in 2 ml of 10 mM TES, 0.25 M sucrose (pH 7.0), and homogenized ... to 1.5 M sodium acetate according to the general method of Thompson et al. (28). High and ...
Val. 266, No. 16, Issue of June 5, PP. 10305-10312,1991 Printed in U.S . A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 01991 by The American Society for Biochemistry and Molecular Biology, Inc
Inhibition of Prostaglandin E2-stimulated cAMP Accumulation by Lipopolysaccharide in Murine PeritonealMacrophages* (Received for publication, June 21,
1990)
Kenji OkonogiS, Tom W. GettysQ, RonaldJ. Uhing, Wallace C. Tarry§, Dolph 0.Adams, and Veronica Prpicn From the Departments of Pathology and §Medicine, Duke University Medical Center, Durham, North Carolina 27710
Treatment of murine peritoneal macrophages with sponsesin phagocyticleukocytes (reviewed in Refs. 1-3). 100 nM prostaglandin E2 (PGE2) produceda rapid bi- Agents elevating cAMP through receptor-mediated activation phasic increase in intracellular cAMP that was maxi- of adenylylcyclase in mononuclear phagocytes include p-admal at 1 min and sustained through 20 min. Pretreat- renergic agonists, H-2 histamine receptors, and prostaglanment of macrophages with 100 ng/ml of lipopolysac- dins El and ES. The activation of CAMP-dependent protein charide (LPS) for60 min prior to PGEz decreased the kinase was shown to mirror the PGE2-induced’ increase in magnitude of cAMP elevation by 50%, accelerated the cAMP with respect to time and PGEz concentration (4, 5). decrease of cAMP to basal levels, and abolished the Incubation of mononuclear phagocytes with the above agents sustained phase of cAMP elevation. The effectof LPS or pharmacological activators of the cAMP cascade also inwas concentration-dependent, with maximal effect at 10 ng/ml in cells incubated in thepresence of 5% fetal hibited antigen presentation(5, 6), cytotoxic activation (7,8), calf serum and at 1 pg/ml in the absence of fetal calf and secretion of various inflammatory mediators. In the case serum. LPS also inhibited cAMP accumulation in cells of antigen presentation, we have demonstrated recently that inhibition of interferon-?-induced class I1 major histocomtreated with 100 ~ L Mforskolin, but the decrease was about half that seen in cells treated with PGE2. LPS patibility complex immune-associated antigen presentationis concentrations that inhibited cAMP accumulation pro- likely to involve suppression of an early transductional event duced a 30%increase in soluble low K,,, cAMP phos- initiated by the cytokine (5). Bacterial endotoxin,a lipopolysaccharide (LPS),is a potent phodiesterase activity while having no effect on parin vitro regulator of the functional responsesof mononuclear ticulatephosphodiesteraseactivity.Thenonspecific phagocytes. Theseresponses include stimulation of tumor phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, as well as the more specific inhibitors rolipram necrosis factor and interleukin-1 secretion (9, 10) and tumorand Ro-20-1724 were effective in inhibiting soluble icidal activity (11), inhibition of interferon-y induction of phosphodiesterase activity in vitro, producing syner- surface class I1 major histocompatibility complex immunegistic elevation of cAMP in PGE2-treated cells, and associated antigens (12,13), and priming for eicosanoid secreblocking the ability of LPS to inhibit accumulationof tion (14). The stimulation of many macrophage responses by CAMP. Separation of the phosphodiesterase isoforms LPS is attenuated by prostaglandins El and E2 and other in the soluble fraction by DEAE chromatography in- CAMP-elevating agents. Themolecular mechanisms that medicated that LPS activated a low K , cAMP phospho- diate cellular responsiveness to LPS have not been clearly diesterase. Theenzyme(s) present in this peak could be defined. In murine macrophages, LPS has been reported to activated 6-fold by cGMPand were potently inhibited by low micromolar concentrationsof Ro-20-1724 and stimulate phosphatidylinositolmetabolism (15), protein phosphorylation (16, 17), and myristoylation of specific proteins rolipram. Usingbothmembranes from LPS-treated (19). cells and membranes incubated with LPS, no decrease (18)and to antagonize stimulated cAMP accumulation In the present studies, we have characterized the inhibition in adenylylcyclase activity could beattributed to LPS. Although effects of LPS on the rate of synthesis of of PGEz-stimulated cAMP accumulationby LPS and invescAMP cannot be excluded, the present evidence is most tigated molecular mechanisms involved in the response. Our of cAMP accumulation consistent witha role for phosphodiesterase activation results indicate that the inhibition in the inhibitory effects of LPS on cAMP accumulation probably does not involve agonist-induced desensitization of the PGEz receptor due to macrophage PGE, synthesis. In in murine peritoneal macrophages. contrast, it is shown that specific phosphodiesterase inhibitors block the inhibition of cAMPaccumulation by LPS. Our results indicate a molecular mechanism whereby LPS counElevation of cAMP has been demonstrated to serve an teracts inhibitory effects against its actions. important inhibitory role in the regulation of functional reMATERIALSANDMETHODS
* This work was supported in partby United States Public Health Service Grants CA 29589 and DK 42486.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked“aduertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported in part by Takeda Chemical Industries. Present address: Biology Research Laboratories, Takeda Chemical Industries, Yodogawa-ku, Osaka 532,Japan. ll To whom all correspondence should be addressed: Dept. of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710.Tel.: 919-684-3300.
Reagents-PGE2 and indomethacin were purchased from Sigma. Forskolin was from Calbiochem. Brewer’s thioglycollate broth and LPS prepared by Westphal phenolic extraction from Escherichia coli The abbreviations used are: LPS,lipopolysaccharide; PGE2, prostaglandinEP;IBMX, 3-isobutyl-1-methylxanthine; FCS,fetal calf serum;Hepes 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid; TES, N-tris~hydroxylmethyl)methyl-2-aminoethanesulfonicacid; Gpp(NH)p, guanyl-5’-yl imidodiphosphate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.
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LPS Inhibition of Stimulated CAMPProduction
026:B6werefromDifco. Fetal calf serum (FCS) was from Sterile Systems, Inc., Logan, UT. RPMI 1640 medium was purchased from GIBCO. Sterile, distilled, nonpyrogenic water was obtained from Abbott. Phosphodiesterase inhibitors and their sources are as follows: anagrelide, Bristol-Myers Company, Wallingford, CT; CI-914, Warner-Lambert Company, Ann Arbor, MI; rolipram, Berlex Laboratories, Cedar Knolls, NJ. The phosphodiesterase inhibitor OPC 3689 was a gift from Dr.H. Hidaka, Nagoya University School of Medicine, Showaku, Japan. The compounds 3-isobutyl-1-methylxanthine (IBMX) and papaverine were purchased from Sigma, and Ro-20-1724 was purchased fromBIOMOL Research Laboratories, Plymouth Meeting, PA.Labeled [32P]cyclicGMP was purchased from ICN Biomedicals, Inc., Costa Mesa, CA. 'zsII-labeledradioimmunoassay kits for prostaglandin E, and 6-ketoprostaglandin F,, were purchased from Du Pont-New England Nuclear. Cell Culture-Specific pathogen-free, inbred C57BL/6 mice (6 weeks old) were purchased from Charles River Laboratories, Inc., Wilmington, MA. Thioglycollate broth-elicited macrophages were obtained by peritoneal lavage with 9 mlof Hanks' balanced salt solution containing 10 mM Hepes (pH 7.4) and 3.5 mM NaHC03 (HHBSS). Thioglycollate broth-elicited cell suspensions contained greater than 90% macrophages, as determined by differential cell count. Cells were washed once, resuspended in RPMI 1640 medium containing 2 mM glutamine, 12.5 units/ml penicillin, and 6.25 pg/ml streptomycin, and plated at a cell density of 3.1 X lo5 cells/cmz. Macrophages were incubated for 2 h at 37 "C in an atmosphere of 5% CO, and washed three times with HHBSS to remove nonadherent cells. The cell monolayers were routinely found to contain greater than 95% macrophages as determined by morphology with Giemsa stain. The macrophages were then cultured in RPMI 1640 medium supplemented with glutamine, penicillin, streptomycin, and 10% FCS for 18 h at 37 "C in 5% CO,. Measurement ofcAMP-Macrophages cultured overnight in RPMI 1640 medium (1.4 X lo6 cells/well) were washed twice with HHBSS, preincubated for 5 min at 37 "C in 5% CO,, and treated with stimuli for the desired periods of time in HHBSS supplemented with or without 5% FCS. Incubation with PGE, or forskolin was performed in a 37"C water bath, and reactions were stopped with an equal volume ofcold methanol. Cells were scraped, transferred to tubes, and lyophilized.Dry residues were resuspended in 1 ml of water, boiled for 5 min, and centrifuged at 2500 X g for 20 min at 4 "C. Cyclic AMP was assayed in clear supernatants by the radioimmunoassay originally described by Brooker et al. (20) and modified by Gettys et al. (21). The assays were calibrated by fitting logistic ogives in relation to log dose by weighted least squares as described by Gettys et al. (22). Determination of Adenylylcyclose Actiuity-Macrophages were cultured overnight and incubated under the conditions described for cAMP measurements. The incubations were stopped by placing the plate on ice-cold water, and cells were immediately scraped, transferred to tubes, pelleted by centrifugation, and retained a t -80 "C until use. Cells (8 X lo6) were suspended in 4 ml of 10 mM TES, 0.25 M sucrose (pH 7.0) and homogenized with 20 strokes of a Dounce homogenizer in an ice bath. A crude membrane preparation was obtained by centrifuging the homogenate at 40,000 X g for 5 min at 4 "C. The supernatantwas decanted, and adenylylcyclaseactivity was determined in the crude membranes as described by Gettys et al. (21). In brief, the reaction proper was started by placing 200 pl of membrane preparation into tubes containing100 plof test mix, producing final concentrations of 4 mM MgCl,, 1 mM ATP, and 1p M GTP. The reaction was stopped by adding 50 pl of cold 25% trichloroacetic acid, and anaddition 650 plof 50 mM phosphate buffer (pH 7.4) wasadded to each tube. All tubes were centrifuged at 3000 rpm for 20 min at 4 "C, and cyclic AMP was assayed in the clear supernatantsas described above. Membranes of cells treated with LPS were prepared in the presence of the same concentration of LPS, and the activity was determined in the presence of LPS. Protein was determined by the method of Bradford (23), and adenylylcyclase activity was expressed as picomoles of cAMP produced/min/mg of protein. Assay of cAMP Phosphodiesterase-Cells (8 x lo6) treated and collected as described for the adenylylcyclase assay were suspended in 2 mlof 10 mM TES, 0.25 M sucrose (pH 7.0), and homogenized with 20 strokes of a Dounce homogenizer in an ice bath. The homogenate was centrifuged at 40,000 X g for 30 min at 4 "C. The supernatant was retained, and themembrane pellet was rinsed gently with 2 ml of 10 mM TES, sucrose (pH 7.0) and resuspended in 1 ml of the same buffer. In separate experiments, crude soluble and particulate fractions were also prepared in the same buffer containing 1 mM
EDTA, 40 pM leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 pglml soybean trypsin inhibitor. Phosphodiesterase activity in the soluble and particulate fractions was measured at a substrateconcentration of 0.125 p~ cAMP to assess low K , activity and at100 p~ to assess high K, activity. The procedure was a modification of the method of Kono (24) as described by Gettys et al. (25) and involved measurement of 32Pformation from [32P]cAMPhydrolysis by phosphodiesterase and snake venom nucleotidase. The reaction was initiated by adding 50 p1 of the crude enzyme or column fractions to 200 pl of reaction mixture containing 50 mM TES (pH 7.5), 0.125 or 100 pM unlabeled CAMP, 2 pCi of [32P]cAMP(-3000 Ci/mmol), and 5 mM MgSO,. In some assays for low K , activity, unlabeled cGMP was . reactions included in the test mix at a concentration of 2.5 p ~ The were carried out at30 "C and stopped by the addition of 250 pl of an equimolar mixture of cAMP and AMP (1mM each) and immediately placed in a boiling HzO bath for 3 min. The tubes were cooled, and 50 pl of 1 mg/ml snake venom in 0.1 M Tris (pH 8) was added. All tubes were incubated for 20 min at 37 "C, and this reaction was terminated by the sequential addition of 50 pl of 5 mM adenosine in 200 mM EDTA and 500 pl of a Norit A charcoal suspension (50 mg/ ml) in 0.1 M &PO,. The tubes were vortexed and centrifuged for 15 min at 3000 X g, and 500 pl of the supernatantwas counted. was prepared according to Preparation of ~*P]cAMP-[~~P]cAMP a modification of the procedure described by Walseth and Johnson (26). Partially purified adenylylcyclase from Bordetella pertussis (27) (3000 Ci/mmol) in the presence was incubated with 1mCi CY-[~'P]ATP of20mM Tris-HC1, 5 mM MgCH3COO-, and 0.1%bovine serum albumin for 90 min at 30 "C. The [3ZP]cAMPformed was purified by C-18 reverse phase high pressure liquid chromatography followed by G-25 (superfine) chromatography in 50 mM NH4HCO3.No contaminating nucleotides were detected after the two chromatographic steps, and inorganic 32Pcontributed less than 0.3% of the totalcounts. The labeled cAMP was stored frozen at -20 "C and was stable for up to 30 days under these conditions. Assay of cGMP Phosphodiesterase-Cyclic GMP phosphodiesterase activity was assayed at a substrate concentration of 1 p~ by the procedure described above using [32P]cGMP. The reaction was stopped after a 20-min incubation with 200 p1 of stop mix containing 1 mM cold cGMP and immediately boiled for 3 min. The [32P]cGMP was purified prior to use by Sephadex G-25 superfine chromatography, and background activity averaged 0.4% of total counts using the purified material. DEAE-Sephacel Chromatography-Extracts from control and LPS-treated macrophages were prepared by homogenizing the cells with 20 strokes of a Dounce homogenizer with 4 ml of ice-cold buffer containing 25 mM Hepes, 1 mM EDTA, 1 pg/ml soybean trypsin inhibitor, 40 p~ leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The extracts were applied to DEAE-Sephacel columns equilibrated with 50 mM sodium acetate buffer (pH 6.5) and washed with 5 bed volumes of this buffer before eluting with a linear gradient of 50 mM to 1.5 M sodium acetate according to thegeneral method of Thompson et al. (28). High and low K, cAMP phosphodiesterase activities, as well as cGMP phosphodiesterase activity, were determined in each 0.5-ml fraction.
RESULTS
Inhibition of PGE2-stimulated CAMP Accumulation by LPS-Treatment of mouse peritoneal macrophages with 100 nM PGEz produced a rapid 3-4-fold increase in intracellular cAMP (Ref. 5 and Fig. 1).A transient decline in cAMP was noted by 5 min, but levels were maintained at twice the basal concentration through 15 min (Fig. 1). Previous work has shown that the sustained phase of cAMP elevation noted in Fig. 1 is maintained for several hours (5). The PGEz-induced increase in cAMP was concentration-dependent, with detectable increases noted at 10 nM and maximal responses seen with concentrations around 100 FM PGEz (data notshown). Pretreatment of macrophages for 60 min with 100 ng/ml of LPS prior to treatment with 100 nM PGEz decreased the magnitude of cAMP elevation and accelerated the decrease of cAMP to basal levels (Fig. 1).The highest concentration of intracellular cAMP in macrophages treated with LPS was half that of control cells, and thesustained phase of increased cAMP noted in control cells was absent in LPS-treated cells
10307
LPS Inhibition of Stimulated CAMPProduction *SERUM
CONTROL
0
5
10 TIME ( r n ~ n )
15
20
LPS ( n e / m l )
FIG. 3. Concentration-dependent suppression of PGEzFIG. 1. PGEz-stimulated cAMP accumulation by macrophages and its suppression by LPS. Cells (1.4 X lo6) cultured stimulated cAMP accumulation by LPS. Cells (1.4 X lo6) were overnight as described under "Materials and Methods" were incu- incubated with LPS at the concentrations indicated in HHBSS in bated with (open circles) or without 100 ng/ml of LPS (closed circles) the absence (left) and in the presence (right) of 5% FCS at 37 "C for in 0.5 ml of HHBSS supplemented with 5% FCS a t 37 "C for 50 min 50 min in a COP atmosphere and then for 10 min in a 37 "C water in 5% CO, atmosphere and then for 10 min in a 37 "C water bath. bath. Cells were then incubated with 100 nM PGE, for 2 min (solid PGE2 (100 nM) was added at time 0, and thereaction was stopped by bar). Control cultures (open bars) received buffer alone. cAMP was adding 0.5 ml of cold methanol after the incubation period indicated. determined as described in the legend to Fig. 1. Values represent the cAMP was assayed by radioimmunoassay as described in the text. mean & standard deviation for triplicate cultures. Values represent the mean & standard deviation for triplicate cultures. .+LPS
IO
30 TIME [rnin)
60
FIG. 2. Time course of LPS suppression of cAMP accumulation stimulated with PGEZ. Cells (1.4 X IO6)were incubated with or without 100 ng/ml of LPS in 0.5 ml of HHBSS supplemented with 5% FCS at 37 "C for 0,20, and 50 min in a CO, atmosphere and then for 10 min in a 37 "C water bath. Cells were then incubated with or without 100 nMPGEZ for 2 min. Reaction was stopped, and cAMP was determined as described in the legend to Fig. 1. Open bar, control; shaded bar, PGE, alone; closed bar, LPS plus PGE,. Values represent the mean f standard deviation for triplicate culture.
CONTROL
II T
O.1pM PGE;,
100pM FOFSKOLIN
FIG. 4. Effect of LPS on PGE,-and forskolin-stimulated cAMP accumulation by macrophages. Cells (1.4 X lo6) were treated with buffer (open bar) or 100 ng/ml of LPS (solid bar) in 0.5 ml of HHBSS supplemented with 5% FCS at 37 "C for 60 min as described in the legend to Fig. 1. cAMP production was stimulated with 100 nM PGE, for 2 min or with 100 PM forskolin for 5 min at 37 "C. Control culture received buffer alone. cAMP was determined as described in the text. Values represent the mean k SD for triplicate cultures.
(Fig. 1).The inhibitory effect of LPS on PGE,-stimulated cAMP accumulation required relatively long exposure times. Suppression of PGE2-induced cAMP inmacrophages by LPS radioimmunoassay, was 43 f 7 pg/106 cells. By contrast, PGE2 was readily evident following 30- or 60-min exposures but not secretion from thioglycollate-elicited macrophages incubated significantly different at 10 min (Fig. 2). In the presence of in the absence or presence of LPS was
