 min with a 3-fold increase in antigen levels (Fig. 3A). A similar pattern was noted for cPLA2 levels, and the increases in the levels of PLAA and cPLA2 coincided with the increase in PLA2 activity (data not shown). RAW264.7 cells stimulated with various concentrations of TNFalpha (100 ng-500 ng) followed by Western blot analysis for PLAA revealed a band of ~72 kDa, which increased in intensity within 10 min, followed by a decrease at 120 min for concentrations of 100 and 200 ng/ml (Fig. 3B). However, TNFalpha concentration of 500 ng/ml resulted in an increase in PLAA within 10 min that continued to increase up to the last time point tested of 2 h after stimulation, with a 50-fold increase in antigen level (Fig. 3B).



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Fig. 3.   Western blot analysis on proteins from IL-1beta (10 ng/ml)-treated murine macrophages RAW264.7 cell line (A) and TNFalpha (100-500 ng/ml)-treated RAW cells (B). 25 µg of total proteins were loaded into each lane of an SDS-12% PAGE, and the blots were probed with polyclonal anti-peptide antibodies to PLAA. Cell lysates were prepared in RIPA buffer with appropriate protease inhibitors as described under "Experimental Procedures." Appropriate secondary antibodies conjugated with horseradish peroxidase were used, followed by enhanced chemiluminescence substrate kit (Pierce) to develop the blots. The blots were scanned to calculate fold increase in the antigen level. Three independent experiments were performed, and a representative blot is shown.

PLAA Is a Phosphorylated Protein Based on Immunoprecipitation Analysis-- Immunoprecipitation studies of PLAA were conducted to ascertain whether the activity of PLAA might be regulated by phosphorylation due to its early up-regulation similar to other signaling molecules. Western blot analysis of phosphorylated proteins (immunoprecipitated with a phosphospecific antibody) using antibodies to PLAA revealed a similarly sized polypeptide both in 0- and 10-min samples after LPS stimulation, indicating that PLAA was phosphorylated before stimulation. We selected the 10-min time point, as maximum PLAA production was noted within the first 30 min of LPS stimulation. The 0-min sample served as a control. No PLAA-specific band was detected in the supernatant portion of the immunoprecipitation reaction (data not shown).

Release of [3H]AA from LPS-stimulated Macrophages Is Reduced in Cells Pretreated with plaa-specific Oligonucleotide-- To establish a more definitive role for PLAA in increasing phospholipase activity and subsequently PGE2 production, RAW264.7 cells were pretreated with antisense plaa oligonucleotide for 4 h prior to stimulation with 10 ng/ml LPS. Results showed that LPS elicited a dramatic decrease in AA release compared with cells that had been pretreated with a scrambled oligonucleotide of similar length and base composition (Fig. 4). Similar results were obtained when the concentration of LPS used to stimulate the cells was increased to 1 ng/ml (data not shown). Our data also indicated that plaa antisense oligonucleotide-treated cells exhibited a reduced level of cPLA2 compared with cells treated with LPS and random oligonucleotide based on Western analysis, indicating a direct effect of PLAA in regulating cPLA2, which controls AA levels in cells.



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Fig. 4.   Antisense oligonucleotide to plaa reduced [3H]AA release from LPS-stimulated RAW cells. RAW264.7 cells were radiolabeled with [3H]AA overnight, and excess unincorporated label was removed. Cells were treated with 25 µg/ml of an antisense oligonucleotide to plaa or a random oligonucleotide of similar base composition and length for 4 h prior to stimulation with LPS. Aliquots of cell supernatants were assayed for percent release of radiolabeled AA using a beta -counter. Cells treated with LPS alone served as a positive control. Three independent experiments were performed, and an arithmetic means ± S.E. are presented. Asterisks denote statistical significance at p < 0.05.

Melittin Induces Expression of COX-2- and TNFalpha -encoding Genes in Macrophages-- The sequence of melittin, which has a high degree of homology within a region of PLAA (aa residues 503-538), prompted us to examine the effect of synthetic melittin on COX-2 and TNFalpha production in macrophages. RAW cells treated with a sub-lethal concentration of melittin (1 µg/ml) resulted in increased abundance of TNFalpha and COX-2 mRNA (Fig. 5). The maximum increase in the message for TNFalpha was noted at 2 h but then declined thereafter. The increase in abundance of COX-2 mRNA occurred at 4 h and was maximally increased at 8 h. As an internal control, a cDNA probe for glyceraldehyde-3-phosphate dehydrogenase was used (Fig. 5). After macrophages were stimulated with melittin, an increase in PGE2 levels occurred in a time-dependent fashion with maximum PGE2 levels observed in 24 h (data not shown). PGE2 levels in melittin-stimulated cells appeared to be primarily produced via PLC, as an inhibitor to PLC reduced PGE2 levels in melittin-treated cells by 77% as compared with sPLA2 and PLD inhibitors, which reduced PGE2 levels by 43 and 37%, respectively (Fig. 6A). Most interestingly, melittin was a potent inhibitor of LPS-induced PGE2 production when both were applied to the cells at the same time. As seen in Fig. 6B, melittin (1 µg/ml) reduced PGE2 production in LPS-stimulated macrophages by ~75%.



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Fig. 5.   Synthetic melittin-stimulated transcription of genes coding for TNFalpha and COX-2 as determined by Northern blot analysis. RAW264.7 cells were treated with 1 µg/ml synthetic melittin for various times. RNA was isolated using TRIzol® reagent, and equal amounts were separated on a 3% formaldehyde/agarose gel. RNA was transferred to nylon membranes and probed with alpha -32P-labeled cDNA probe for TNFalpha . The blots were exposed to film overnight. After exposure, the blots were stripped (50% formamide + 2× SSC, 2 h 68 °C) to remove probe and reprobed with alpha -32P-labeled cDNA probe for COX-2 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) to normalize for RNA loading. Three independent experiments were performed, and the results from a representative experiment are shown.



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Fig. 6.   Melittin induced PGE2 production via PLC and reduced LPS-induced PGE2 production in RAW cells. RAW264.7 cells were treated concurrently with 1 µg/ml synthetic melittin and a phospholipase inhibitor to either sPLA2 (LY311727 (25 µM)), PLC (D609 (50 µM)), or PLD (propranolol (70 µM)) (A). RAW264.7 cells were treated with 10 ng/ml LPS alone or with 1 µg/ml synthetic melittin and LPS at the same time (B). The supernatants were collected and analyzed for PGE2 levels using an enzyme immunoassay. Three independent experiments were performed, and an arithmetic means ± S.E. are shown. Asterisks denote statistical significance at p < 0.05.

PGE2 Is Formed in LPS-stimulated Macrophages Via Various Phospholipases-- The paucity of literature as to the identity of the specific phospholipases involved in LPS-induced AA release from phospholipids prompted us to initiate studies to ascertain the contribution of various phospholipases in LPS-induced PGE2 production in macrophages. LPS-stimulated RAW264.7 cells concurrently treated with the sPLA2 inhibitor LY311727, PLC inhibitor D609, or PLD inhibitor propranolol showed significant decreases (~90%) in PGE2 production, as compared with cells treated with LPS alone (Fig. 7). To address the specificity of these various inhibitors, we examined cox-2 gene expression. It is evident from Fig. 8A that sPLA2 inhibitor LY311727 abrogated LPS-induced cox-2 gene expression in macrophages when compared with cells treated with LPS alone (Fig. 8C). However, the PLC inhibitor D609 (data not shown) and PLD inhibitor propranolol did not alter LPS-induced cox-2 gene expression, as indicated by both Northern and Western blot analyses (Fig. 8, B and C). In addition to the various inhibitors, we also tested the effects of pretreatment of LPS-stimulated cells with an antisense oligonucleotide to cPLA2 upon PGE2 production. As shown in Fig. 7, pretreatment of cells with a specific antisense oligonucleotide to cPLA2 reduced LPS-stimulated PGE2 production by ~60%, as compared with cells treated with LPS alone. We further demonstrated that cells pretreated with cPLA2 antisense oligonucleotide prior to LPS stimulation reduced cPLA2 levels based on Western blot analysis; however, the levels of COX-2 remained unaffected (data not shown), indicating specificity of the antisense oligonucleotide used. The data in Fig. 7 pointed to the role of both cPLA2 and sPLA2 as well as PLC and PLD in PG production in LPS-stimulated macrophages.



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Fig. 7.   Various phospholipase inhibitors reduced PGE2 levels in RAW264.7 cells. RAW cells were treated with LPS (10 ng/ml) and LY311727 (25 µM), propranolol (70 µM), D609 (50 µM), BTEE (10 µM), TPCK (10 µM), or with antisense cPLA2 oligonucleotide for 24 h as described under "Experimental Procedures." Supernatants from variously treated cells were collected and assayed for PGE2 levels using an enzyme immunoassay kit (Amersham Pharmacia Biotech) as described under "Experimental Procedures." Arithmetic means ± S.E. are plotted, and asterisks indicate statistically significant differences at p < 0.05, compared with LPS alone from four different experiments.



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Fig. 8.   Inhibitors to sPLA2 reduced cox-2 gene expression in RAW cells, whereas inhibitors to PLD did not, based on Northern and Western blot analysis. RAW264.7 cells were treated with LPS (10 ng/ml) and indicated inhibitors at the same time for various time points. RNA was isolated using TRIzol® reagent, and equal amounts of RNA were separated on a formaldehyde/agarose gel. RNA was transferred to nylon membranes and probed with alpha -32P-labeled cDNA probe for COX-2. Equal amounts of protein (15 µg/ml) isolated from cells were separated on an SDS-12% PAGE gel and transferred to a nitrocellulose membrane. Membranes were then probed with COX-2 antibodies, followed by appropriate secondary antibody, and the blots developed with a chemiluminescence substrate. A, Northern and Western blots of LPS- and LY311727 (25 µM)-treated cells. B, Northern and Western blots of LPS- and propranolol (70 µM)-treated cells. C, Northern and Western blots of cells treated with LPS alone (10 ng/ml). Four independent experiments were performed, and the results from a representative experiment are shown.

Protease Inhibitors and sPLA2 Inhibitor Abrogated cox-2 Gene Expression by Preventing LPS-induced Translocation of NF-kappa B in the Nucleus-- To define the mechanisms(s) that leads to down-regulation of cox-2 gene expression in LPS-stimulated cells by protease inhibitors (e.g. TPCK and BTEE) and LY311727, we determined their effect on the activation of transcription factor NF-kappa B. Both protease inhibitors and LY311727 decreased PGE2 production in LPS-stimulated cells by 60-97% (Fig. 7). These protease inhibitors also abrogated cox-2 gene expression in LPS-induced cells (data not shown). Both TPCK and BTEE significantly reduced LPS-induced NF-kappa B (p50/p65 heterodimer) that was translocated into the nucleus as compared with cells stimulated with LPS alone (data not shown). Likewise, the sPLA2 inhibitor (LY311727) reduced translocation of p50/p65 in LPS-induced macrophages as observed for protease inhibitors TPCK and BTEE (data not shown). In contrast, no significant effect on NF-kappa B p50/p65 heterodimer translocation was observed in macrophages treated with propranolol and LPS (Fig. 9A) when compared with cells treated with LPS alone (Fig. 9B). Similarly, no effect on NF-kappa B translocation was observed when cells were treated with a PLC inhibitor, D609 (data not shown).



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Fig. 9.   PLD inhibitor did not alter NF-kappa B translocation in LPS-stimulated macrophages. Gel shift analysis of RAW264.7 cells treated with propranolol (70 µM) and LPS (10 ng/ml) (A) or LPS alone (B) for indicated times. Gel shift nuclear extracts from stimulated RAW cells were isolated according to protocols outlined under "Experimental Procedures" and incubated with gamma -32P-labeled consensus oligonucleotide of NF-kappa B for 20 min prior to separation on a nondenaturing 4% PAGE. After electrophoresis, the gel was dried and exposed to film overnight. Arrows indicate p50/50 homodimer and p50/p65 heterodimer bands of NF-kappa B. Three independent experiments were performed, and the results from a representative experiment are shown.

Inflammatory Mediators Are Up-regulated in Biopsy Specimens from Patients with Inflammation-- We have screened over 200 patient tissue specimens for antigen levels of PLAA, cPLA2, COX-2, and sPLA2 as well as for PLA2 activity. Representative biopsy specimens were from patients with Crohn's disease (including both quiescent and actively diseased tissue samples), unclassified IBD samples, inflamed colon samples, and normal samples. In addition to these biopsy specimens, patients with other types of intestinal inflammation, including Clostridium difficile colitis, melanosis coli, and ischemic colitis were also analyzed. Western blot analysis of these specimens for PLAA, cPLA2, COX-2, and sPLA2 revealed that actively diseased and inflamed tissues contained elevated levels of these antigens as compared with quiescent and normal tissue sections (Fig. 10, A-D). The increase in these antigen levels coincided with an increase in PLA2 activity in these tissues, which was 3-5-fold higher (data not shown) compared with that of patients in which no apparent inflammation was evident. Likewise, of the 14 analyzed cases of actively diseased IBD, 71% had elevated levels of these antigens as compared with normal controls (data not shown).



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Fig. 10.   Up-regulation of inflammatory mediators in patients with IBD. Representative specimens from patients diagnosed with IBD (CD) consisting of both actively inflamed tissue and quiescent tissue and from patients with normal tissue were analyzed for cPLA2 (A), COX-2 (B), PLAA (C), and sPLA2 (D) by Western blot analysis. A and B were from the same patient, whereas the CD and uncategorized IBD samples in D were from different patients. Equal concentrations of protein (50 µg/ml) were loaded onto an SDS-12% PAGE, and the blots were probed with the indicated antibodies. Appropriate secondary antibodies conjugated to horseradish peroxidase were used, followed by enhanced chemiluminescence substrate kit (Pierce) to develop the blots.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

For an inflammatory response to occur in the host, there must be an initial event (e.g. infection and trauma). This event either triggers an explosive cellular infiltration response that may result in injury to the tissues of the host or a protective innate immune response. This protective immune response may become uncontrolled due to predisposing factors, such as the genetics of the individual in cases of UC or CD (7). Patients with UC and CD exhibit increased permeability of their intestinal mucosa (6), which allows bacterial products, such as LPS, to penetrate the lamina propria from the intestinal lumen and to bind to its receptor, CD14, on macrophages to induce transcriptional activation of a number of genes. Likewise, antiglycan antibodies to the cell wall mannan of Saccharomyces cerevisiae and mycobacterial antigens are highly correlated with Crohn's disease (56-58). LPS is a known stimulator of the immune response and of the pro-inflammatory cytokines IL-1beta and TNFalpha , which can lead to an inflammatory state. LPS also increases the production of PGs, which are synthesized from AA liberated from cell membranes by phospholipases. Several phospholipases have been characterized (e.g. PLA2, PLC, and PLD), but the contribution of these to PG production has not been thoroughly studied. Furthermore, it is unknown how various phospholipases are regulated in a cell. These studies therefore were designed to demonstrate the contribution of various phospholipases in PG production and to determine the role of a regulatory protein (PLAA) in controlling phospholipase activity in a cell.

Among various genes activated by LPS in host cells, we sought to delineate the role of PLAA, a G-like regulatory protein (59), in PGE2 production, as its level was up-regulated within minutes in LPS-stimulated macrophages (Fig. 1A). To define the role of PLAA in eicosanoid formation, we used plaa antisense oligonucleotide, and we demonstrated that it decreased levels of AA release in LPS-stimulated cells (Fig. 4). In addition, the plaa antisense oligonucleotide-pretreated cells reduced LPS-induced cPLA2 levels, indicating the direct role of PLAA in controlling cPLA2. These data demonstrated the importance of PLAA in the regulation of eicosanoid formation at the initial rate-limiting step of substrate (AA) formation. The ability of PLAA to increase sPLA2 activity in rheumatoid arthritis as well as in cell extracts of BC3H1 cells has been reported, thus making PLAA an important regulatory component in inflammation (1, 60, 61). Furthermore, the possibility that PLAA may regulate additional phospholipases (e.g. PLC and PLD) cannot be ruled out, and these studies are currently under investigation. The ability of melittin, which exhibits homology within the sequence of PLAA (62), to produce PGE2 via activation of PLC and PLD, in addition to PLA2 (Fig. 6A), points to the possible role of PLAA in regulating various phospholipases.

In addition to LPS, stimulation of macrophages was examined with the pro-inflammatory cytokines IL-1beta and TNFalpha , which also resulted in increased levels of PLAA at early (within 10 min) time points (Fig. 3, A and B). However, saturation of cells with the highest tested concentration of TNFalpha (500 ng/ml) also resulted in PLAA expression at a later time point of 120 min (Fig. 3B). These findings, in conjunction with the reported ability of PLAA to increase cytokine production (63-65), led to the possibility of an autocrine role of PLAA, which could result in perpetuating the inflammatory response. The rapid induction of the plaa gene in macrophages by LPS, IL-1beta , and TNFalpha coincided with an early increase in cPLA2 and PGE2 levels, with concomitant increases in the PLA2 activity (Figs. 1B, 2, and 3A), indicating the role of PLAA and cPLA2 in initiating AA metabolism. The role of cPLA2 in the production of early phase PGE2 has been reported in mast cells and fibroblasts (17, 21). However, some studies have indicated a role for cPLA2 in COX-2-dependent, delayed PGE2 production (23, 26, 66).

Most reports on PLAA have focused primarily on cell culture, with few clinical correlations. Likewise, most Western analysis on PLAA has utilized antibodies to melittin, rather than to PLAA. This has resulted in some confusion regarding the size of PLAA, as antibodies to melittin resulted in the identification of PLAA in IL-1beta -stimulated rabbit distal colon cells at ~45 kDa plausibly representing a degraded version of PLAA (67), whereas antibodies to PLAA synthetic peptides we have generated resulted in the identification of a band of ~72 kDa, which is the correct size of the full-length PLAA (44). The ability of the plaa gene to be up-regulated rapidly after induction, and its homology with numerous G protein beta -subunits, which regulate the activities of many cellular enzymes (59), prompted us to determine whether PLAA requires phosphorylation for its activity. Immunoprecipitation studies with cells stimulated with LPS revealed PLAA to be phosphorylated both at 0- and 10-min time points, indicating that although phosphorylation may be needed for the function of PLAA, it does not regulate its activity, as is the case with many other intermediate signaling molecules.

The purported role of melittin as an sPLA2 activator is widely known (45, 68-71). Melittin is also highly hemolytic to sheep red blood cells; however, downstream effects of melittin on cells have not been described (72). The homology of PLAA to melittin led us to ascertain the effects melittin had directly on macrophages, which could then be correlated back to PLAA. As shown in Fig. 5, melittin caused increases in abundance of message for both TNFalpha and COX-2 as measured by Northern blot analysis. PGE2 levels were elevated in macrophages treated with melittin, and these increased levels were mediated primarily by PLC and to a lesser extent by PLD and PLA2 (Fig. 6A).

Melittin also blocked many of the toxic effects of Lipid A, such as pyrogenicity in the rabbit model and lethality of chick embryos by preventing Lipid A from binding its receptor (73). However, the effects melittin has on LPS-induced PGE2 production have not been examined. Interestingly, melittin reduced PGE2 levels in LPS-stimulated RAW cells by ~75% (Fig. 6B). The exact mechanism of reduced PGE2 production by melittin in LPS-stimulated cells is currently unknown, but it has been hypothesized that melittin could prevent binding of LPS to its receptor (73). Alternatively, melittin has been reported to be a noncompetitive inhibitor of PLA2 (62). Based on these reports (62, 73), melittin would be expected to antagonize the effect of LPS on PLA2 activity and subsequent PGE2 production. Synthetic peptides of PLAA that share this region of homology with melittin could be developed to block LPS-induced PGE2 production to help abrogate the immune response.

Since it is unknown whether PLAA regulates more than one kind of phospholipase and since the relative contribution of each of the phospholipases upon PG production has not been thoroughly investigated in the literature, we sought to establish the involvement of PLA2, PLC, and PLD in PG synthesis. Through the use of various inhibitors and antisense oligonucleotides, our results indicated that several phospholipases are involved in PG production as inhibitors to sPLA2, cPLA2, PLC, and PLD significantly reduced PG production in LPS-stimulated macrophages (Fig. 7). Since our studies demonstrated the role of cPLA2 early in AA metabolism, we used methyl arachidonyl fluorophosphonate (5 µM) to show whether cPLA2 was also involved in late PG production, as cPLA2 antigen levels remained high at 8-24 h based on Western blot analysis (Fig. 1B). Our results indicated that this inhibitor could itself induce PGE2 production (data not shown). These results were recently substantiated by Lin and Chen (74) who indicated that cPLA2 inhibitor methyl arachidonyl fluorophosphonate induced PGE2 production via activation of COX-2. To circumvent this problem, we used a cPLA2 antisense oligonucleotide, which reduced LPS-induced PGE2 production in macrophages by 60% after 24 h (Fig. 7), indicating cPLA2 to be involved in both early and late phase production of PGE2 (21). Our data also supported the role of sPLA2 in late PGE2 production based on Western blot analysis (Fig. 1D) and the ability of cPLA2 antisense oligonucleotide to block only 60% of PGE2 production (Fig. 7). At present, we are investigating cross-talk between cPLA2 and sPLA2 that leads to sustained PGE2 production over a period of 24 h. The ability of various phospholipases (e.g. PLA2, PLC, and PLD) to reduce significantly PG production also suggests some communication among these phospholipases as well, with recent studies by Ueno et al. (75) indicating cross-talk between sPLA2 and PLD.

The sPLA2 inhibitor LY311727 reduced PGE2 production in RAW cells (Fig. 7); however, we noted that its effect was partly nonspecific as this inhibitor also reduced cox-2 gene expression (Fig. 8). On the contrary, both the PLC and PLD inhibitors did not alter cox-2 gene expression but significantly reduced PGE2 levels in LPS-stimulated macrophages (Figs. 7 and 8). These results indicated that COX-2 production was not regulated by availability of AA substrate and that the cox-2 gene was independently expressed in LPS-stimulated macrophages. The presence of two binding sites for NF-kappa B upstream of the cox-2 gene prompted us to ascertain whether the nonspecific effect of LY311727 on cox-2 gene expression might be due to interference with NF-kappa B activation. Indeed, sPLA2 inhibitor LY311727 reduced NF-kappa B translocation and may in part explain the effects this inhibitor had upon reduced PGE2 production. Protease inhibitors that prevent the degradation of Ikappa B, the inhibitor component of the NF-kappa B complex, should reduce translocation of NF-kappa B and subsequently the cox-2 gene expression (35, 38). We therefore examined the role of transcription factor NF-kappa B on cox-2 gene expression using protease inhibitors BTEE and TPCK. BTEE and TPCK significantly impaired NF-kappa B translocation and cox-2 gene expression with a concomitant decrease in PGE2 production (data not shown). Both PLC and PLD inhibitors did not alter NF-kappa B translocation as compared with cells stimulated with LPS alone, indicating the specificity of these inhibitors (Fig. 9). Taken together, these data showed that in addition to PLA2, PLC and PLD are important phospholipases involved in PGE2 production.

Our results indicated that PLAA potentially could play an important role in many inflammatory diseases such as IBD. Reports that purified PLAA can induce synthesis of the proinflammatory cytokines IL-1beta and TNFalpha led to the possibility that both an autocrine and paracrine loop can form, which would help perpetuate the aberrant immune response in IBD (7, 63-65). However, the purified PLAA used in the above-mentioned studies was isolated using anti-melittin affinity chromatography and exhibited a size of 28 kDa, which probably represented a degraded version of the full-length PLAA (76). We recently reported purification of full-length human PLAA (44), which will now be used to demonstrate possible autocrine and paracrine roles of this regulatory molecule.

To substantiate that PLAA is a major contributor to the inflammation seen in IBD, we examined biopsy specimens from patients with CD, UC, and from other patients undergoing endoscopy for increases in PLAA, cPLA2, COX-2, and sPLA2 antigen levels (Fig. 10, A-D). Most IBD cases examined were from patients with CD; however, similar results were seen in patients with UC. We have analyzed over 200 samples consisting of both normal, IBD, and non-IBD inflamed tissue, with results indicating that only those tissues that were pathologically inflamed showed increased levels of the above proteins and elevated PLA2 activity. These results corroborate our tissue culture studies and support the role of PLAA in inflammation. In addition, the level of PLC and PLD in biopsy specimens is currently under investigation to confirm their role in inflammation. Since many of the responses seen in IBD, such as elevated cytokine and eicosanoid production, are common to many other inflammatory diseases, we would expect that regulation of PLAA would not only help patients with intestinal inflammation but improve inflammation in patients with other general types of inflammation such as allergies, asthma, and arthritis. Given that PLAA is an upstream mediator of PLA2 activity, and the ability of PLAA to regulate other phospholipases is unknown, it is important that we determine in the future the direct effect of PLAA on other phospholipases. Furthermore, since PLC and PLD significantly contribute to PG production, we can target both PLAA and these phospholipases as therapeutic tools to control inflammation.


    ACKNOWLEDGEMENTS

We thank X-J. Xu for providing technical help and Mardelle Susman for editorial assistance.


    FOOTNOTES

* This work was supported in part by a grant from the Crohn's and Colitis Foundation of America and American Heart Association (to A. K. C.).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.

Dagger Supported by The James W. McLaughlin Predoctoral Fellowship Fund.

To whom correspondence should be addressed: Dept. of Microbiology and Immunology, 301 University Blvd., University of Texas Medical Branch, Galveston, TX 77555-1070. Tel.: 409-747-0578; Fax: 409-747-6869; E-mail: achopra@utmb.edu.

Published, JBC Papers in Press, November 27, 2000, DOI 10.1074/jbc.M006690200


    ABBREVIATIONS

The abbreviations used are: IL, interleukin; PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; PLAA, phospholipase A2-activating protein; PG(s), prostaglandin(s); LPS, lipopolysaccharide; TNFalpha , tumor necrosis factor alpha ; AA, arachidonic acid; IBD, inflammatory bowel diseases; CD, Crohn's disease; UC, ulcerative colitis; aa, amino acids; ELISA, enzyme-linked immunosorbent assay; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; BTEE, N-benzoyl-L-tyrosine ethyl ester; PBS, phosphate-buffered saline; TBS, Tris-buffered saline.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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