Effects of Ethanol on the Chemotactic Peptide-Induced Second Messenger Generation and Superoxide Production in Polymorphonuclear Leukocytes Eva Nilsson, Tommy Andersson, Maria Fallman, Kerstin Rosendahl, and Jan Palmblad

Granulocyte Laboratory. Department of Medicine. Karolinska Institute. and Department ofChemistry. Stockholm Soder Hospital; Department of Cell Biology. University ofLinkiiping. Sweden

A number ofclinical and experimental studies have shown that the administration of ethanol may confer an increase in morbidity and mortality in infections (reviewed in [1, 2]). Although several mechanisms may be involved in this enhanced susceptibility to infectious agents, attention has been focused on the effects of ethanol on the function of the polymorphonuclear leukocyte (PMNL), the principal effector cell in host defense against bacteria and fungi. Among a variety of antimicrobial activities of PMNL, the ability to produce oxygen radicals plays a pivotal role for its defense activity [3, 4]. The agonist-induced transmembrane signaling in PMNL consists of a complex series of reactions involving two interrelated pathways: changes in intracellular calcium concentrations ([Ca 2+]i) and the activation of protein kinase C. After the binding of the agonist to a surface receptor, phosphatidylinositol 4,5-bisphosphate is hydrolyzed by phospholipase C and phosphatidylcholine by phospholipase D into two putative intracellular messengers, diacylglycerol and, in the case of phospholipase C, inositol 1,4,5-trisphosphate [Ins(l,4,5)P3]. Ins( 1,4,5)P3 mediates the release of calcium from intracellular stores, whereas diacylglycetol is considered to be the endogenous activator of protein kinase C [5,

Received 2 February 1992; revised 28 April 1992. Grant support: Swedish Medical Research Council (19X-5991. 19P8884). Swedish Alcohol Research Fund. Swedish Cancer Association. King GustafV Memorial Foundation. Swedish Association against Rheumatism. and Funds of P. and A. Hedlund. C. Bergh. S. Svensson. M. Bergwall, Karolinska Institute. and S6dersjukhuset. Reprints or correspondence: Dr. Eva Nilsson. Department of Medicine. Stockholm SOder Hospital. S-118 83 Stockholm. Sweden. The Journal of Infectious Diseases


© 1992 by The University of Chicago. All rights reserved.


6]. Although the exact mechanism has not yet been unraveled, activation of superoxide ion production presumably results from subsequent calcium ion- or protein kinase Cdependent phosphorylation or both [7-9]. In vitro, ethanol inhibits formylpeptide- but not PMA-induced superoxide ion production in PMNL [10, 11]. The effect of ethanol on intracellular Ca2+ concentrations and on the Ca2+ flow has not yet been studied in PMNL, apart from our observation that the formylpeptide-activated initial rise in cytosolic Ca2+ appeared normal in ethanol-treated PMNL [10]. However, in other cell lines that have been studied, such as neural cells and macrophages, ethanol has been shown to have a modulating effect on the influx of Ca2+ through specific calcium channels and on [Ca2+]i [12, 13]. In addition, ethanol has been widely used to detect phospholipase D activity, since it catalyzes a transphosphatidyl reaction, causing the formation of phosphatidylethanol instead ofphosphatidic acid (PA) and its degradation product, diacylglycerol (DAG), derived from phosphatidyl choline. Against this background, we decided to analyze various aspects of the chemotactic peptide-induced second messenger system in PMNL after treatment in vitro with ethanol and relate this to the effect on superoxide ion generation. We also tested whether propranolol, which inhibits the breakdown ofPA to DAG [14, 15] and which has been shown to reduce mortality in infections in ethanol-treated rabbits [2], would modulate oxidative and calcium responses ofethanolimpaired PMNL.

Materials and Methods Chemicals. The chemicals and their sources were as follows: Hanks' balanced salt solution (HBSS), SBL (Stockholm); Percoli and Ficoll-Paque, Pharmacia (Uppsala, Sweden); iono-

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The generation of oxygen radicals by polymorphonuclear leukocytes (PMNL) plays a pivotal role for host defense. Since ethanol reduced FMLP- but not PMA-induced superoxide ion (02) formation by PMNL, the effects of ethanol on second messenger systems in PMNL were studied. FMLP induced a biphasic rise in cytosolic calcium concentrations, [Ca2 +]j. Ethanol treatment abolished the second phase (believed to reflect Ca2+ influx), an effect also observed in PMNL treated with La 3+ or suspended in Ca 2 +-free buffer. The FMLP-induced inositol trisphosphate generation was unaffected by ethanol, whereas diacylglycerol formation was, as expected, markedly reduced. Propranolol, an inhibitor of diacylglycerol formation from phosphatidic acid, caused a prolonged transmembrane influx of Ca2+ and partially reversed the inhibitory effect of ethanol on FMLP-induced O 2 production. Thus, the ability of ethanol to inhibit FMLP-induced O 2 generation in neutrophils seems to be due to both impaired influx of Ca2+ across the plasma membrane and reduced phospholipase D-mediated generation of phosphatidic acid.

JID 1992; 166 (October)

Effects of Ethanol on PMNL

Table 1. Effect of treatment with I% ethanol on increase in FMLP (0.1 ~M)-induced inositol triphosphate production. Time after stimulation Treatment Buffer (controls) Ethanol


10 s

30 s

100 116 ± 12

208 ± 35 218 ± 29

255 ± 34 231 ± 35

NOTE. Data are mean ± SE for 8 experiments. Values represent increase in inositol triphosphate (% of basal level).

nm. Measurements were made at 37°C with continuous stirring of the cell suspension. After a stable baseline had been established, the stimulus was added and emitted light recorded. The stability of the baseline was not affected by ethanol, La3+, or propranolol (data not shown). The signal was calibrated by the addition ofEGTA, TRIS buffer, Triton X-IOO, and CaCI2, after which calcium concentrations were calculated [22, 23]. Determination ofinositolphosphateformation. Phosphoinositides and inositol phosphates were labeled by incubating cells (5 X 107/mL) with myo-[2-3H]inositol (50 p.,CijmL) in a physiologically balanced salt solution (except that the Ca 2+ concentration was reduced to 0.5 mM to avoid aggregation) for 2 h in an atmosphere of 5%CO 2 and 95% air at 37°C [24]. For the determination of ethanol-induced changes in the agonist-induced formation of inositol phosphates, the labeled cells were treated with ethanol before FMLP stimulation (see table I). The stimulations were terminated by adding ice-cold TCA (final concentration, 15%, vol/vol). Inositol phosphates were extracted by a previously described procedure [25]. Samples were first put on ice for 15 min and then centrifuged, and the supernatants were washed three times with a fivefold excess of diethyl ether. The washed extracts were adjusted to pH 7.5 with TRIS (final concentration, 0.20 M), and the inositol phosphates were separated stepwise by elution from small Dowex anion-exchange columns (Sigma, St. Louis). After addition of67% Aquasol, the radioactivity of the different fractions was determined by liquid scintillation counting. To exclude effects on the calcium-releasing isomer, we made extractions of and quantitative determinations of Ins( IA,5)P3 using a commercially available kit (Amersham). Preincubated cells were stimulated with FMLP (I p.,M) for lOs. Cell reactions were terminated by ice-cold TCA. After centrifugation, supernatants were extracted with diethyl ether (saturated with water). Samples and known standards of Ins( I A,5)P 3 were incubated with a [3H]Ins( I A,5)P3 tracer and a specific Ins( 1A,5)P 3-binding protein. After centrifugation, the supernatants were discarded and the pellets resuspended in distilled water and transferred to scintillation vials. After the addition of2 mL of Ready Gel, the tritium levels were counted in a Rack-Beta liquid scintillation counter (LKB, Bromma, Sweden). The ratio of bound [3H]Ins(I A,5)P 3 in the samples relative to a blank correlated inversely with the Ins( 1A,5)P3 contents of the samples in a lin/ log manner. Determination of1.2-diacy/g(vcerolformation. To determine the chemotactic peptide-induced changes in cellular diacylglycerol concentrations, neutrophils were washed and resuspended

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phore A23187, ionomycin, and Fura-2 acetoxymethylester (Fura-2 AM), Calbiochem (La Jolla, CA); ['Y-32P]ATP, Hyperfilm multipurpose, and n-myo-inositol-I A,5-trisphosphate CH) assay, Amersham (Amersham, UK); octylglycoside and imidazole, Boehringer (Mannheim, Germany); 1,2-diacylglycerol kinase purified from Escherichia coli, Lipidex (Middleton, WI); silica gel 60, thin-layer chromatography plates, diethyl ether, and trichloroacetic acid (TCA), Merck (Darmstadt, Germany); propranolol, ICI (Macclesfield, UK); Ready Gel, Beckman Instruments (Stockholm); Aquasol, NEN (Boston). All other chemicals were from Sigma (St. Louis). If not otherwise stated, FMLP was used at a final concentration of 0.1 p.,M. The concentration of ethanol used here is given as vol/vol percentages. Thus, I % ethanol corresponds to 7.9 mg of ethanol/ mL or 0.17 M. This concentration was used in most experiments to delineate its effects. When required, other ethanol concentrations (obtainable in vivo) were also analyzed. The concentration of cytochalasin B, when used, was 5 p.,g/mL. Cell isolation. Heparinized blood samples were obtained from healthy members of the staff. None were on medication. Granulocytes were isolated by a one-step Percoll technique [16], followed by lysis of residual erythrocytes with 0.155 M ammonium chloride. With this technique, platelets were removed by an initial centrifugation step, and >95% ofthe cells were neutrophils as determined from stained smears. Platelets represented -- 1 in 10 granulocytes [17]. When studying inositol trisphosphate (InsP 3) and 1,2-diacylglycerol formation, we prepared PMNL by Ficoll-Paque gradient separation [18]. Cells were suspended in HBSS at pH 7.4 and kept at 4°C until used. Each experiment was started by a IS-min preincubation at 37°C. The concentrations of ethanol used here did not influence the pH of the suspensions. Cell viability, as determined from trypan blue staining and lactate dehydrogenase release, was not influenced by I%ethanol treatment [10]. Measurement ofsuperoxide production. Superoxide ion production was analyzed by the superoxide dismutase-inhibitable cytochrome c reduction method [19] at 37°C with continuous stirring of the granulocytes. The concentration of cytochrome c was 50 p.,M and that of superoxide dismutase was 3 p.,M. When La3+ as LaCl 3 was used, P04 was excluded from the medium to prevent the formation of complexes [20]. Absorbance was read at 550 nm, and superoxide ion production was calculated as nanomoles of reduced cytochrome c by use of an absorption coefficient of 21.1 mAr1cm- 1. Measurements of cytosolic free Ca2+. Cytosolic free Ca 2+ [Ca 2+]i was calculated from the changes in Fura-2 fluorescence as described previously [10, 21, 22]. Neutrophils (5 X 106 cells/ mL) in HBSS supplemented with 20 mM HEPES, pH 7.4, were incubated at 37°C with 0.5 p.,M Fura-2 AM for 30 min. Loaded cells were washed twice and reconstituted in HBSS (with or without Ca 2+ at 1.27 mM). To the calcium-free HBSS medium, I mM EGTA was added to decrease the extracellular calcium concentration to the nanomolar range. When La3+ as LaCI3 was used, P04 was excluded from the medium to prevent complex formation [20]. Subsequently, cells were warmed at 37°C for 15 min and added to quartz cuvettes in a spectrofluorometer. Fluorescence was excited at 340 nm, and the emitted light was read at 510


Nilsson et al.



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1ID 1992; 166 (October)


in a physiologically balanced salt solution. The cells were then stimulated with the chemotactic peptide FMLP, as described in the legend to figure I. The stimulations were terminated by adding an ice-cold solution of chloroform/methanol (I :2, vol/vol), and the cellular lipids were thereafter extracted [26]. The assay used was essentially the same as the one previously described [27, 28]. Each sample, which had been evaporated under nitrogen, was reconstituted in 20 ~L of a mixture containing 5 mM cardiolipin, 225 mM octylglycoside, and I mM diethylenetriaminepentaacetic acid and then sonicated for 15 s and left for an additional 10 min at room temperature. The samples containing the reconstituted lipids were then mixed with 50 ~L ofa reaction solution (containing 100 mM imidazole-HCl [pH 6.6], 100 mM NaCI, 25 mM MgCI2 , and 2 mM EGTA), 2 ~L of a solution containing 100 mM dithiothreitol and 1 mM diethylenetriaminepentaacetic acid, 13 ~L ofdistilled water, and 5 ~L ofa 1,2-diacylglycerol kinase solution (final activity, 0.02 units). The samples were then incubated for 5 min at 26°C, after which the enzymatic reactions were initiated by adding I0 ~L of a 10 mM [-y_ 32 p]ATP solution (specific activity, 70,000 cprn/nmol). After 15 min the reactions were stopped by adding 2 mL of chloroform/methanol (1:2, vol/vol), and the lipids were then extracted as above. The samples were evaporated under nitrogen and resuspended in 30 ~L ofchloroform before application onto a silica gel 60 thin-layer chromatography plate. The plates were developed with chloroform/methanol/acetic acid (65: 15:5, vol/ vol), air dried, and subjected to autoradiography. The spots corresponding to phosphatidic acid were scraped into scintillation vials, and after addition of 5 mL of Aquasol to each vial, the radioactivities were determined in an LKB scintillation counter. Statistical calculations. Statistical calculations were made with Student's t test of paired samples.


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Effects of ethanol on the chemotactic peptide-induced second messenger generation and superoxide production in polymorphonuclear leukocytes.

The generation of oxygen radicals by polymorphonuclear leukocytes (PMNL) plays a pivotal role for host defense. Since ethanol reduced FMLP- but not PM...
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