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Mechanisms that account for the selective release of arachidonic acid from intact cells by secretory phospholipase A2
Biochimica et Biophysica Acta 1393 (1998) 253^266
Mechanisms that account for the selective release of arachidonic acid from intact cells by secretory phospholipase A2 Alfred N. Fonteh
a;
*, James M. Samet c , Marc Surette Floyd H. Chilton a;b
1;a
, William Reed c ,
a b
Department of Internal Medicine (Division of Pulmonary and Critical Care Medicine), Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA c Center for Environmental Medicine and Lung Biology, CB #7310, Mason Farm Road, University of North Carolina, Chapel Hill, NC 27599-7310, USA Received 2 June 1998; accepted 9 June 1998
Abstract The current study examined mechanisms that account for the selective release of arachidonic acid (AA) from cells by secretory phospholipase A2 (sPLA2 ). Initial studies demonstrated that low concentrations of group I and group III PLA2 isotypes and an sPLA2 -enriched extract from bone marrow-derived mast cells (BMMC) selectively released AA from mast cells. Much higher concentrations of group II PLA2 were required to release comparable quantities of AA. Group I PLA2 also selectively released AA from another mast cell line (CFTL-15) and a monocytic cell line (THP-1). In contrast, high concentrations of group I PLA2 were required to release fatty acids from a promyelocytic cell line (HL-60) and this release was not selective for AA. Binding studies revealed that cell types (BMMC, CFTL-15 and THP-1) which selectively released AA also had the capacity to specifically bind group I PLA2 . However, group II PLA2 , which did not selectively release AA from cells, also did not specifically bind to these same cell types. Additional studies revealed that sPLA2 binding to the mast cell receptor was attenuated after stimulation with antigen or ionophore A23187. Reverse transcriptase^polymerase chain reaction analyses indicated the presence of mRNA for the sPLA2 receptor in BMMC, CFTL-15 and THP-1 and the absence of this mRNA in HL-60. Final studies demonstrated that p-aminophenyl-K-D-mannopyranoside BSA, a known ligand of the sPLA2 receptor, also selectively released AA from mast cells but not from HL-60 cells. These experiments indicated that receptor occupancy alone (without PLA2 activity) is sufficient to induce the release of AA from mast cells. Together, these data reveal that specific isotypes of sPLA2 have the capacity to selectively release AA from certain cells by their capacity to bind to sPLA2 receptors on the cell surface. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Arachidonic acid; Mast cell; Secretory phospholipase A2 ; Secretory phospholipase A2 receptor
Abbreviations: AA, arachidonic acid; OA, oleic acid; LA, linoleic acid; SA, stearic acid; sPLA2 , secretory phospholipase A2 ; cPLA2 , cytosolic phospholipase A2 ; Ag, antigen; NICI^GC/MS, negative ion chemical ionization^gas chromatography/mass spectrometry; [2 H8 ], octadeuterated; [2 H3 ], trideuterated; BMMC, bone marrow-derived mast cells; FCS, fetal calf serum; BSA, bovine serum albumin; RT^ PCR, reverse transcriptase^polymerase chain reaction * Corresponding author. Fax: +1-336-716-7277; E-mail: [email protected] 1 Present address: Centre Hospitalier de Quebec, Centre de Recherche en Rhumatologie et Immunologie, T 1-49 2705 Laurier, St Foy, Quebec G1V 4G2, Canada. 0005-2760 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 5 - 2 7 6 0 ( 9 8 ) 0 0 0 7 9 - 4
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1. Introduction Arachidonic acid (AA), a polyunsaturated fatty acid located at the sn-2 position of glycerophospholipids, serves as the precursor for several important lipid mediators (termed eicosanoids) of in£ammation [1]. Before AA is converted to eicosanoids, it must be hydrolyzed from phospholipids by a family of enzymes collectively known as phospholipase A2 (PLA2 ). PLA2 activities that may participate in this event include cytosolic, high-molecular-mass PLA2 (cPLA2 , 85^110 kDa) [2^9], several isotypes of secretory, low-molecular-mass PLA2 (s) (sPLA2 , V14 kDa) [10^12] and calcium-independent PLA2 activities [13^15]. The existence of multiple PLA2 s in cells and tissues has generated considerable research e¡ort to identify the PLA2 that is responsible for AA release and eicosanoid biosynthesis during in£ammation. Although the sPLA2 family of enzymes has been studied for the past six decades, there has not been a complete elucidation of their physiologic or pathophysiologic roles in mammals. Potential biological activities for sPLA2 include antibacterial e¡ects, key components in digestion, enzymes that generate arachidonic acid utilized for eicosanoid generation, potential regulators of severe illnesses such as sepsis, pancreatitis and organ injury and pro-in£ammatory components in diseases such as rheumatoid arthritis and asthma [16^27]. With regard to AA mobilization for the synthesis of bioactive eicosanoids, sPLA2 s have received only modest attention primarily due to the fact that these enzymes, when assayed in cell-free systems utilizing chemically pure substrates, show no selectivity for the hydrolysis of phospholipids containing AA when compared to phospholipids containing other more abundant fatty acids such as oleic acid and linoleic acid [28^30]. However, we have recently demonstrated that partially puri¢ed sPLA2 from human lung or sPLA2 isolated from snake venom can selectively release AA from human bronchial epithelial cells and from mast cells, respectively [31,32]. These ¢ndings suggest that arachidonate-containing phospholipids may be presented to sPLA2 in intact cells in a manner that facilitates the selective release of AA. Alternatively, binding of sPLA2 to putative receptors on cell surfaces may induce the selective release of AA by other PLA2
enzymes [33,34]. In addition to the aforementioned results, several other studies utilizing inhibitors, antisense oligonucleotides, cytokines or speci¢c antibodies have implicated sPLA2 as the rate-limiting step in the mobilization of AA leading to eicosanoid generation in certain cells [35^40]. All these ¢ndings have raised fundamental questions as to how sPLA2 causes AA release with subsequent eicosanoid generation in mammalian cells. The major objective of the current study was to examine the molecular events that facilitate the selective mobilization of AA by sPLA2 isotypes from membranes of intact mammalian cells. 2. Materials and methods 2.1. Materials Octadeuterated [2 H8 ]AA and trideuterated [ H3 ]SA were purchased from Cayman (Ann Arbor, MI). L-K-1-Palmitoyl-2-[1-14 C]arachidonoyl-sn-glycero-3-phosphocholine was purchased from American Radiolabel (St. Louis, MO). 125 I as sodium salt (NaI) was purchased from Amersham (Arlington Heights, IL). Essentially fatty acid free human serum albumin (HSA), Naja naja PLA2 , bee venom PLA2 , porcine pancreatic PLA2 , essential and non-essential amino acids, clone SPE-7 monoclonal anti-dinitrophenyl (IgE anti-DNP) a¤nity puri¢ed antibody, heat-inactivated fetal calf serum (FCS), ionophore A23187 and p-aminophenyl-K-D-mannopyranoside bovine serum albumin (BSA), were purchased from Sigma (St. Louis, MO). Recombinant human synovial £uid PLA2 was kindly supplied by Dr. L. Marshall (SmithKline Beecham, King of Prussia, PA). Acid extracts of BMMC highly enriched with PLA2 (0.18 þ 0.04 to 1.25 þ 0.24 nmol/mg/min) was obtained as previously described [40]. Iodobeads iodination reagents, penta£uorobenzyl bromide (20% in acetonitrile) and diisopropylethylamine (20% in acetonitrile), were purchased from Pierce (Rockford, IL). RPMI 1640 cell culture media was purchased from Gibco (Grand Island, NY). Hanks' balanced salt solution (HBSS) with or without calcium was purchased from Specialty Media (Lavallete, NJ). [3 H]AA-labeled E. coli membranes were purchased from New England Nuclear (Boston, MA). The un2
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saturated fatty acid composition of the E. coli membrane (determined by NICI-GC/MS and used to calculate PLA2 activities) was 7.8 þ 3.7 pmol/nmol Pi , 17.3 þ 7.9 pmol/nmol Pi and 4.7 þ 3.6 pmol/nmol Pi (n = 3) for LA, OA and AA, respectively. HPLC grade organic solvents were purchased from Fisher Scienti¢c (Norcross, GA). 2.2. Cell culture and activation Mouse bone marrow mast cells (BMMC) were obtained from CBA/J mice (Jackson, Bar Harbor, ME) and grown in RPMI 1640 culture medium (Gibco) supplemented with 10% (v/v) fetal calf serum (FCS), 50 WM 2-mercaptoethanol, 1% essential amino acids, 1% non-essential amino acids, 2 mM L-glutamine, 5 Wg/ml gentamycin and 1% (v/v) penicillin/streptomycin, for 4 weeks. The culture medium was supplemented twice a week with 50% WEHI supernatant £uid as a source of IL-3. The CFTL-15 mast cell line was similarly maintained in RPMI supplemented with 15% FCS, 1% (v/v) penicillin/streptomycin and 25% WEHI supernatant £uids. The human monocytic leukemia cells (THP-1) and the human promyelocytic leukemic cell line (HL-60) were maintained in RPMI supplemented with 10% FCS, 5 Wg/ml gentamycin and 1% (v/v) penicillin/streptomycin. In experiments designed to measure PLA2 secretion and AA release, BMMC were harvested after 3 weeks in culture and placed in newly-prepared culture media containing 50% WEHI for 24 h. During this period, BMMC were sensitized with 0.5 Wg/ml mouse IgE anti-DNP. Subsequently, cells were removed from culture and placed in HBSS containing 1 mM calcium, 0.2 mg/ml gelatin and 0.02 mg/ml HSA (HBSS/gelatin bu¡er). Cell viability ( s 95%) was determined by Trypan Blue exclusion. BMMC were then stimulated with DNP (2 Wg/ml), p-aminophenyl-K-D-mannopyranoside BSA (50 WM) or with ionophore A23187 (1 WM) for 5 min at 37³C. For kinetic studies, BMMC were incubated with antigen for di¡erent periods of time and stimulation was stopped by adding 0.5 ml ice-cold HBSS/gelatin buffer to each tube. Cells were then removed from supernatant £uids by centrifugation (400Ug, 5 min at 4³C). A portion of the supernatant £uid was utilized to measure PLA2 activity as described below. Four volumes of ethanol and 100 ng [2 H8 ]AA and
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100 ng [2 H3 ]SA were added to the remainder of the supernatant £uids as internal standards for the determination of mole quantities of fatty acids by negative ion chemical ionization^gas chromatography/mass spectroscopy (NICI^GC/MS). In experiments where fatty acid release from cells was determined after the addition of exogenous sPLA2 , cells were removed from growth media and placed in HBSS/gelatin bu¡er. After incubation at 37³C for 5 min, di¡erent concentrations of sPLA2 (0^100 nM or 0^40 Wg BMMC acid extract) were added to the cells and the incubation allowed to proceed for 5 min. The amount of sPLA2 added to these cells did not compromise cell viability. Cells were then removed from supernatant £uids by centrifugation (400Ug, 5 min at 4³C). Mole quantities of fatty acids were determined in supernatant £uids by NICI^GC/MS as described below. 2.3. Determination of PLA2 activity in supernatant £uids Portions of supernatant £uids (0.5 ml) were utilized for PLA2 activity determination. The PLA2 reaction was initiated by the addition of 0.02 WCi (9.7 nmol) [3 H]AA-labeled E. coli membranes (New England Nuclear) and CaCl2 (10 mM ¢nal concentration). After incubation (90 min at 37³C) in a water bath, the reaction was stopped by extracting lipids by the method of Bligh and Dyer [41]. Free fatty acids were isolated from phospholipids by TLC on silica gel G developed in hexane/ethyl ether/formic acid (90:60:6 v/v). The radioactivity in lipids was located using a radiochromatogram imaging system (Bioscan, Washington, DC). Free AA and phospholipids were isolated using TLC zonal scraping, and the quantity of radioactivity was determined by liquid scintillation counting. PLA2 activity was calculated and expressed as pmol AA released per hour. 2.4. Quantitation of free fatty acids in supernatant £uids After the addition of deuterated fatty acids as internal standards to supernatant £uids, solvents were removed from extracts using a stream of nitrogen. Fatty acids were then converted to penta£uorobenzyl esters and the mole quantities of free fatty acids de-
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termined by NICI^GC/MS using a Hewlett Packard model 5989 GC/MS instrument [42]. Carboxylate anions (m/z) were monitored at 279, 281, 286, 303 and 311 for LA, OA, [2 H3 ]SA, AA and [2 H8 ]AA, respectively, in the single ion monitoring mode. 2.5. Iodination of sPLA2 and binding assays The purity of PLA2 from Naja naja was veri¢ed by reverse phase HPLC using a diphenyl column [40]. PLA2 activity was determined using [3 H]AA-labeled E. coli membranes as described above. In addition to activity determination, eluted proteins were monitored at 280 nm and peak purity determined spectrophotometrically using a UV diode array detector. Iodination of group I PLA2 and group II PLA2 was performed using an Iodo-Bead iodination reagent kit (Pierce). Brie£y, 300 WCi Na125 I was added to V20 Wg PLA2 in 0.1 ml, 0.5 M NaH2 PO4 (pH 7.5) bu¡er. Iodination was initiated by adding an Iodo-Bead to the reaction mixture. The reaction was terminated after 2 min at room temperature by removing the reaction mixture from the bead. After the addition of 2 mg BSA in 20 Wl PBS as a protein carrier, [125 I]PLA2 was isolated using a Pharmacia PD-10 Sephadex G-25 column. Proteins were eluted from the column with 0.1 M NaH2 PO4 . Radioactivity in fractions containing [125 I]PLA2 was determined by gamma counting, and these fractions were stored at 4³C until required for binding assays. Binding assays were performed using 1 million cells in 0.4 ml HBSS containing 1 mg/ml HSA. Saturation binding was determined using various concentrations of [125 I]sPLA2 (0.05^4 nM). Non-speci¢c binding was determined with labeled ligands in the presence of 200-fold excess unlabeled PLA2 . For studies designed to examine speci¢c binding, cells were incubated with 2 nM iodinated PLA2 without or with 400 nM of unlabeled PLA2 in a ¢nal volume of 0.4 ml. Binding was performed at 20³C for 2 h. Binding was stopped by rapidly removing the cells from the binding media by centrifugation (400Ug, 5 min at 4³C). Cells were then washed (2U) using HBSS containing 0.1 mg/ml HSA. Bound receptorligand was trapped on a Whatman GF/C ¢lter that had been pre-soaked in PBS containing 1% polyethyleneimine (Sigma) using a vacuum manifold [43]. The ¢lters were then washed (3U) using HBSS containing
0.1 mg/ml HSA and bound radioactivity determined. Speci¢c binding was obtained after the subtraction of non-speci¢c binding and a¤nity binding constants determined by Scatchard analysis of sPLA2 using the KELL equilibrium binding data analysis software by Biosoft (Milltown, NJ). For competitive binding experiments, cells were incubated with 0^ 100 nM unlabeled sPLA2 in the presence of 1 nM [125 I]sPLA2 . Bound ligand was then determined using the KELL equilibrium binding data analysis software. 2.6. Reverse transcriptase^polymerase chain reaction of sPLA2 receptors RNA was isolated from 5 million cells using 4 M guanidine thiocyanate containing 50 mM sodium citrate (pH 7.0) and 0.5% Sarkosyl. After shearing with an insulin syringe, the lysate was layered over a cushion of 5.7 M CsCl containing 50 mM EDTA. RNA was isolated by centrifugation (80 000 rpm, 2 h at 15³C). Reverse transcriptase (RT) reactions were performed for 40^60 min at 39³C using 100 ng puri¢ed RNA, 50 units of RNAsin, 250 pmol random hexamers, 500 units of murine leukemia virus reverse transcriptase (Gibco BRL) in a ¢nal volume of 50 Wl to synthesize cDNA copies of total RNA isolated from cells. Subsequently, steady-state mRNA levels were quantitated by polymerase chain reaction (PCR) ampli¢cation of cDNA templates obtained from the RT reaction in a ¢nal volume of 50 Wl containing primer mixtures, 2.5 mM dNTP and 1.25 units of Taq polymerase. By analyzing product formation at di¡erent ampli¢cation cycles, it was determined that 32 ampli¢cation cycles was within the linear range of the PCR for human and mouse sPLA2 receptors products. Speci¢c 18^20-mer oligonucleotide primers were designed using the Primer software package (Scienti¢c and Educational Software, State Line, PA) based on sequences published in the GenBank database (Seq. ID: 565645 and Seq. ID: 565653) for human and mouse sPLA2 receptors, respectively [44]. Oligonucleotide primers were synthesized using an Applied Biosystems 391 DNA Synthesizer (Forster City, CA). On the basis of a 50% GC content, the sense and antisense sequences for the human sPLA2 receptor are 5P-GGA TAG CTC TTC AGG ACC AA-3P and 5P-GCT TCA GCT
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TCT CTC CAT GT-3P, respectively. Likewise, the sense and antisense primers for the mouse sPLA2 receptors are 5P-TCT TGC CAC CAC TGT GTT TG-3P and 5P-ATT ATC CAG AGC GAG AGC CT-3P, respectively. The 299- and 360-basepair PCR products for mouse and human sPLA2 receptors, respectively, were separated on 2% agarose gels. The authenticity of the PCR ampli¢cation products obtained using CFTL-15 and THP-1 mRNA that are representative of mouse and human PCR products, respectively, was determined by microsequencing (University of North Carolina DNA Sequencing Facility). 2.7. Statistical analysis All data are expressed as the means þ S.E.M. of separate experiments. Statistics (P-values) were obtained from Student's t-test for paired samples. Notations used on ¢gures and legends are * for P 6 0.05. Kd and Bmax values were obtained from Scatchard analysis of saturation binding data analyzed using the KELL equilibrium binding data analysis program for Macintosh computers (Biosoft, Milltown, NJ) [45,46]. 3. Results 3.1. Kinetics of PLA2 secretion and AA release into supernatant £uids by stimulated BMMC Initial studies examined the secretion of PLA2 activity into supernatant £uids of antigen-stimulated BMMC. Stimulation of BMMC resulted in a rapid increase of PLA2 activity in supernatant £uids (data not shown). Maximum PLA2 activity was found in supernatant £uids within 0.5 min of BMMC stimulation and represented a signi¢cant increase over basal levels (basal activity 166.7 þ 22.8 pmol/h to 758.3 þ 55.6 pmol/h within 0.5 min, n = 4, P 6 0.05). Subsequently, there was a decrease in PLA2 activity in supernatant £uids to V55% of this maximum activity within 5 min. This activity has previously been demonstrated to be acid-stable, DTT-sensitive and partially neutralized by an antibody directed against sPLA2 [31]. We have previously demonstrated that antigen-induced AA release from BMMC is also
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transient and closely mirrors the observed increase in PLA2 activity found in supernatant £uids [31]. 3.2. Hydrolysis of AA from BMMC by di¡erent PLA2 isotypes To further elucidate the link between sPLA2 activity and AA levels in supernatant £uids, BMMC were incubated with di¡erent amounts (0^100 nM) of two group I PLA2 s (Naja naja and porcine pancreas), a group II PLA2 (synovial £uid) or a group III PLA2 (bee venom). Incubation of BMMC with group I PLA2 resulted in a dose-dependent increase in AA in supernatant £uids. Signi¢cant increases in the levels of AA in supernatant £uids were measured with group I PLA2 at concentrations as low as 0.05 nM. There was a 20^40-fold increase in amounts of AA in supernatant £uids with both group I PLA2 s at concentrations ranging from 10 to 100 nM (Fig. 1A,B). In contrast to AA, no statistically signi¢cant change in the mole quantities of other unsaturated fatty acids was measured in supernatant £uids after the addition of the two group I PLA2 isotypes at concentrations 6 50 nM to mast cells. However, porcine pancreatic PLA2 at s 50 nM released signi¢cant amounts of all three fatty acids from BMMC. In contrast to group I PLA2 s, signi¢cant increases in AA levels in supernatant £uids were observed only at the highest concentration of enzyme when BMMC were incubated with a group II PLA2 (Fig. 1C). Similar to group I PLA2 isotypes, group III PLA2 potently mobilized AA from BMMC (Fig. 1D). These data demonstrated that group I PLA2 and group III PLA2 s were V50^100-fold more potent in releasing AA from BMMC than was group II PLA2 . In a cell free assay system utilizing E. coli membranes labeled with [3 H]AA as a substrate, Naja naja PLA2 , porcine pancreas PLA2 , synovial £uid PLA2 and bee venom PLA2 , all at the same concentration hydrolyzed 2.0 þ 0.01, 2.98 þ 0.04, 2.89 þ 0.10 and 3.29 þ 0.03 Wmol/min per mg of arachidonate, respectively. Thus, there was no correlation between the capacity of sPLA2 isotypes to hydrolyze AA from E. coli membranes and the capacity of these same enzymes to release AA from mast cells. Together, these data revealed that low levels of group I PLA2 and group III PLA2 have the capacity to selectively hydrolyze AA from BMMC.
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Fig. 1. Hydrolysis of fatty acids from BMMC by sPLA2 isotypes. BMMC were incubated with various concentrations of group I PLA2 from Naja naja (A) or porcine pancreas (B), recombinant group II PLA2 (synovial £uid) (C) or group III PLA2 from bee venom (D) for 5 min at 37³C. Cell pellets were removed by centrifugation and mole quantities of fatty acids (LA, R; OA, b ; AA, F) released into supernatant £uids by were determined by NICI^GC/MS. These data are the mean þ S.E.M. of more than ¢ve separate experiments. *P 6 0.05.
3.3. Release of fatty acids from di¡erent cell lines by group I PLA2 To determine whether group I PLA2 selectively hydrolyzed AA or other unsaturated fatty acids from cells other than BMMC, three di¡erent cell types were incubated with di¡erent amounts of group I PLA2 (Naja naja) and the mole quantities of a variety of fatty acids were measured by NICI^ GC/MS. Fig. 2A shows that group I PLA2 (0.5^50 nM) induced a marked hydrolysis of AA from CFTL-15 mast cells. As with BMMC, levels of other
unsaturated fatty acids were initially (untreated) high and were increased only by the highest concentration of group I PLA2 (100 nM). These high levels of cellassociated LA and OA are likely derived from the cell culture media and accumulate within the cells because of the low rates of incorporation of these fatty acids into cellular phospholipids [1]. In THP-1 cells, there was a signi¢cant increase in the levels of AA in supernatant £uids at the higher concentrations of group I PLA2 and no changes in the levels of LA and OA at all concentrations tested (Fig. 2B). In contrast to the other cell types, incubation of HL-
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60 cells with group I PLA2 resulted a non-selective release of all unsaturated fatty acids. For example, group I PLA2 concentrations greater than 5 nM induced signi¢cant increases in the levels of LA, OA and AA found in supernatant £uids of HL-60 cells (Fig. 2C). The rank order of quantities of AA released by group I PLA2 from the di¡erent cell types was CFTL-15 s BMMC s THP-1 s HL-60. Taken together, these data revealed that some cells (BMMC, CFTL-15 and THP-1) have the capacity to selectively release AA into their supernatant £uids when they are incubated with low levels of group I PLA2 while another cell line (HL-60) does not selectively release AA under similar conditions. 3.4. In£uence of endogenous sPLA2 isotypes on AA release from mast cells.
Fig. 2. Release of fatty acids from mouse and human cell lines. CFTL-15 (A), THP-1 (B) and HL-60 (C) cells were incubated with di¡erent concentrations of group I PLA2 (Naja naja) for 5 min at 37³C and mole quantities of fatty acids (LA, R; OA, b ; AA, F) released into supernatant £uids determined as by NICI^GC/MS. These data are the mean þ S.E.M. of eight separate experiments *P 6 0.05.
Recent studies suggest that several sPLA2 isotypes including a group V PLA2 are found in mast cells [47,48]. Consequently, it was important to determine whether endogenous sPLA2 isotypes within mast cells selectively release AA when provided exogenously to mast cells. To accomplish this, sPLA2 isotypes were extracted from BMMC and then this sPLA2 was provided to mast cells. Basal levels of LA (213.90 þ 31.30 pmol/5 million mast cells, n = 5) and OA (1006.06 þ 164.35 pmol/5 million cells, n = 5) in supernatant £uids were not altered when BMMC were treated with 40 Wg of BMMC extract. By contrast, there was a signi¢cant increase in the levels of AA from 288.92 þ 59.78 (no treatment) to 650.51 þ 183.50 pmol/5 million BMMC (P 6 0.05, n = 5) after treatment with endogenous sPLA2 . Unsaturated fatty acid levels were also examined in the supernatant £uids of another mast cell line (CFTL15) that did not rapidly metabolize AA but also expressed the sPLA2 receptor. As shown in Fig. 3, addition of acid stable PLA2 isotypes also induced a marked and selective release of AA from CFTL-15 mast cells. In summary, endogenous PLA2 isotypes from BMMC as well as group I and group III PLA2 isotypes when provided exogenously all induce the selective release of AA from certain cell. 3.5. Binding of [125 I]sPLA2 to cells A mechanism that could account for the selective
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Fig. 3. E¡ect of BMMC acid extract on fatty acid release from mast cells. CFTL-15 mast cells were incubated with various concentrations (0^40 Wg) of acid extracts from BMMC for 5 min at 37³C. Cell pellets were removed by centrifugation and mole quantities of fatty acids (LA, R; OA, b ; AA, F) released into supernatant £uids were determined by NICI^GC/MS. These data are the mean þ S.E.M. of ¢ve separate experiments. *P 6 0.05.
release of AA from certain cells is the association of PLA2 isotypes (such as group I) with speci¢c receptors on the surfaces of these cells. To test this hypothesis, saturation binding studies were performed on cells using iodinated group I PLA2 (Naja naja). As shown in Fig. 4, BMMC, CFTL-15 and THP-1 speci¢cally bind group I PLA2 . By contrast, no speci¢c binding was observed when iodinated group I PLA2 was incubated with HL-60 cells. Scatchard analysis resulted in binding constants (Kd s) of
V0.56 þ 0.14 nM (n = 5), 17.16 þ 7.38 nM (n = 3) and 4.13 þ 1.9 nM (n = 3), for BMMC, CFTL-15 and THP-1 cells, respectively (Table 1). The receptor density on cells was of the order CFTL15 s BMMC s THP-1. Hill constants of V1 and similarities in Kd s of 125 I-group I PLA2 binding to all three cell types suggest that only one form of the binding site is involved. Together, these data suggest that certain cell types (BMMC, CFTL-15 and THP1) have sPLA2 receptors on their surface while others such as HL-60 cells do not. In another set of studies, microsomal membranes were prepared from all cell lines and utilized for binding and a¤nity labeling studies. Microsomal fractions from BMMC, CFTL-15 and THP-1 bound 125 I-group I PLA2 and the bound ligand could be competitively reduced by an excess of unlabeled group I PLA2 ligand. Speci¢c binding of [125 I]-group I PLA2 to cell membranes was calculated at 1.91 pg/ Wg, 1.46 pmol/Wg and 0.25 pmol/Wg protein for BMMC, CFTL-15 and THP-1 cells, respectively. In contrast to BMMC, CFTL-15 and THP-1 cells, HL60 cell membranes did not contain speci¢c binding sites for group I PLA2 (data not shown). Together, data from both whole cells and membrane fractions suggest that BMMC, CFTL-15 and THP-1 have membrane-associated sPLA2 receptors while this receptor is absent from HL-60 cells. Binding studies were also performed using iodinated group II PLA2 in the presence or absence of 200-fold unlabeled ligand. No speci¢c binding was observed for all the four cell lines incubated with group II PLA2 (data not shown). Competitive binding studies were also carried out using iodinated group I PLA2 and unlabeled group II or group III PLA2 . Unlabeled group II PLA2 did not in£uence
Table 1 Binding of [125 I]sPLA2 to cells Cell types
Kd (nM)
Bmax (pmol/million cells)
Hill coe¤cient
BMMC CFTL-15 THP-1 HL-60
0.56 þ 0.14 (n = 5) 17.16 þ 7.38 (n = 3) 4.13 þ 1.95 (n = 3) No regressiona
7.98 þ 2.6 200.03 þ 154.8 18.30 þ 12.82 No regressiona
0.90 þ 0.05 0.90 þ 0.08 0.74 þ 0.06 No regressiona
One million cells in 0.4 ml bu¡er were incubated with various concentrations of [125 I]sPLA2 for 2 h at 20³C. Speci¢c binding was determined as described in Section 2. Kd , Bmax and Hill coe¤cients were estimated using the KELL equilibrium binding data analysis program. These data are the means þ S.E.M. of ¢ve (BMMC) or three (CFTL-15, THP-1) separate experiments. a Implies regression failed during data analysis.
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Fig. 4. Speci¢c binding of group I PLA2 to cells. BMMC, CFTL-15, THP-1 and HL-60 (1 million cells in 0.4 ml HBSS containing 1 mg/ml HSA) were incubated with various concentrations of 125 I-group I PLA2 in the absence for total binding or in the presence of 200-fold excess of cold group I PLA2 for non-speci¢c binding determination. Binding was performed at 20³C for 2 h after which, cells were removed from the binding media by centrifugation. Unbound ligand was removed by three washes of the cells followed by ¢ltration as described in Section 2. These data are representative of ¢ve separate experiments. Amount of ligand speci¢cally bound to cells are shown for BMMC (a), CFTL-15 (b), THP-1 (F) and HL-60 (E). Scatchard analysis of these data are shown in the inset.
the binding of group I PLA2 to BMMC. By contrast, group III PLA2 dose-dependently inhibited the binding of the iodinated group I PLA2 to BMMC with an IC50 of V0.4 nM. Taken together, these data suggested that group III PLA2 and group I PLA2 occupy the same binding sites on the surface of BMMC. 3.6. Determination of sPLA2 receptor mRNA in cells In subsequent studies, RT-PCR was utilized to determine the presence of sPLA2 receptor mRNA in BMMC, CFTL-15, THP-1 and HL-60 cells. RTPCR analysis of sPLA2 receptor mRNA levels indicated that BMMC, CFTL-15 and THP-1 cells expressed sPLA2 receptor mRNA. The 299 bp and 360 bp products of the RT-PCR reactions from CFTL-15 mast cells and from THP-1 cells were sub-
sequently sequenced and shown to be homologous to the predicted sequences for mouse and human sPLA2 receptors, respectively. In contrast, no sPLA2 receptor mRNA was detected after forty PCR ampli¢cation cycles of HL-60 cell cDNA (Fig. 5). These data suggest that CFTL-15, BMMC and THP-1 express sPLA2 receptor mRNA while this receptor mRNA is absent from HL-60 cells. 3.7. In£uence of immunologic and non-immunologic activation on group I PLA2 binding to BMMC The above studies revealed that several cell types including BMMC express sPLA2 receptor mRNA and possess a functional receptor on their membranes under resting conditions. Subsequent studies determined whether the capacity to bind the sPLA2
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indicates that sPLA2 (released from the cell after stimulation) has bound to the sPLA2 receptor and this complex has been internalized. 3.8. E¡ect of p-aminophenyl-K-D-mannopyranoside BSA on arachidonic acid release from mast cells
Fig. 5. RT^PCR of sPLA2 receptor mRNA. RNA was extracted from 5 million cells as described in Section 2. The presence or absence of mRNA for mouse or human sPLA2 receptor was determined after RT by PCR ampli¢cation using speci¢c primers for mouse sPLA2 receptor (BMMC and CFTL-15) or human sPLA2 receptor (THP-1 and HL-60). PCR products were isolated using 2% agarose gels and visualized by ethidium bromide staining followed by densitometer scans of the 299 bp and 360 bp products for mouse and human sPLA2 receptor mRNA, respectively. These data are representative of four separate experiments.
receptor was in£uenced by antigen or ionophore stimulation of BMMC. In control cells, speci¢c binding of 125 I-group I PLA2 was 8.31 þ 4.57 (n = 3) fmol/ million cells. Pre-stimulation of BMMC with antigen or with ionophore A23187 resulted in a 84.92 þ 9.22% and a 64.17 þ 3.46% decrease in [125 I]group I PLA2 binding, respectively. These data suggest that sPLA2 receptors are in£uenced by stimulation of BMMC. Previous studies have demonstrated that the sPLA2 receptor is internalized after receptor occupancy and thus the reduction in exogenous sPLA2 binding observed after BMMC stimulation
Nicolas and colleagues [49] have recently shown that p-aminophenyl-K-D-mannopyranoside BSA binds to the sPLA2 receptor in the same carbohydrate recognition domains as sPLA2 . Thus, p-aminophenyl-K-D-mannopyranoside BSA was added to mast cells to determine whether receptor occupancy alone (without PLA2 activity) could elicit the release of AA from mast cells. Fig. 6 shows that p-aminophenyl-K-D-mannopyranoside BSA induces the selective release of AA from BMMC. These data suggest that receptor occupancy alone is enough to initiate the selective release of AA from mast cells. To show that p-aminophenyl-K-D-mannopyranoside BSA induced the selective release of AA from mast cells as a result of sPLA2 binding, we examined its e¡ects on a cell line (HL-60) that does not express the sPLA2 receptor. Unstimulated levels of LA, OA and AA in supernatant £uids of HL-60 cells were
Fig. 6. In£uence of p-aminophenyl-K-D-mannopyranoside BSA on fatty acid release from BMMC. BMMC were incubated without (white bars) or with 50 WM p-aminophenyl-K-D-mannopyranoside BSA (black bars) for 5 min at 37³C. Cell pellets were removed by centrifugation and mole quantities of fatty acids (LA, OA, AA) released into supernatant £uids were determined by NICI^GC/MS. These data are the mean þ S.E.M. of three separate experiments. *P 6 0.05.
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140.11 þ 63.31, 1809.26 þ 622.51, 53.95 þ 12.32 pmol/ 5 million cells (n = 3, performed in duplicate), respectively. Levels of these unsaturated fatty acids were not altered in supernatant £uids of HL-60 cells incubated with p-aminophenyl-K-D-mannopyranoside BSA. These data suggest that the association of the sPLA2 receptor ligand with a cell surface receptor is a critical requirement for the selective release of AA from cells. 4. Discussion The present study demonstrates that secretory PLA2 isotypes bind to surface receptors on certain cells and this event facilitates the selective release of AA from these cells. The following six lines of evidence support this hypothesis. (1) Following antigen activation, mast cells rapidly release sPLA2 and this enzyme then reassociates with the cell. (2) Exogenously-provided sPLA2 isotypes (at concentrations 6 1 nM) or acid extracts from sPLA2 -containing cells selectively release AA from certain but not all cells. (3) Cells that selectively and potently release AA in response to sPLA2 are those that have the capacity to speci¢cally bind sPLA2 . (4) Those cells that selectively release AA in response to sPLA2 and speci¢cally bind sPLA2 contain mRNA for a sPLA2 receptor. (5) Mast cell activation by antigen or ionophore causes a reduction in the capacity of exogenously-provided group I PLA2 to bind to the mast cell surface, suggesting that the PLA2 receptor is either occupied or internalized after cell activation. (6) Another known ligand of the sPLA2 receptor, p-aminophenyl-K-D-mannopyranoside BSA, also induces the selective release of AA from sPLA2 receptor-expressing cells but not from cells that do not express the sPLA2 receptor. Initial studies in the present work demonstrated that an acid-stable, DTT-sensitive PLA2 is released from mast cells within 30 s of antigen activation. The levels of this activity then declines as a function of time, suggesting that this PLA2 activity reassociates with the cell. The increase and subsequent decline of sPLA2 on the outside of mast cells is closely mirrored by the selective release of AA (and not other polyunsaturated fatty acids) from mast cells. The concurrent appearance of this sPLA2 and AA in superna-
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tant £uids raised key questions as to the relationship between these two events. As mentioned in Section 1, previous studies have demonstrated that sPLA2 hydrolyzes several fatty acids including oleic acid, linoleic acid and AA from several membrane phospholipid systems [28^ 30]. Therefore, the selective release of AA from BMMC was initially inconsistent with the concept that sPLA2 is responsible for the mobilization of AA from intact cells. To better understand the selective hydrolysis of AA from BMMC, mast cells were incubated with four di¡erent isotypes that are representative of group I, a group II and a group III PLA2 . Addition of two group I sPLA2 s or a group III sPLA2 caused a selective release AA from BMMC at very low concentrations ( 6 1 nM). Human synovial group II PLA2 also selectively released AA from BMMC but at 100-fold higher concentrations. These data suggested that there were molecular events which occurred at the cell surface of mast cells that facilitated the selective hydrolysis of AA by sPLA2 from these cells. In the last 8 years, di¡erent subtypes of membrane receptors for sPLA2 have been identi¢ed in a variety of cell types by determining their a¤nities for various types of sPLA2 s [43,44,49^55]. Work by Arita and colleagues described the existence of a speci¢c receptor family termed PLA2 -I receptor that is abundant in brain and several other tissues and has high a¤nity for the binding of pancreatic-type PLA2 [50]. Lambeau and colleagues have suggested that sPLA2 receptors can be divided into two classes termed Ntype receptors and M-type receptors [56]. Isotypes of M-type sPLA2 receptors from di¡erent mammalian species have been puri¢ed, cloned and expressed [51,57]. Despite the fact that these receptors subtypes show somewhat di¡erent PLA2 binding pro¢les, their amino acid sequences are very similar with homology as high as 82% [55]. Although occupancy of the sPLA2 receptor has been suggested to enhance cell proliferation and induce chemokinetic cell migration, much remains to be learned about the physiological rami¢cations of this molecular event. Based on the selective hydrolysis of AA from mast cells at very low concentrations of sPLA2 , we postulated that the selective hydrolysis of AA was modulated through cellular receptors. Lambeau and colleagues report that a major di¡erence between N- and M-
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type receptors is their capacity to bind bee venom sPLA2 [56]. For example, the N-type receptor associates very tightly with both pancreatic sPLA2 and bee venom sPLA2 while the rabbit muscle M-type receptor tightly binds pancreatic and human synovial £uid sPLA2 but does not associate with bee venom sPLA2 . Our ¢nding showed that bee venom, at extremely low concentrations, hydrolyzed AA from BMMC suggesting that this mast cell expresses a protein that is similar if not identical to N-type receptor. A major question that arose from these studies was whether the selective release of AA by certain sPLA2 isotypes was a characteristic of the BMMC or was shared by other cell types. To examine this question, several cell types were incubated with exogenous group I sPLA2 . These studies revealed that AA was selectively released from some cells but not others. Cells that selectively released AA responded to a much lower concentration of group I sPLA2 than HL-60 cells which did not selectively release AA. These ¢ndings raised the critical question of whether the cells that selectively release AA are the cells which contained the sPLA2 receptor. This hypothesis was tested by searching for evidence of the expression of the sPLA2 receptor and mRNA utilizing binding and RT^PCR experiments, respectively. These experiments revealed that cells which selectively release AA also possess binding sites on their cell surfaces for group I PLA2 and express mRNA for an sPLA2 receptor. Additional experiments demonstrated that binding sites are lost from the surface of BMMC after challenge with ionophore or antigen. Studies from several laboratories suggest that mast cells have multiple sPLA2 isotypes that are secreted after stimulation [40,47,48]. For example, we have shown that two major acid-stable PLA2 are released from antigenstimulated BMMC [40]. Therefore, decreased binding of exogenous sPLA2 to mast cells may be due to the fact that sPLA2 receptors are occupied by endogenous ligands. Alternatively, sPLA2 receptors may be internalized during mast cell activation. In either case, these data support the contention that sPLA2 receptors are utilized during cell stimulation. The major ¢nding of this study is that receptor occupancy translates into the selective release of AA by an enzyme (sPLA2 ) that normally shows little
preference for unsaturated fatty acids. There are at least two potential explanations for how this shift in fatty acid selectivity might occur. One explanation is that the receptor itself is located in a part of the membrane that is highly enriched in AA. This hypothesis is supported by experiments in recent manuscripts which demonstrate that most of the AA released from mast cells after the addition of sPLA2 is hydrolyzed from phosphatidylethanolamine [31]. The fact that phosphatidylethanolamine is enriched in AA and is hydrolyzed during sPLA2 receptor occupancy suggest that the receptor may reside in a region of the plasma membrane highly enriched in phosphatidylethanolamine. An alternative hypothesis is that the selective release of AA by receptor occupancy does not involve the hydrolytic activity of sPLA2 at all, but involves an indirect mechanism by which sPLA2 simply acts as an agonist that stimulates signal transduction events leading to the selective release of AA. This idea is supported by several studies which suggest that sPLA2 which has been rendered catalytically inactive by inhibitors or mutations can still stimulate cellular and physiological responses [53,58]. Importantly, we provide direct evidence for this hypothesis in this paper by showing that another catalytically inactive ligand of the sPLA2 receptor, p-aminophenyl-K-D-mannopyranoside BSA, induces the selective release of AA from mast cells and not from HL-60 cells. Our preliminary studies (data not shown) suggest that sPLA2 occupancy of its receptor leads to the activation of cPLA2 and subsequent release of AA. In conclusion, it may no longer be accurate to assume that sPLA2 does not participate in AA metabolism based on the capacities of these enzymes to hydrolyze fatty acids utilizing chemically-pure substrates in cell free systems. The current study suggest that other molecular events that occur in whole cells, such a receptor occupancy, may markedly enhance the capacity of sPLA2 to selectively release AA (by either direct or indirect mechanisms). Recent studies indicate that sPLA2 released from mast cells can participate in transcellular prostaglandin production in ¢broblasts [39]. The molecular events described in the present study may facilitate the role of sPLA2 in AA metabolism during in£ammatory reactions by involving not only cells that release sPLA2 (such
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as mast cells) but also by neighboring cells which contain sPLA2 receptors. Acknowledgements We are grateful for the expert technical assistance of Dennis Swan. This work was supported in part by NIH grants AI 24985, AI 24985 S1 and P01-HL50395. M.S. was supported by a Centennial Fellowship from the Medical Research Council of Canada.
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Mechanisms that account for the selective release of arachidonic acid from intact cells by secretory phospholipase A2 Alfred N. Fonteh
a;
*, James M. Samet c , Marc Surette Floyd H. Chilton a;b
1;a
, William Reed c ,
a b
Department of Internal Medicine (Division of Pulmonary and Critical Care Medicine), Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA c Center for Environmental Medicine and Lung Biology, CB #7310, Mason Farm Road, University of North Carolina, Chapel Hill, NC 27599-7310, USA Received 2 June 1998; accepted 9 June 1998
Abstract The current study examined mechanisms that account for the selective release of arachidonic acid (AA) from cells by secretory phospholipase A2 (sPLA2 ). Initial studies demonstrated that low concentrations of group I and group III PLA2 isotypes and an sPLA2 -enriched extract from bone marrow-derived mast cells (BMMC) selectively released AA from mast cells. Much higher concentrations of group II PLA2 were required to release comparable quantities of AA. Group I PLA2 also selectively released AA from another mast cell line (CFTL-15) and a monocytic cell line (THP-1). In contrast, high concentrations of group I PLA2 were required to release fatty acids from a promyelocytic cell line (HL-60) and this release was not selective for AA. Binding studies revealed that cell types (BMMC, CFTL-15 and THP-1) which selectively released AA also had the capacity to specifically bind group I PLA2 . However, group II PLA2 , which did not selectively release AA from cells, also did not specifically bind to these same cell types. Additional studies revealed that sPLA2 binding to the mast cell receptor was attenuated after stimulation with antigen or ionophore A23187. Reverse transcriptase^polymerase chain reaction analyses indicated the presence of mRNA for the sPLA2 receptor in BMMC, CFTL-15 and THP-1 and the absence of this mRNA in HL-60. Final studies demonstrated that p-aminophenyl-K-D-mannopyranoside BSA, a known ligand of the sPLA2 receptor, also selectively released AA from mast cells but not from HL-60 cells. These experiments indicated that receptor occupancy alone (without PLA2 activity) is sufficient to induce the release of AA from mast cells. Together, these data reveal that specific isotypes of sPLA2 have the capacity to selectively release AA from certain cells by their capacity to bind to sPLA2 receptors on the cell surface. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Arachidonic acid; Mast cell; Secretory phospholipase A2 ; Secretory phospholipase A2 receptor
Abbreviations: AA, arachidonic acid; OA, oleic acid; LA, linoleic acid; SA, stearic acid; sPLA2 , secretory phospholipase A2 ; cPLA2 , cytosolic phospholipase A2 ; Ag, antigen; NICI^GC/MS, negative ion chemical ionization^gas chromatography/mass spectrometry; [2 H8 ], octadeuterated; [2 H3 ], trideuterated; BMMC, bone marrow-derived mast cells; FCS, fetal calf serum; BSA, bovine serum albumin; RT^ PCR, reverse transcriptase^polymerase chain reaction * Corresponding author. Fax: +1-336-716-7277; E-mail: [email protected] 1 Present address: Centre Hospitalier de Quebec, Centre de Recherche en Rhumatologie et Immunologie, T 1-49 2705 Laurier, St Foy, Quebec G1V 4G2, Canada. 0005-2760 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 5 - 2 7 6 0 ( 9 8 ) 0 0 0 7 9 - 4
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1. Introduction Arachidonic acid (AA), a polyunsaturated fatty acid located at the sn-2 position of glycerophospholipids, serves as the precursor for several important lipid mediators (termed eicosanoids) of in£ammation [1]. Before AA is converted to eicosanoids, it must be hydrolyzed from phospholipids by a family of enzymes collectively known as phospholipase A2 (PLA2 ). PLA2 activities that may participate in this event include cytosolic, high-molecular-mass PLA2 (cPLA2 , 85^110 kDa) [2^9], several isotypes of secretory, low-molecular-mass PLA2 (s) (sPLA2 , V14 kDa) [10^12] and calcium-independent PLA2 activities [13^15]. The existence of multiple PLA2 s in cells and tissues has generated considerable research e¡ort to identify the PLA2 that is responsible for AA release and eicosanoid biosynthesis during in£ammation. Although the sPLA2 family of enzymes has been studied for the past six decades, there has not been a complete elucidation of their physiologic or pathophysiologic roles in mammals. Potential biological activities for sPLA2 include antibacterial e¡ects, key components in digestion, enzymes that generate arachidonic acid utilized for eicosanoid generation, potential regulators of severe illnesses such as sepsis, pancreatitis and organ injury and pro-in£ammatory components in diseases such as rheumatoid arthritis and asthma [16^27]. With regard to AA mobilization for the synthesis of bioactive eicosanoids, sPLA2 s have received only modest attention primarily due to the fact that these enzymes, when assayed in cell-free systems utilizing chemically pure substrates, show no selectivity for the hydrolysis of phospholipids containing AA when compared to phospholipids containing other more abundant fatty acids such as oleic acid and linoleic acid [28^30]. However, we have recently demonstrated that partially puri¢ed sPLA2 from human lung or sPLA2 isolated from snake venom can selectively release AA from human bronchial epithelial cells and from mast cells, respectively [31,32]. These ¢ndings suggest that arachidonate-containing phospholipids may be presented to sPLA2 in intact cells in a manner that facilitates the selective release of AA. Alternatively, binding of sPLA2 to putative receptors on cell surfaces may induce the selective release of AA by other PLA2
enzymes [33,34]. In addition to the aforementioned results, several other studies utilizing inhibitors, antisense oligonucleotides, cytokines or speci¢c antibodies have implicated sPLA2 as the rate-limiting step in the mobilization of AA leading to eicosanoid generation in certain cells [35^40]. All these ¢ndings have raised fundamental questions as to how sPLA2 causes AA release with subsequent eicosanoid generation in mammalian cells. The major objective of the current study was to examine the molecular events that facilitate the selective mobilization of AA by sPLA2 isotypes from membranes of intact mammalian cells. 2. Materials and methods 2.1. Materials Octadeuterated [2 H8 ]AA and trideuterated [ H3 ]SA were purchased from Cayman (Ann Arbor, MI). L-K-1-Palmitoyl-2-[1-14 C]arachidonoyl-sn-glycero-3-phosphocholine was purchased from American Radiolabel (St. Louis, MO). 125 I as sodium salt (NaI) was purchased from Amersham (Arlington Heights, IL). Essentially fatty acid free human serum albumin (HSA), Naja naja PLA2 , bee venom PLA2 , porcine pancreatic PLA2 , essential and non-essential amino acids, clone SPE-7 monoclonal anti-dinitrophenyl (IgE anti-DNP) a¤nity puri¢ed antibody, heat-inactivated fetal calf serum (FCS), ionophore A23187 and p-aminophenyl-K-D-mannopyranoside bovine serum albumin (BSA), were purchased from Sigma (St. Louis, MO). Recombinant human synovial £uid PLA2 was kindly supplied by Dr. L. Marshall (SmithKline Beecham, King of Prussia, PA). Acid extracts of BMMC highly enriched with PLA2 (0.18 þ 0.04 to 1.25 þ 0.24 nmol/mg/min) was obtained as previously described [40]. Iodobeads iodination reagents, penta£uorobenzyl bromide (20% in acetonitrile) and diisopropylethylamine (20% in acetonitrile), were purchased from Pierce (Rockford, IL). RPMI 1640 cell culture media was purchased from Gibco (Grand Island, NY). Hanks' balanced salt solution (HBSS) with or without calcium was purchased from Specialty Media (Lavallete, NJ). [3 H]AA-labeled E. coli membranes were purchased from New England Nuclear (Boston, MA). The un2
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saturated fatty acid composition of the E. coli membrane (determined by NICI-GC/MS and used to calculate PLA2 activities) was 7.8 þ 3.7 pmol/nmol Pi , 17.3 þ 7.9 pmol/nmol Pi and 4.7 þ 3.6 pmol/nmol Pi (n = 3) for LA, OA and AA, respectively. HPLC grade organic solvents were purchased from Fisher Scienti¢c (Norcross, GA). 2.2. Cell culture and activation Mouse bone marrow mast cells (BMMC) were obtained from CBA/J mice (Jackson, Bar Harbor, ME) and grown in RPMI 1640 culture medium (Gibco) supplemented with 10% (v/v) fetal calf serum (FCS), 50 WM 2-mercaptoethanol, 1% essential amino acids, 1% non-essential amino acids, 2 mM L-glutamine, 5 Wg/ml gentamycin and 1% (v/v) penicillin/streptomycin, for 4 weeks. The culture medium was supplemented twice a week with 50% WEHI supernatant £uid as a source of IL-3. The CFTL-15 mast cell line was similarly maintained in RPMI supplemented with 15% FCS, 1% (v/v) penicillin/streptomycin and 25% WEHI supernatant £uids. The human monocytic leukemia cells (THP-1) and the human promyelocytic leukemic cell line (HL-60) were maintained in RPMI supplemented with 10% FCS, 5 Wg/ml gentamycin and 1% (v/v) penicillin/streptomycin. In experiments designed to measure PLA2 secretion and AA release, BMMC were harvested after 3 weeks in culture and placed in newly-prepared culture media containing 50% WEHI for 24 h. During this period, BMMC were sensitized with 0.5 Wg/ml mouse IgE anti-DNP. Subsequently, cells were removed from culture and placed in HBSS containing 1 mM calcium, 0.2 mg/ml gelatin and 0.02 mg/ml HSA (HBSS/gelatin bu¡er). Cell viability ( s 95%) was determined by Trypan Blue exclusion. BMMC were then stimulated with DNP (2 Wg/ml), p-aminophenyl-K-D-mannopyranoside BSA (50 WM) or with ionophore A23187 (1 WM) for 5 min at 37³C. For kinetic studies, BMMC were incubated with antigen for di¡erent periods of time and stimulation was stopped by adding 0.5 ml ice-cold HBSS/gelatin buffer to each tube. Cells were then removed from supernatant £uids by centrifugation (400Ug, 5 min at 4³C). A portion of the supernatant £uid was utilized to measure PLA2 activity as described below. Four volumes of ethanol and 100 ng [2 H8 ]AA and
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100 ng [2 H3 ]SA were added to the remainder of the supernatant £uids as internal standards for the determination of mole quantities of fatty acids by negative ion chemical ionization^gas chromatography/mass spectroscopy (NICI^GC/MS). In experiments where fatty acid release from cells was determined after the addition of exogenous sPLA2 , cells were removed from growth media and placed in HBSS/gelatin bu¡er. After incubation at 37³C for 5 min, di¡erent concentrations of sPLA2 (0^100 nM or 0^40 Wg BMMC acid extract) were added to the cells and the incubation allowed to proceed for 5 min. The amount of sPLA2 added to these cells did not compromise cell viability. Cells were then removed from supernatant £uids by centrifugation (400Ug, 5 min at 4³C). Mole quantities of fatty acids were determined in supernatant £uids by NICI^GC/MS as described below. 2.3. Determination of PLA2 activity in supernatant £uids Portions of supernatant £uids (0.5 ml) were utilized for PLA2 activity determination. The PLA2 reaction was initiated by the addition of 0.02 WCi (9.7 nmol) [3 H]AA-labeled E. coli membranes (New England Nuclear) and CaCl2 (10 mM ¢nal concentration). After incubation (90 min at 37³C) in a water bath, the reaction was stopped by extracting lipids by the method of Bligh and Dyer [41]. Free fatty acids were isolated from phospholipids by TLC on silica gel G developed in hexane/ethyl ether/formic acid (90:60:6 v/v). The radioactivity in lipids was located using a radiochromatogram imaging system (Bioscan, Washington, DC). Free AA and phospholipids were isolated using TLC zonal scraping, and the quantity of radioactivity was determined by liquid scintillation counting. PLA2 activity was calculated and expressed as pmol AA released per hour. 2.4. Quantitation of free fatty acids in supernatant £uids After the addition of deuterated fatty acids as internal standards to supernatant £uids, solvents were removed from extracts using a stream of nitrogen. Fatty acids were then converted to penta£uorobenzyl esters and the mole quantities of free fatty acids de-
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termined by NICI^GC/MS using a Hewlett Packard model 5989 GC/MS instrument [42]. Carboxylate anions (m/z) were monitored at 279, 281, 286, 303 and 311 for LA, OA, [2 H3 ]SA, AA and [2 H8 ]AA, respectively, in the single ion monitoring mode. 2.5. Iodination of sPLA2 and binding assays The purity of PLA2 from Naja naja was veri¢ed by reverse phase HPLC using a diphenyl column [40]. PLA2 activity was determined using [3 H]AA-labeled E. coli membranes as described above. In addition to activity determination, eluted proteins were monitored at 280 nm and peak purity determined spectrophotometrically using a UV diode array detector. Iodination of group I PLA2 and group II PLA2 was performed using an Iodo-Bead iodination reagent kit (Pierce). Brie£y, 300 WCi Na125 I was added to V20 Wg PLA2 in 0.1 ml, 0.5 M NaH2 PO4 (pH 7.5) bu¡er. Iodination was initiated by adding an Iodo-Bead to the reaction mixture. The reaction was terminated after 2 min at room temperature by removing the reaction mixture from the bead. After the addition of 2 mg BSA in 20 Wl PBS as a protein carrier, [125 I]PLA2 was isolated using a Pharmacia PD-10 Sephadex G-25 column. Proteins were eluted from the column with 0.1 M NaH2 PO4 . Radioactivity in fractions containing [125 I]PLA2 was determined by gamma counting, and these fractions were stored at 4³C until required for binding assays. Binding assays were performed using 1 million cells in 0.4 ml HBSS containing 1 mg/ml HSA. Saturation binding was determined using various concentrations of [125 I]sPLA2 (0.05^4 nM). Non-speci¢c binding was determined with labeled ligands in the presence of 200-fold excess unlabeled PLA2 . For studies designed to examine speci¢c binding, cells were incubated with 2 nM iodinated PLA2 without or with 400 nM of unlabeled PLA2 in a ¢nal volume of 0.4 ml. Binding was performed at 20³C for 2 h. Binding was stopped by rapidly removing the cells from the binding media by centrifugation (400Ug, 5 min at 4³C). Cells were then washed (2U) using HBSS containing 0.1 mg/ml HSA. Bound receptorligand was trapped on a Whatman GF/C ¢lter that had been pre-soaked in PBS containing 1% polyethyleneimine (Sigma) using a vacuum manifold [43]. The ¢lters were then washed (3U) using HBSS containing
0.1 mg/ml HSA and bound radioactivity determined. Speci¢c binding was obtained after the subtraction of non-speci¢c binding and a¤nity binding constants determined by Scatchard analysis of sPLA2 using the KELL equilibrium binding data analysis software by Biosoft (Milltown, NJ). For competitive binding experiments, cells were incubated with 0^ 100 nM unlabeled sPLA2 in the presence of 1 nM [125 I]sPLA2 . Bound ligand was then determined using the KELL equilibrium binding data analysis software. 2.6. Reverse transcriptase^polymerase chain reaction of sPLA2 receptors RNA was isolated from 5 million cells using 4 M guanidine thiocyanate containing 50 mM sodium citrate (pH 7.0) and 0.5% Sarkosyl. After shearing with an insulin syringe, the lysate was layered over a cushion of 5.7 M CsCl containing 50 mM EDTA. RNA was isolated by centrifugation (80 000 rpm, 2 h at 15³C). Reverse transcriptase (RT) reactions were performed for 40^60 min at 39³C using 100 ng puri¢ed RNA, 50 units of RNAsin, 250 pmol random hexamers, 500 units of murine leukemia virus reverse transcriptase (Gibco BRL) in a ¢nal volume of 50 Wl to synthesize cDNA copies of total RNA isolated from cells. Subsequently, steady-state mRNA levels were quantitated by polymerase chain reaction (PCR) ampli¢cation of cDNA templates obtained from the RT reaction in a ¢nal volume of 50 Wl containing primer mixtures, 2.5 mM dNTP and 1.25 units of Taq polymerase. By analyzing product formation at di¡erent ampli¢cation cycles, it was determined that 32 ampli¢cation cycles was within the linear range of the PCR for human and mouse sPLA2 receptors products. Speci¢c 18^20-mer oligonucleotide primers were designed using the Primer software package (Scienti¢c and Educational Software, State Line, PA) based on sequences published in the GenBank database (Seq. ID: 565645 and Seq. ID: 565653) for human and mouse sPLA2 receptors, respectively [44]. Oligonucleotide primers were synthesized using an Applied Biosystems 391 DNA Synthesizer (Forster City, CA). On the basis of a 50% GC content, the sense and antisense sequences for the human sPLA2 receptor are 5P-GGA TAG CTC TTC AGG ACC AA-3P and 5P-GCT TCA GCT
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TCT CTC CAT GT-3P, respectively. Likewise, the sense and antisense primers for the mouse sPLA2 receptors are 5P-TCT TGC CAC CAC TGT GTT TG-3P and 5P-ATT ATC CAG AGC GAG AGC CT-3P, respectively. The 299- and 360-basepair PCR products for mouse and human sPLA2 receptors, respectively, were separated on 2% agarose gels. The authenticity of the PCR ampli¢cation products obtained using CFTL-15 and THP-1 mRNA that are representative of mouse and human PCR products, respectively, was determined by microsequencing (University of North Carolina DNA Sequencing Facility). 2.7. Statistical analysis All data are expressed as the means þ S.E.M. of separate experiments. Statistics (P-values) were obtained from Student's t-test for paired samples. Notations used on ¢gures and legends are * for P 6 0.05. Kd and Bmax values were obtained from Scatchard analysis of saturation binding data analyzed using the KELL equilibrium binding data analysis program for Macintosh computers (Biosoft, Milltown, NJ) [45,46]. 3. Results 3.1. Kinetics of PLA2 secretion and AA release into supernatant £uids by stimulated BMMC Initial studies examined the secretion of PLA2 activity into supernatant £uids of antigen-stimulated BMMC. Stimulation of BMMC resulted in a rapid increase of PLA2 activity in supernatant £uids (data not shown). Maximum PLA2 activity was found in supernatant £uids within 0.5 min of BMMC stimulation and represented a signi¢cant increase over basal levels (basal activity 166.7 þ 22.8 pmol/h to 758.3 þ 55.6 pmol/h within 0.5 min, n = 4, P 6 0.05). Subsequently, there was a decrease in PLA2 activity in supernatant £uids to V55% of this maximum activity within 5 min. This activity has previously been demonstrated to be acid-stable, DTT-sensitive and partially neutralized by an antibody directed against sPLA2 [31]. We have previously demonstrated that antigen-induced AA release from BMMC is also
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transient and closely mirrors the observed increase in PLA2 activity found in supernatant £uids [31]. 3.2. Hydrolysis of AA from BMMC by di¡erent PLA2 isotypes To further elucidate the link between sPLA2 activity and AA levels in supernatant £uids, BMMC were incubated with di¡erent amounts (0^100 nM) of two group I PLA2 s (Naja naja and porcine pancreas), a group II PLA2 (synovial £uid) or a group III PLA2 (bee venom). Incubation of BMMC with group I PLA2 resulted in a dose-dependent increase in AA in supernatant £uids. Signi¢cant increases in the levels of AA in supernatant £uids were measured with group I PLA2 at concentrations as low as 0.05 nM. There was a 20^40-fold increase in amounts of AA in supernatant £uids with both group I PLA2 s at concentrations ranging from 10 to 100 nM (Fig. 1A,B). In contrast to AA, no statistically signi¢cant change in the mole quantities of other unsaturated fatty acids was measured in supernatant £uids after the addition of the two group I PLA2 isotypes at concentrations 6 50 nM to mast cells. However, porcine pancreatic PLA2 at s 50 nM released signi¢cant amounts of all three fatty acids from BMMC. In contrast to group I PLA2 s, signi¢cant increases in AA levels in supernatant £uids were observed only at the highest concentration of enzyme when BMMC were incubated with a group II PLA2 (Fig. 1C). Similar to group I PLA2 isotypes, group III PLA2 potently mobilized AA from BMMC (Fig. 1D). These data demonstrated that group I PLA2 and group III PLA2 s were V50^100-fold more potent in releasing AA from BMMC than was group II PLA2 . In a cell free assay system utilizing E. coli membranes labeled with [3 H]AA as a substrate, Naja naja PLA2 , porcine pancreas PLA2 , synovial £uid PLA2 and bee venom PLA2 , all at the same concentration hydrolyzed 2.0 þ 0.01, 2.98 þ 0.04, 2.89 þ 0.10 and 3.29 þ 0.03 Wmol/min per mg of arachidonate, respectively. Thus, there was no correlation between the capacity of sPLA2 isotypes to hydrolyze AA from E. coli membranes and the capacity of these same enzymes to release AA from mast cells. Together, these data revealed that low levels of group I PLA2 and group III PLA2 have the capacity to selectively hydrolyze AA from BMMC.
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Fig. 1. Hydrolysis of fatty acids from BMMC by sPLA2 isotypes. BMMC were incubated with various concentrations of group I PLA2 from Naja naja (A) or porcine pancreas (B), recombinant group II PLA2 (synovial £uid) (C) or group III PLA2 from bee venom (D) for 5 min at 37³C. Cell pellets were removed by centrifugation and mole quantities of fatty acids (LA, R; OA, b ; AA, F) released into supernatant £uids by were determined by NICI^GC/MS. These data are the mean þ S.E.M. of more than ¢ve separate experiments. *P 6 0.05.
3.3. Release of fatty acids from di¡erent cell lines by group I PLA2 To determine whether group I PLA2 selectively hydrolyzed AA or other unsaturated fatty acids from cells other than BMMC, three di¡erent cell types were incubated with di¡erent amounts of group I PLA2 (Naja naja) and the mole quantities of a variety of fatty acids were measured by NICI^ GC/MS. Fig. 2A shows that group I PLA2 (0.5^50 nM) induced a marked hydrolysis of AA from CFTL-15 mast cells. As with BMMC, levels of other
unsaturated fatty acids were initially (untreated) high and were increased only by the highest concentration of group I PLA2 (100 nM). These high levels of cellassociated LA and OA are likely derived from the cell culture media and accumulate within the cells because of the low rates of incorporation of these fatty acids into cellular phospholipids [1]. In THP-1 cells, there was a signi¢cant increase in the levels of AA in supernatant £uids at the higher concentrations of group I PLA2 and no changes in the levels of LA and OA at all concentrations tested (Fig. 2B). In contrast to the other cell types, incubation of HL-
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60 cells with group I PLA2 resulted a non-selective release of all unsaturated fatty acids. For example, group I PLA2 concentrations greater than 5 nM induced signi¢cant increases in the levels of LA, OA and AA found in supernatant £uids of HL-60 cells (Fig. 2C). The rank order of quantities of AA released by group I PLA2 from the di¡erent cell types was CFTL-15 s BMMC s THP-1 s HL-60. Taken together, these data revealed that some cells (BMMC, CFTL-15 and THP-1) have the capacity to selectively release AA into their supernatant £uids when they are incubated with low levels of group I PLA2 while another cell line (HL-60) does not selectively release AA under similar conditions. 3.4. In£uence of endogenous sPLA2 isotypes on AA release from mast cells.
Fig. 2. Release of fatty acids from mouse and human cell lines. CFTL-15 (A), THP-1 (B) and HL-60 (C) cells were incubated with di¡erent concentrations of group I PLA2 (Naja naja) for 5 min at 37³C and mole quantities of fatty acids (LA, R; OA, b ; AA, F) released into supernatant £uids determined as by NICI^GC/MS. These data are the mean þ S.E.M. of eight separate experiments *P 6 0.05.
Recent studies suggest that several sPLA2 isotypes including a group V PLA2 are found in mast cells [47,48]. Consequently, it was important to determine whether endogenous sPLA2 isotypes within mast cells selectively release AA when provided exogenously to mast cells. To accomplish this, sPLA2 isotypes were extracted from BMMC and then this sPLA2 was provided to mast cells. Basal levels of LA (213.90 þ 31.30 pmol/5 million mast cells, n = 5) and OA (1006.06 þ 164.35 pmol/5 million cells, n = 5) in supernatant £uids were not altered when BMMC were treated with 40 Wg of BMMC extract. By contrast, there was a signi¢cant increase in the levels of AA from 288.92 þ 59.78 (no treatment) to 650.51 þ 183.50 pmol/5 million BMMC (P 6 0.05, n = 5) after treatment with endogenous sPLA2 . Unsaturated fatty acid levels were also examined in the supernatant £uids of another mast cell line (CFTL15) that did not rapidly metabolize AA but also expressed the sPLA2 receptor. As shown in Fig. 3, addition of acid stable PLA2 isotypes also induced a marked and selective release of AA from CFTL-15 mast cells. In summary, endogenous PLA2 isotypes from BMMC as well as group I and group III PLA2 isotypes when provided exogenously all induce the selective release of AA from certain cell. 3.5. Binding of [125 I]sPLA2 to cells A mechanism that could account for the selective
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Fig. 3. E¡ect of BMMC acid extract on fatty acid release from mast cells. CFTL-15 mast cells were incubated with various concentrations (0^40 Wg) of acid extracts from BMMC for 5 min at 37³C. Cell pellets were removed by centrifugation and mole quantities of fatty acids (LA, R; OA, b ; AA, F) released into supernatant £uids were determined by NICI^GC/MS. These data are the mean þ S.E.M. of ¢ve separate experiments. *P 6 0.05.
release of AA from certain cells is the association of PLA2 isotypes (such as group I) with speci¢c receptors on the surfaces of these cells. To test this hypothesis, saturation binding studies were performed on cells using iodinated group I PLA2 (Naja naja). As shown in Fig. 4, BMMC, CFTL-15 and THP-1 speci¢cally bind group I PLA2 . By contrast, no speci¢c binding was observed when iodinated group I PLA2 was incubated with HL-60 cells. Scatchard analysis resulted in binding constants (Kd s) of
V0.56 þ 0.14 nM (n = 5), 17.16 þ 7.38 nM (n = 3) and 4.13 þ 1.9 nM (n = 3), for BMMC, CFTL-15 and THP-1 cells, respectively (Table 1). The receptor density on cells was of the order CFTL15 s BMMC s THP-1. Hill constants of V1 and similarities in Kd s of 125 I-group I PLA2 binding to all three cell types suggest that only one form of the binding site is involved. Together, these data suggest that certain cell types (BMMC, CFTL-15 and THP1) have sPLA2 receptors on their surface while others such as HL-60 cells do not. In another set of studies, microsomal membranes were prepared from all cell lines and utilized for binding and a¤nity labeling studies. Microsomal fractions from BMMC, CFTL-15 and THP-1 bound 125 I-group I PLA2 and the bound ligand could be competitively reduced by an excess of unlabeled group I PLA2 ligand. Speci¢c binding of [125 I]-group I PLA2 to cell membranes was calculated at 1.91 pg/ Wg, 1.46 pmol/Wg and 0.25 pmol/Wg protein for BMMC, CFTL-15 and THP-1 cells, respectively. In contrast to BMMC, CFTL-15 and THP-1 cells, HL60 cell membranes did not contain speci¢c binding sites for group I PLA2 (data not shown). Together, data from both whole cells and membrane fractions suggest that BMMC, CFTL-15 and THP-1 have membrane-associated sPLA2 receptors while this receptor is absent from HL-60 cells. Binding studies were also performed using iodinated group II PLA2 in the presence or absence of 200-fold unlabeled ligand. No speci¢c binding was observed for all the four cell lines incubated with group II PLA2 (data not shown). Competitive binding studies were also carried out using iodinated group I PLA2 and unlabeled group II or group III PLA2 . Unlabeled group II PLA2 did not in£uence
Table 1 Binding of [125 I]sPLA2 to cells Cell types
Kd (nM)
Bmax (pmol/million cells)
Hill coe¤cient
BMMC CFTL-15 THP-1 HL-60
0.56 þ 0.14 (n = 5) 17.16 þ 7.38 (n = 3) 4.13 þ 1.95 (n = 3) No regressiona
7.98 þ 2.6 200.03 þ 154.8 18.30 þ 12.82 No regressiona
0.90 þ 0.05 0.90 þ 0.08 0.74 þ 0.06 No regressiona
One million cells in 0.4 ml bu¡er were incubated with various concentrations of [125 I]sPLA2 for 2 h at 20³C. Speci¢c binding was determined as described in Section 2. Kd , Bmax and Hill coe¤cients were estimated using the KELL equilibrium binding data analysis program. These data are the means þ S.E.M. of ¢ve (BMMC) or three (CFTL-15, THP-1) separate experiments. a Implies regression failed during data analysis.
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Fig. 4. Speci¢c binding of group I PLA2 to cells. BMMC, CFTL-15, THP-1 and HL-60 (1 million cells in 0.4 ml HBSS containing 1 mg/ml HSA) were incubated with various concentrations of 125 I-group I PLA2 in the absence for total binding or in the presence of 200-fold excess of cold group I PLA2 for non-speci¢c binding determination. Binding was performed at 20³C for 2 h after which, cells were removed from the binding media by centrifugation. Unbound ligand was removed by three washes of the cells followed by ¢ltration as described in Section 2. These data are representative of ¢ve separate experiments. Amount of ligand speci¢cally bound to cells are shown for BMMC (a), CFTL-15 (b), THP-1 (F) and HL-60 (E). Scatchard analysis of these data are shown in the inset.
the binding of group I PLA2 to BMMC. By contrast, group III PLA2 dose-dependently inhibited the binding of the iodinated group I PLA2 to BMMC with an IC50 of V0.4 nM. Taken together, these data suggested that group III PLA2 and group I PLA2 occupy the same binding sites on the surface of BMMC. 3.6. Determination of sPLA2 receptor mRNA in cells In subsequent studies, RT-PCR was utilized to determine the presence of sPLA2 receptor mRNA in BMMC, CFTL-15, THP-1 and HL-60 cells. RTPCR analysis of sPLA2 receptor mRNA levels indicated that BMMC, CFTL-15 and THP-1 cells expressed sPLA2 receptor mRNA. The 299 bp and 360 bp products of the RT-PCR reactions from CFTL-15 mast cells and from THP-1 cells were sub-
sequently sequenced and shown to be homologous to the predicted sequences for mouse and human sPLA2 receptors, respectively. In contrast, no sPLA2 receptor mRNA was detected after forty PCR ampli¢cation cycles of HL-60 cell cDNA (Fig. 5). These data suggest that CFTL-15, BMMC and THP-1 express sPLA2 receptor mRNA while this receptor mRNA is absent from HL-60 cells. 3.7. In£uence of immunologic and non-immunologic activation on group I PLA2 binding to BMMC The above studies revealed that several cell types including BMMC express sPLA2 receptor mRNA and possess a functional receptor on their membranes under resting conditions. Subsequent studies determined whether the capacity to bind the sPLA2
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indicates that sPLA2 (released from the cell after stimulation) has bound to the sPLA2 receptor and this complex has been internalized. 3.8. E¡ect of p-aminophenyl-K-D-mannopyranoside BSA on arachidonic acid release from mast cells
Fig. 5. RT^PCR of sPLA2 receptor mRNA. RNA was extracted from 5 million cells as described in Section 2. The presence or absence of mRNA for mouse or human sPLA2 receptor was determined after RT by PCR ampli¢cation using speci¢c primers for mouse sPLA2 receptor (BMMC and CFTL-15) or human sPLA2 receptor (THP-1 and HL-60). PCR products were isolated using 2% agarose gels and visualized by ethidium bromide staining followed by densitometer scans of the 299 bp and 360 bp products for mouse and human sPLA2 receptor mRNA, respectively. These data are representative of four separate experiments.
receptor was in£uenced by antigen or ionophore stimulation of BMMC. In control cells, speci¢c binding of 125 I-group I PLA2 was 8.31 þ 4.57 (n = 3) fmol/ million cells. Pre-stimulation of BMMC with antigen or with ionophore A23187 resulted in a 84.92 þ 9.22% and a 64.17 þ 3.46% decrease in [125 I]group I PLA2 binding, respectively. These data suggest that sPLA2 receptors are in£uenced by stimulation of BMMC. Previous studies have demonstrated that the sPLA2 receptor is internalized after receptor occupancy and thus the reduction in exogenous sPLA2 binding observed after BMMC stimulation
Nicolas and colleagues [49] have recently shown that p-aminophenyl-K-D-mannopyranoside BSA binds to the sPLA2 receptor in the same carbohydrate recognition domains as sPLA2 . Thus, p-aminophenyl-K-D-mannopyranoside BSA was added to mast cells to determine whether receptor occupancy alone (without PLA2 activity) could elicit the release of AA from mast cells. Fig. 6 shows that p-aminophenyl-K-D-mannopyranoside BSA induces the selective release of AA from BMMC. These data suggest that receptor occupancy alone is enough to initiate the selective release of AA from mast cells. To show that p-aminophenyl-K-D-mannopyranoside BSA induced the selective release of AA from mast cells as a result of sPLA2 binding, we examined its e¡ects on a cell line (HL-60) that does not express the sPLA2 receptor. Unstimulated levels of LA, OA and AA in supernatant £uids of HL-60 cells were
Fig. 6. In£uence of p-aminophenyl-K-D-mannopyranoside BSA on fatty acid release from BMMC. BMMC were incubated without (white bars) or with 50 WM p-aminophenyl-K-D-mannopyranoside BSA (black bars) for 5 min at 37³C. Cell pellets were removed by centrifugation and mole quantities of fatty acids (LA, OA, AA) released into supernatant £uids were determined by NICI^GC/MS. These data are the mean þ S.E.M. of three separate experiments. *P 6 0.05.
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140.11 þ 63.31, 1809.26 þ 622.51, 53.95 þ 12.32 pmol/ 5 million cells (n = 3, performed in duplicate), respectively. Levels of these unsaturated fatty acids were not altered in supernatant £uids of HL-60 cells incubated with p-aminophenyl-K-D-mannopyranoside BSA. These data suggest that the association of the sPLA2 receptor ligand with a cell surface receptor is a critical requirement for the selective release of AA from cells. 4. Discussion The present study demonstrates that secretory PLA2 isotypes bind to surface receptors on certain cells and this event facilitates the selective release of AA from these cells. The following six lines of evidence support this hypothesis. (1) Following antigen activation, mast cells rapidly release sPLA2 and this enzyme then reassociates with the cell. (2) Exogenously-provided sPLA2 isotypes (at concentrations 6 1 nM) or acid extracts from sPLA2 -containing cells selectively release AA from certain but not all cells. (3) Cells that selectively and potently release AA in response to sPLA2 are those that have the capacity to speci¢cally bind sPLA2 . (4) Those cells that selectively release AA in response to sPLA2 and speci¢cally bind sPLA2 contain mRNA for a sPLA2 receptor. (5) Mast cell activation by antigen or ionophore causes a reduction in the capacity of exogenously-provided group I PLA2 to bind to the mast cell surface, suggesting that the PLA2 receptor is either occupied or internalized after cell activation. (6) Another known ligand of the sPLA2 receptor, p-aminophenyl-K-D-mannopyranoside BSA, also induces the selective release of AA from sPLA2 receptor-expressing cells but not from cells that do not express the sPLA2 receptor. Initial studies in the present work demonstrated that an acid-stable, DTT-sensitive PLA2 is released from mast cells within 30 s of antigen activation. The levels of this activity then declines as a function of time, suggesting that this PLA2 activity reassociates with the cell. The increase and subsequent decline of sPLA2 on the outside of mast cells is closely mirrored by the selective release of AA (and not other polyunsaturated fatty acids) from mast cells. The concurrent appearance of this sPLA2 and AA in superna-
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tant £uids raised key questions as to the relationship between these two events. As mentioned in Section 1, previous studies have demonstrated that sPLA2 hydrolyzes several fatty acids including oleic acid, linoleic acid and AA from several membrane phospholipid systems [28^ 30]. Therefore, the selective release of AA from BMMC was initially inconsistent with the concept that sPLA2 is responsible for the mobilization of AA from intact cells. To better understand the selective hydrolysis of AA from BMMC, mast cells were incubated with four di¡erent isotypes that are representative of group I, a group II and a group III PLA2 . Addition of two group I sPLA2 s or a group III sPLA2 caused a selective release AA from BMMC at very low concentrations ( 6 1 nM). Human synovial group II PLA2 also selectively released AA from BMMC but at 100-fold higher concentrations. These data suggested that there were molecular events which occurred at the cell surface of mast cells that facilitated the selective hydrolysis of AA by sPLA2 from these cells. In the last 8 years, di¡erent subtypes of membrane receptors for sPLA2 have been identi¢ed in a variety of cell types by determining their a¤nities for various types of sPLA2 s [43,44,49^55]. Work by Arita and colleagues described the existence of a speci¢c receptor family termed PLA2 -I receptor that is abundant in brain and several other tissues and has high a¤nity for the binding of pancreatic-type PLA2 [50]. Lambeau and colleagues have suggested that sPLA2 receptors can be divided into two classes termed Ntype receptors and M-type receptors [56]. Isotypes of M-type sPLA2 receptors from di¡erent mammalian species have been puri¢ed, cloned and expressed [51,57]. Despite the fact that these receptors subtypes show somewhat di¡erent PLA2 binding pro¢les, their amino acid sequences are very similar with homology as high as 82% [55]. Although occupancy of the sPLA2 receptor has been suggested to enhance cell proliferation and induce chemokinetic cell migration, much remains to be learned about the physiological rami¢cations of this molecular event. Based on the selective hydrolysis of AA from mast cells at very low concentrations of sPLA2 , we postulated that the selective hydrolysis of AA was modulated through cellular receptors. Lambeau and colleagues report that a major di¡erence between N- and M-
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type receptors is their capacity to bind bee venom sPLA2 [56]. For example, the N-type receptor associates very tightly with both pancreatic sPLA2 and bee venom sPLA2 while the rabbit muscle M-type receptor tightly binds pancreatic and human synovial £uid sPLA2 but does not associate with bee venom sPLA2 . Our ¢nding showed that bee venom, at extremely low concentrations, hydrolyzed AA from BMMC suggesting that this mast cell expresses a protein that is similar if not identical to N-type receptor. A major question that arose from these studies was whether the selective release of AA by certain sPLA2 isotypes was a characteristic of the BMMC or was shared by other cell types. To examine this question, several cell types were incubated with exogenous group I sPLA2 . These studies revealed that AA was selectively released from some cells but not others. Cells that selectively released AA responded to a much lower concentration of group I sPLA2 than HL-60 cells which did not selectively release AA. These ¢ndings raised the critical question of whether the cells that selectively release AA are the cells which contained the sPLA2 receptor. This hypothesis was tested by searching for evidence of the expression of the sPLA2 receptor and mRNA utilizing binding and RT^PCR experiments, respectively. These experiments revealed that cells which selectively release AA also possess binding sites on their cell surfaces for group I PLA2 and express mRNA for an sPLA2 receptor. Additional experiments demonstrated that binding sites are lost from the surface of BMMC after challenge with ionophore or antigen. Studies from several laboratories suggest that mast cells have multiple sPLA2 isotypes that are secreted after stimulation [40,47,48]. For example, we have shown that two major acid-stable PLA2 are released from antigenstimulated BMMC [40]. Therefore, decreased binding of exogenous sPLA2 to mast cells may be due to the fact that sPLA2 receptors are occupied by endogenous ligands. Alternatively, sPLA2 receptors may be internalized during mast cell activation. In either case, these data support the contention that sPLA2 receptors are utilized during cell stimulation. The major ¢nding of this study is that receptor occupancy translates into the selective release of AA by an enzyme (sPLA2 ) that normally shows little
preference for unsaturated fatty acids. There are at least two potential explanations for how this shift in fatty acid selectivity might occur. One explanation is that the receptor itself is located in a part of the membrane that is highly enriched in AA. This hypothesis is supported by experiments in recent manuscripts which demonstrate that most of the AA released from mast cells after the addition of sPLA2 is hydrolyzed from phosphatidylethanolamine [31]. The fact that phosphatidylethanolamine is enriched in AA and is hydrolyzed during sPLA2 receptor occupancy suggest that the receptor may reside in a region of the plasma membrane highly enriched in phosphatidylethanolamine. An alternative hypothesis is that the selective release of AA by receptor occupancy does not involve the hydrolytic activity of sPLA2 at all, but involves an indirect mechanism by which sPLA2 simply acts as an agonist that stimulates signal transduction events leading to the selective release of AA. This idea is supported by several studies which suggest that sPLA2 which has been rendered catalytically inactive by inhibitors or mutations can still stimulate cellular and physiological responses [53,58]. Importantly, we provide direct evidence for this hypothesis in this paper by showing that another catalytically inactive ligand of the sPLA2 receptor, p-aminophenyl-K-D-mannopyranoside BSA, induces the selective release of AA from mast cells and not from HL-60 cells. Our preliminary studies (data not shown) suggest that sPLA2 occupancy of its receptor leads to the activation of cPLA2 and subsequent release of AA. In conclusion, it may no longer be accurate to assume that sPLA2 does not participate in AA metabolism based on the capacities of these enzymes to hydrolyze fatty acids utilizing chemically-pure substrates in cell free systems. The current study suggest that other molecular events that occur in whole cells, such a receptor occupancy, may markedly enhance the capacity of sPLA2 to selectively release AA (by either direct or indirect mechanisms). Recent studies indicate that sPLA2 released from mast cells can participate in transcellular prostaglandin production in ¢broblasts [39]. The molecular events described in the present study may facilitate the role of sPLA2 in AA metabolism during in£ammatory reactions by involving not only cells that release sPLA2 (such
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as mast cells) but also by neighboring cells which contain sPLA2 receptors. Acknowledgements We are grateful for the expert technical assistance of Dennis Swan. This work was supported in part by NIH grants AI 24985, AI 24985 S1 and P01-HL50395. M.S. was supported by a Centennial Fellowship from the Medical Research Council of Canada.
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