outer-membrane porins from gram-negative bacteria stimulate platelet-activating-factor biosynthesis...

Eur. J. Biochern. 214, 685-693 (1993) 0 FEBS 1993

Outer-membrane porins from Gram-negative bacteria stimulate platelet-activating-factor biosynthesis by cultured human endothelial cells Maria A. TUFANO', Luigi BIANCONE*, Fabio ROSSANO', Ciro CAPASSO', Adone BARONI', Antonella DE MARTINOZ, Eugenio L. IORI04, Luigi SILVESTR03 and Giovanni CAMUSSIZ,4 ' Istituto di Micobiologia, Facolth di Medicina e Chirurgia, Seconda Universiti di Napoli, Italy

Laboratorio di Immunopatologia, Cattedra di Nefrologia, Universith di Torino, Italy Res Pharma Pharmacological Research s.r.l., Torino, Italy Dipartimento di Biochimica e Biofisica, Facoltii di Medicina e Chimrgia, Seconda Universith di Napoli, Italy

(Received December 24, 1992March 22,1993) - EJB 92 1849/6

Porins are a family of hydrophobic proteins located in the outer membrane of the cell wall in Gram-negative bacteria. The effect of porins on the biosynthesis of platelet-activating factor (PAF) by cultured human umbilical-cord-vein-derived endothelial cells (HUVEC) was investigated. The results demonstrate that porins were able to induce a dose-dependent synthesis of PAF in HUVEC. PAF, synthesized after stimulation with porins, was mainly cell associated and the synthesis peaked at 15 min, decreasing rapidly thereafter. Experiments with radiolabeled precursors demonstrated that PAF, a 1 -O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, was synthesized via the remodeling pathway involving the acetylation of 1 -O-alkyl-2-lyso-sn-glyceryl-3-phosphorylcholine (2-lysoPAF) generated from l-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase-A2 activity. The activation of phospholipase A2 in HUVEC stimulated by porins was detected by observing the mobilization of [i4C]arachidonic acid. In addition, the activity of acetyl-CoA :l-alkyl-sn-glycero-3- phosphorylcholine 2-0-acetyltransferase was transiently increased in porin-stimulated HUVEC and, after incubation with [3H]CoASAc or [3H]acetate, the [3H]acetyl group was incorporated into newly synthesized PAF. Porins, by forming transmembrane channels, induced a sustained influx of extracellular 45Ca2+ into the cytosol. The activation of PAF synthesis by porins depended on this influx rather than on intracellular calcium mobilization, since PAF synthesis did not occur in the absence of extracellular Ca".

It is now recognized that vascular endothelial cells actively participate in the genesis of inflammatory and systemic alterations occurring in sepsis sustained by Gram- negative bacteria. The endothelial-derived vasodilators and the vascular leak resulting from endothelial-cell contraction, either from cytokine-mediated cytoskeletal reorganization or from endothelial injury, may produce blood stasis favoring leukocyte-endothelial adhesion. This may contribute to the systemic alteration which is characteristic of endotoxicheptic shock [l]. Dynamic changes in the surface adhesion molecules of both endothelium and leukocytes are required for sequential adhesion and transmigration of leukocytes through the vascular wall. These changes are promoted by the active

Correspondence to G. Camussi, Laboratorio di Immunopato- logia, Divisione de Nefrologia e Dialisi, Ospedale S. G. Battista, Molinette, Corso Dogliotti 14, 1-10 126, Torino, Italy

Abbreviations. HUVEC, human umbilical-cord-vein-derived endothelial cells; PAF, platelet-activating factor; C16-PAF, 1 -0-alkyl- 2-acetyl-sn-glyceryl-3-phosphorylcholine; 2-lysoPAF, l-O-alkyl-2- lyso-sn-glyceryl-3-phosphorylcholine; 2-lysoPtdCho, 2-lysophos- phatidylcholine ; LPS, lipopolysaccharide ; PMN, polymorphonuclear neutrophils ; lL, interleukin ; TNF, tumor-necrosis factor ; acetyl-CoA:l-alkyl-sn-glycero-3-phosphorylcholine 2-0-acetyltransferase, CoASAc 2-lysoPAF acetyltransferase ; SD, standard deviation.

Enzyme. Acetyl-CoA : 1 -alkyl-sn-glycero-3-phosphorylcholine 2- 0-acetyltransferase (EC 2.3.1.67).

synthesis of leukocyte-activating mediators, such as interleukin-1 (interleukin, IL), IL-8, monocyte chemotactic protein-1 or platelet-activating factor (PAF), by endothelial cells [2].

PAF (1 -O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine), an ether phospholipid with a wide spectrum of biological activities (for a review, see [3,4]) is a mediator of inflammation and shock induced by Gram-negative sepsis [5 -71.

PAF synthesis may occur either via the remodeling pathway, through acetylation of 1 -O-alkyl-2-lyso-sn-glyceryl-3- phosphorylcholine (2-lysoPAF) generated from 1 -5-alkyl-2- acyl-sn-glyceryl-3-phosphorylcholine by phospholipase-A2 activity, or via the de now biosynthetic pathway that involves the synthesis of 1 -O-alkyl-2-acetyl-sn-glycerol, which is then converted to PAF by a unique CDPcho1ine:l-alkyl-2- acetyl-sn-glycerol-cholinephosphoryltransferase [8]. In sepsis, sustained by Gram-negative bacteria, the synthesis of PAF may be triggered either by bacterial endotoxin or by endotoxin-induced cytokines. Indeed, tumor-necrosis factor (TNF), the primary mediator of endotoxin-induced inflammation [9], as well as IL-1 and IL-8 are able to stimulate PAF synthesis by activating the remodeling pathway. This occurs not only in polymorphonuclear neutrophils (PMN) and macrophages but also in vascular endothelial cells [lo- 121. In addition, human umbilical-cord-vein-derived endothelial cells (HUVEC) were shown to produce PAF after sti-

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mulation with other mediators generated during the inflammatory reactions such as thrombin, angiotensin 11, vasopressin, leukotriene C4, leukotriene D4, histamine, bradykinin, elastase and cathepsin G [13-161. PAF synthesized by vascular endothelium remains mainly associated with the cell and may promote leukocyte adhesion [14]. Moreover, PAF is, on a molar basis, 1000-10000-fold more potent than histamine in increasing vascular permeability [17], due to its effect on the endothelial-cell cytoskeleton [ 181.

The aim of the present study was to investigate whether bacterial components, other than endotoxin, were able to directly stimulate PAF synthesis in HUVEC. Porins, hydrophobic proteins of the outer-membrane layer of the Gram-negative bacterial cell wall [19, 201, were selected for these studies on the basis of our recent observation that they may stimulate PAF synthesis by PMN [21] and human mesangial cells [22]. Porins, which form transmembrane channels (pores) for the passive diffusion of small solutes through the outer membrane of the Gram-negative bacterial cell wall [23, 241, may play a role in bacterial virulence because of their ability to insert into the membrane of nucleated cells [25, 261.

The results obtained show that porins stimulate the synthesis of PAF from HUVEC by a mechanism that involves the influx of extracellular Caz+ and the activation of the enzymes involved in the remodeling pathway.

MATERIALS AND METHODS Materials

Cycloheximide, polymyxin B, phospholipase A2, lipase Al , bovine serum albumin (fraction V), porcine heparin, endothelial cell-growth factor, CoASAc, sphingomyelin, 2-ly- sophosphatidylcholine (2-lysoPtdCho), PtdCho, and F‘tdEtn were from Sigma ; 4-bromodiphenacylbromide was from Car10 Erba ; collagenase from Clostridium histolyticum was from Boehringer Mannheim; human-factor-VIII antiserum was from Nordic Immunology; anti-human leukocyte antigens class I, anti-human leukocyte antigens class I1 and anti- common antigens (CD45) mouse mAb were purchased from Beckton-Dickinson; synthetic C16-PAF (1 -0-hexadecyl-2- acetyl-sn-glyceryl-3-phosphorylcholine), 2-lysoPAF (1-0- octadecyl-2-lyso-sn-glyceryl-3-phosphorylcholine), were purchased from Bachem Feinchemikalien; SDZ 63675 (San- doz), WEB 2170 (Boehringer) and CV 3988 (Takeda Chemi- cal Industries) were used as specific PAF-receptor antagonists. [3H]CoASAc (1 Ci/mmol), [3H]acetate ([3H]CH3C0,Na; 2.5 Ci/mmol), [3H]methylcholine (75 Ci/mmol) and [‘“Clar- achidonic acid (55.5 Ci/mol) were purchased from Amity. 45Ca2+ (30 Ci/g Ca”) was obtained from Amersham.

Preparation of porins A strain of Salmonella typhimurium SH5014, kindly pro-

vided by M. Nurminen (Central Public Health Laboratory, Helsinki, Finland), was used as a source of porins. Porins, extracted by the method of Nurminen [27], were further purified by gel filtration in the presence of detergent (SDS) [28] to remove lipopolysaccharide (LPS) contamination. Purified porins, solubilized at 100°C for 5 min in a sample buffer containing 62.5 mM Tris/HCl, pH 6.8, 2% SDS, 5% 2-mer- captoethanol, 10% glycerol and 0.001 % bromophenol blue, were analyzed by SDS/polyacrylamide (16%) gel electrophoresis [26]. Gels were stained with Coomassie blue. As shown

Fig. 1. SDS/polyacrylamide (16 %) gel electrophoresis of purified porins. The porin preparation (lane B) showed a single band with apparent molecular mass 34-36 kDa after staining with Coomassie blue. The molecular-mass markers (lane A) were phosphorylase B, 106 m a ; bovine serum albumin, 80 kDa; ovalbumin, 49.5 kDa; car- bonic anhydrase, 32.5 kDa; soybean trypsin inhibitor, 27.5 kDa; ly- sozyme 18.5 kDa. For details of SDS/polyacrylamide gel electrophoresis, see Materials and Methods.

in Fig. 1, a single band with apparent molecular mass 34- 36 kDa was detectable. The bioactivity of the porin preparation was tested using the release of from human PMN purified as previously described [21]. Briefly, 2.5 X lo6 PMN were incubated for 1 h at 37°C with 250 pCi Na51Cr04 (specific activity 300-500 mCi/g Cr; New England Nuclear), washed three times with Eagle’s modified Dulbecco Medium (Gibco) and resuspended with 1 ml of the same medium. Af- ter incubation with or without porins, the cells were centri- fuged and the amount of ”Cr released into the supernatant was calculated. The radioactivity in the supernatant, plus the value for a sample extracted from the cell pellet treated with 5% Triton X-100, was assumed to represent 100% radioactivity [29]. This value was corrected for any spontaneous release of radioactivity. After a 1-h incubation with 50 pg/ml NaTrO,, the porin preparation used in the present study induced 47 -50% T r release. LPS contamination, detected by the Limulus assay [30] in the final preparations of porins, was only 1 pg/pg porin. In selected experiments, porins were incubated with 5 pglml polymyxin B (Sigma) at room tem- perature for 1 h to neutralize the biological activity of the traces of contaminating LPS as described by Blanchard et al. 1311.

Endothelial cell culture HUVEC were isolated by treatment of human umbilical-

cord veins with 0.2% collagenase from C. histolyticum (20 min, 37°C) and cultured as previously described until they reached confluence [13]. These cells were characterized by morphologic criteria and positive immunofluorescence for factor-VIII antigen and class-I, but not class-11, histocompati- bility antigens, as previously described [13]. To study the ability to release PAF, HUVEC subcultures were grown on

687

35-mm-diameter petri wells until the cell number reached 4.6 X lo5 ? 3.2 X 104, mean +_ SD, 11 determinations, after 3-4 days). Cells were then washed three times with RPMI- 1640 medium containing 0.25% bovine serum albumin in order to remove fetal calf serum. HUVEC were incubated in 2 ml RPMI-1640 medium at 37°C for the indicated time with the following different stimuli: 0.1 -20 pg/ml porins or 0.1 - 20 pg/ml polymyxin-B-treated porins. In selected experiments, HUVEC were incubated with 1 pM 4-bromodiphenacylbromide in the absence or presence of 10 pM 2-lysoPAF, or with 10 pM 2-lysoPAF alone, for 30 min at 37°C and sub- sequent treatment with porins for 15 min at 37°C.

The release of cytosolic lactate-dehydrogenase activity was determined by a spectrophotometric method. Lactate- dehydrogenase release was expressed as a percentage of the total cellular content, which was determined by treating HUVEC with 0.01% Triton X-100 for 1 h at room temper- ature.

The role of extracellular Caz+ in porin-induced PAF synthesis was evaluated using H W E C incubated in a balanced- salt solution (0.159 M NaC1, pH 7.2, 6.0 mM KCl, 0.8 mM MgSO, , 11.1 mM glucose and 0.25% bovine serum albumin prepared in double-distilled water containing 2.5 pM Ca” in the absence or presence of various concentrations of CaC1, as described by Ludwig et al. [32]. In some experiments, EDTA or EGTA was added (10 mM, final concentration) to HUVEC incubated in Ca2+-containing Tyrode’s buffer 10 min prior to porin addition. The extracellular-Ca” uptake by porin-stimulated HUVEC was evaluated using ,TaZ+ [33]. Cells (5 X lO’/ml>, incubated in the balanced-salt solution without CaCl, and containing 0.2 mM EGTA, were treated with porins. The reaction was started by adding 2 mM CaClZ, 0.5 pCi 45Ca2+ and the different agonists. Termination was achieved by adding 2.5 mM EGTA and 5 pM ruthenium red (final concentration) on ice to remove cell-surface-bound Ca2+. After washing twice with the balanced-salt solution, the pellet was solubilized with formic acid and the radioactivity was measured in the presence of 4 ml Instagel.

Assay and quantification of PAF The synthesis of PAF was studied using the incorporation

of radioactive precursors as previously described, and quantified using the bioassay. To study the incorporation of radioactive precursors, 5 X lo5 HUVEC were incubated in lml RPMI 1640 for 30 min with 30 pCi [3H]CoASAc, 30 pCi [3H]acetate or 2.5 pCi [3H]methylcholine before stimulation. The supernatants and cell pellets were extracted according to a modification of the Bligh and Dyer procedure [34], with formic acid added to lower the pH of the aqueous phase to three [35] and lipids were fractionated by TLC on aluminium-sheet silica-gel plates (silica gel 60, F254, 0.2-rnm thickness, Merck Darmstadt) using as solvent chlorofodmetha- noVacetic acidwater (50: 25 :8 : 4, by vol.). The plates were cut into 0.5-cm sections and the radioactivity of each was measured. Radiolabeled PAF was used as a standard. To discriminate between the presence of an ester or ether group at the sn-1 position, the TLC-extracted radiolabeled lipids, comigrating with PAF, were treated with phospholipase C (type XIII) from Bacillus cereus (Sigma) for 2 h [36]. The l-radyl-2-[3H]acetylglycerols, obtained by phospholipase-C treatment were then acetylated at position 3 by incubation for 16 h at 37°C with 0.5 ml acetic anhydride and 0.1 ml pyridine. The radiolabeled 1 -radyl-2,3-diacetylglycerols, obtained with this reaction, were extracted with hexane/diethyl

ether (1 : 1, by vol.) and analyzed by TLC using silica-gel plates developed with hexane/diethyl ethedformic acid (90: 60:6, by vol.) that allows the separation of 1-alkyl-2,3- diacetylglycerol from 1 -acyl-2,3-diacetylglycerol as described by Triggiani et al. [36]. The quantification of PAF bioactivity, which was shown to be related to 1-alkyl-2-acetyl-3-glycerylphosphorylcholine rather than the 1 -acyl-2-acetyl-3-glycerylphosphorylcholine [4], was performed on the basis of aggregating activity for washed rabbit platelets [ l l , 131. PAF was quantified after extraction and purification by TLC (silica gel 60, F254, Merck) and HPLC (pPorasi1 column, Millipore chromatographic division, Waters) [37]. PAF was characterized by a comparison with synthetic PAF according to the following criteria: (a) the induction of platelet aggregation by a pathway independent from both ADP-mediated and arachidonic acid/thromboxane-A2-mediated path- ways; (b) the specificity of platelet aggregation as inferred from the inhibitory effect of 5 pM WEB 2170 [38] and 5 pM CV 3988 171, two different PAF-receptor antagonists: (c) TLC and HPLC behavior and physicochemical characteristics, such as inactivation by strong bases [39] and phospholipase A2 [40], but resistance to phospholipase A1 [40], acids, weak bases and heating for 5 min in boiling water [39]; (d) chemical identity with the synthetic C16-PAF evaluated by a newly developed technique based on HPLC-tandem MS [37]. An API I11 (Perkin Elmer SCIEX) mass spectrometer, with an ionspray articulated source interfaced to a HPLC syringe pump (Applied Biosystems 140A), was used. Briefly, each sample purified by TLC was resuspended in 60 pl acetoni- trile/water (90: 10, by vol.) with 0.1 % trifluoracetic acid. A 50-pl sample was injected into the HPLC column, a reverse- phase wide-pore butyl column (Hypersil W-Butyl, 100 mm X 1 mm, Shandon), using a mobile-phase gradient of methanol with 0.1% trifluoracetic acid (A) and water with 0.1% trifluoracetic acid (B). The concentration of A was increased from 30% to 80% in 20 min, with a linear gradient followed by an isocratic elution at 80% A for 15 min at a flow rate of 50 pl/min.

MS analysis was performed using HPLC-tandem MS. Daughter ions, in the range m/z 40-560, were obtained in the positive mode from parent ions with m/z 524, which cor- respond to the protonated molecular ion of C16-PAF. Frag- mentation was obtained by collision with argon and a collision-gas thickness of 2.5 X 10“ atoms/cm with an impact en- ergy of 70 eV. Standard C16-PAF was purified and analyzed using the same technique.

Acetyltransferase enzymic assays For the measurement of acetyltransferase activity, cell-

extract preparation and enzyme assays were as previously described [lo]. Briefly, cells were sonicated in 1 ml buffer containing 0.24 M sucrose, 1 mM dithiothreitol and 50 pg cell-lysate proteins and mixed with 40 pM 2-lysoPAF, 0.2 pCi [3H]CoASAc and 200 pM cold CoASAc in 0.5 ml buffer containing 20 mM Tris/HCL, pH 6.8. After a 15-min incubation at 37°C the reaction mixture was analyzed by TLC and [3H]PAF was quantified. Acetyltransferase activity was expressed as nmol [3H]acetate transferred to 2-lysoPAF/ min of incubatiodmg protein present in the lysate.

Release of [14C]arachidonic acid The release of [‘4C]arachidonic acid from cellular lipids,

mainly phospholipids, was measured using the method de-

688

Table 1. Incorporation of radiolabeled precursors into PAF molecules synthesized by porin-stimulated HUVEC. 5 X 105 HUVEC were incubated for 30 min with 30 pCi t3H]acetate, 30 pCi [3H]CoASAc or 2.5 pCi [3H]methylcholine and stimulated with I 0 lg/ml porin for 15 min. Control cells were incubated at 37°C for the same period of time in the absence of stimuli (None). The supernatants and the cell pellets were extracted according to the modified method [35] of Bligh and Dyer [34] and lipids were fractionated by TLC using aluminium sheets as silica-gel plates with chlorofodmethanoUacetic acidwater (50: 25:8:4, by vol.) as solvent. The plates were cut into sections of 0.5 cm each and measured for radioactivity. Radiolabeled PAF was used as a standard. Results are expressed as the mean 2 SD of 3 experiments. One-way analysis of variance, with Dunnett's multiple comparison test, was performed between the control (None) and experimental groups.

Addition Incorporation of radiolabel from

[3H]acetate [3H]CoASAc ["Hlmethyl- choline

None 438 2 37 1 1 2 0 2 145 556 2 232 Porins 3531 2 560' 4456 2 220' 687 2 144

' P<O.O5.

scribed by Hirata et al. [41]. 5 X lo5 cells were incubated in 2 ml with 0.15 pCi ['4C]arachidonic acid (55.5 Ci/mol) at 37°C for 1 h. The cells were washed twice with modified Grey's buffer and incubated in 2 ml of the same buffer. The release was measured after treatment with different stimuli at 37°C as described for the release of PAF.

Statistical analysis Data, within different experimental groups, were ana-

lyzed by the Student's t test or by one-way analysis of variance with Dunnett's or Newman-Keuls multiple comparison test where appropriate. Values are given as the mean 2 standard deviation (SD). Values of P<0.05 were considered sta- tistically significant.

RESULTS The incorporation of radioactive precursors into newly

synthesized PAF was studied after stimulation with porins (Table 1). The amound of radiolabel incorporated into cells after a 30-min incubation was 0.1% and 0.46% for r3H]CoA- SAC and [3H]acetate, respectively. No further incremeht for radiolabel incorporation was obtained by increasing the incubation time up to 120 min (data not shown). Fig. 2 shows the TLC profiles of radiolabeled lipids extracted from nonstimulated and porin-stimulated HUVEC and compares the incorporation of [3H]CoASAc to that of [3H]acetate. The TLC analysis of lipid fractions, extracted 15 min after addition of porins to HUVEC previously incubated with [3H]CoASAc or with [3H]acetate, demonstrated, for both conditions, the presence of one main peak of radioactivity for the sample that migrated with synthetic C16-PAF. This peak was absent in the lipid fractions extracted from untreated HUVEC. For the [3H]acetate incubation, an increase in the amount of radiolabel was found for lipids other than PAF and at the solvent front (Fig. 2A) compared to incubation with [3H]CoASAc (Fig. 2B). In the latter case, no radiolabel was

6 .

1 2 3 - -- I A PAF -

0 5' 1 0

Distance (cm) 6

0 5 1 0 1 5' Distance (crn)

rr I

Distance (cm) Fig. 2. Representative TLC analysis of radiolabeled lipids extracted from nonstimulated and porin-stimulated HUVEC. The TLC analysis of PAF synthesized by HUVEC is shown. Cells were incubated for 30 min with 30 pCi [3H]CoASAc (A), 30 pCi [3H]- acetate (B) or 2.5 pCi [3H]methylcholine (C) and stimulated for 15 min with 10 pg/ml porins. The lipids that were extracted from 5 X lo5 cells were analyzed by TLC using chlorofodmethanoUace- tic acidwater (50: 25:s: 4, by vol.) as solvent. The plates were di- vided into 0.5-cm sections and the radioactivity was measured as described in Materials and Methods. The results obtained with porin- stimulated HUVEC (-) and control nonstimulated HUVEC (---) are shown. The chromatographic behaviours of synthetic C16- PAF (PAF), PtdCho (l), PtdSer (2) and PtdEtn (3) are also indicated in A. These data are representative of three different experiments.

detected at the solvent front. In contrast, incubation of HUVEC with [3H]methylcholine did not result in enhanced incorporation into PAF molecules (Table 1) or other classes of phospholipid (Fig. 2C) after porin stimulation.

Since several studies [36, 42-44] have documented the heterogeneity of the acetylated. glycerophospholipds produced by HUVEC, concerning the type of group present at the sn-1 position, the 3H-labeled lipids, extracted and purified by TLC, were modified into 1 -radyl-2,3-diacetyl-glycerols as described in Materials and Methods. When the labeled 1- radyl-2,3-diacetylglycerols (11 800 cmp) were separated by TLC, the radioactive products appeared in two distinct re-

689

I

0 5 1 0

Distance (cm) Fig. 3. Representative TLC analysis of radiolabeled l-radyl-2,3- diacetylglycerols obtained by treating radiolabeled lipids, migrating with C-l6PAF, with phospholipase C followed by acetylation at the sn-3 position (see Material and Methods). Radiola- beled l-radyl-2,3-diacetyl-glycerols (11 800 cpm) were analyzed by TLC with hexane/diethyl ethedformic acid (90: 60:6, by vol.) as the solvent. Peak I comigrates with 1 -palmitoyl-2,3-diacetylglycerol and accounted for approximately 42% of the total radioactivity applied to TLC ; peak I1 comigrated with l-O-hexadexyl-2,3-diacetylglycerol and accounted for approximately 25 % of the total radioactivity.

gions of the chromatogram, corresponding to the R, of radiolabeled l-palmitoyl-2,3-diacetylglycerol (I) and 1 -0-hexadecyl-2,3-diacetylglycerol (11) (Fig. 3). The major radiolabeled compound produced by HUVEC was the 1 -acyl-derivative of PAF (42% total label applied to TLC) which was 1000-fold less acive than the alkyl derivative in terms of platelet activation [4]. The compound migrating with the 1- alkyl-derivative accounted for approximately 25% of the radiolabeled 1 -radyl-2,3-diacetylglycerols submitted to TLC. The l-alkyl derivative of PAF was shown to be the compo- nent active in platelet activation [4]. Indeed, as shown in Fig. 4A and Fig. 5, HUVEC synthesized bioactive PAF in response to porin stimulation, as detected by washed-rabbit- platelet aggregation. PAF remained almost completely associated with the cells since no significant release of PAF was detected in the supernatant.

PAF synthesis started 10 min after the addition of porins, peaked at 15 min and decreased thereafter. After 60 min no significant activity could be detected (Fig. 4A). Porin-induced PAF synthesis was dose dependent (Fig. 5). PAF synthesis was detected at 5 pg/ml porin and reached a plateau of maximal synthesis at 10 pg/ml. The effect of porin did not depend on LPS contamination since polymyxin-B-treated porins were still active (data not shown).

PAF bioactive material had biological and physicochemical characteristics identical to synthetic C16-PAF (data not shown). It induced platelet aggregation in an ADP-independent and arachidonic-acid-independent way, which was specifically inhibited by the PAF-receptor antagonists WEB 2170 [38] and CV3988 [7]. PAF activity was destroyed after base-catalyzed methanolysis or treatment with phospholipase A2, indicating the presence of an ester linkage at the sn-2 position [39, 401. Treatment with phospholipase A1 did not inhibit PAF activity using washed rabbit platelets, suggesting that the PAF detected by the bioassay has an ether bond at the sn-1 position [40]. This result also confirms that the acyl- PAF, synthesized by HUVEC, was not detected by the bioassay. PAF activity was resistant to treatment with acids or

0 5 1 0 1 5 2 0 3 0 6 0

Time (min)

51

0 1 0 2 0 3 0

Time (min)

Fig. 4. Time course of PAF synthesis and acetyltransferase activity induced by porins. (A) 5 X lo5 HUVEC were stimulated with 10 I g / d porin (cell-associated PAF, dotted columns; released PAF, white columns). Nonstimulated cells were used as a control (cell- associated PAF, dark columns; released PAF, lined columns). In this figure, PAF concentration refers to 1 ml of supernatant and to the corresponding cell aliquot to allow comparison of the amount of PAF released to that remaining associated with the cell. Vertical bars indicate the standard deviation of the mean for three different experiments performed in duplicate. (B) Time course for acetyltransferase activity assayed in 5 X lo5 HUVEC stimulated with 10 pg/ml porin (A) and without porin (W) as a nonstimulated control. Results are expressed as the mean 5 S D for a single typical experiment performed in triplicate. Three experiments were performed with similar results.

5-1

~

J Y - LL a

0 ’bL==$=s 0. 1 0 2 0

[Porins] (pg/ml)

Fig.5. Dosdresponse of PAF synthesis and release by HUVEC stimulated for 15 min with porins. The cell-associated PAF (A) and PAF released into the supernatant (M) by 5 X 1 W cells treated with different concentrations of porin were measured. Results are the mean 5 SD of three experiments.

weak bases [39]. After base-catalyzed methanolysis or diges- tion with phospholipase A2, treatment with acetic anhydride restored 80-90% of the biological activity [45]. The PAF obtained from HUVEC had the same R, (0.21) as synthetic PAF and migrated between 2-lysoPtdCho (R, = 0.11) and sphingomyelin (R, = 0.29) using TLC analysis with chloro- form/methanol/water (65 : 35 : 6, by vol.) as the solvent. The

690

A 184 I

524 1

"." too 200 300 400 500

dz

524 1

T

0 1 0 2 0 3 0

Time (min) Fig.7. Time course of the release of ['4C]arachidonic acid assayed in 5x105 HUVEC stimulated with 10 j@ml porin (A) and without porin as a nonstimulated control (m). Results are expressed as the mean ? SD of a single typical experiment performed in triplicate. Three experiments were performed with similar results.

100 200 300 400 500

mlz Fig.6. MS analysis of PAF bioactive material obtained from porin-stimulated HUVEC. Spectra of daughter ions from parent species with mlz 524 obtained from a representative sample of 5 X l@ HUVEC treated with 10 Fg/ml porin for 15 min (A) and synthetic C16-PAF used as a standard (B). In the inset, the chroma- tograms of the daughter ions with mlz 184, from parent species with mlz 524, of the corresponding samples are shown. The fragmentation spectra are identical in A and B and only one peak with superimpos- able retention time, can be observed in each chromatogram.

same retention time as synthetic C16-PAF was observed using HPLC analysis (pporasil column) developed with chlo- rofodmethanoVwater (60 : 55 : 5, by vol.). No PAF activity was detected in any other TLC or HPLC fraction. The MS analysis [37] confirmed that PAF bioactive material con- tained 1 -alkyl-PAF. The tandem-MS spectra, obtained from a porin-stimulated HUVEC sample at the retention time for C16-PAF (29.9-30.2 min), exhibited a fragmentation pattern with a molecular ion at mlz 524 and a molecular ion corresponding to phosphorylcholine at mlz 184. This was identical to the spectrum of synthetic C16-PAF submitted to the same extraction and purification procedures (Fig. 6).

The viability of HUVEC after treatment with the experimental concentrations of porin was >95% and the release of lactate dehydrogenase was < 1 %. A significant reduction of cell viability was observed for concentrations greater" than 50-100 pglml porin and after 1 h of treatment (data not shown). When HUVEC were incubated with 50 pglml porin they synthesized 4.6 n g / d PAF with kinetics (peak at 15 min) comparable to that observed with non-cytotoxic concentrations of porins (see Fig. 5).

Enzymic pathway of porin-induced PAF biosynthesis

The incorporation of radiolabeled acetate but not radiolabeled choline into newly synthesized PAF suggests that for HUVEC the synthesis of PAF induced by porins occurs mainly via the remodeling pathway rather than the de novo pathway. The activation of phospholipase A2 is the first step involved in PAF biosynthesis via the remodeling pathway.

As shown in Table 2 and Fig. 7, HUVEC released ['"Clar- achidonic acid after stimulation with porins. The basal level of ['"C]arachidonic acid released by control cells was 0.54% the radiolabel incorporated during the initial incubation. Af- ter stimulation, HUVEC released about 6% of the total label incorporated. 4-Bromodiphenacylbromide, an inhibitor of phospholipase A2 [46], markedly reduced the release of [I4C]arachidonic acid as well as the synthesis of PAF by porin-stimulated HUVEC (Table 2). The inhibition of phospholipase A2 by 4-bromodiphenacylbromide possibly prevented the mobilization of 2-lysoPAF, the substrate for the acetyl-CoA : 1-alkyl-sn-glycero-3-phosphorylcholine 2-0- acetyltransferase (CoASAc:2-lysoPAF acetyltransferase). The second step involved in PAF synthesis by the remodeling pathway is the acetylation of 2-lysoPAF generated from membrane 1 -O-alkyl-2-acyl-sn-glyceryl-2-phosphorylcholine by phospholipase A2. Indeed, the addition of 2-lysoPAF to HUVEC treated with 4-bromodiphenacylbromide restored the synthesis of PAF induced by porins (Table 2). The activity of CoASAc :2-lysoPAF acetyltransferase was studied in parallel with the synthesis of PAF. In nonstimulated HUVEC, basal activity was 0.51 It 0.26 nmollmidmg protein. This enzymic activity increased about eightfold during the synthesis of PAF after stimulation with porins. The increase in CoA- SAC : 2-lysoPAF acetyltransferase activity induced by porins was transient and rapidly decreased at 20-30 min (Fig. 4B); the kinetics could be superimposed on those for PAF synthesis (Fig. 4A).

Role of extracellular Ca" in porin-induced PAF synthesis

In the present study, we also investigated the role of extracellular divalent cations on the synthesis of PAF, by study- ing the dependency on extracellular Ca" concentration and the effect of EDTA or EGTA addition to HUVEC 10 min before stimulation with porins. The results showed that the synthesis of PAF is dependent on Ca2+ concentration (Fig. 8). and is prevented by chelation of extracellular divalent cations or by stimulation of the cells in Ca2+-free balanced-salt solution (Table 3). Addition of 1.4 mM Ca" to HUVEC incubated in Ca2+-free balanced-salt solution (see Materials and Methods), 10 min before stimulation with 10 pg/mI porin, restored PAF synthesis. These experiments suggest that the triggering of PAF synthesis by porins may depend on extra-

691

Table 2. Release of ['4Clarachidonic acid, CoASAc:2-lysoPAF acetyltransferase activity and PAF synthesis by HUVEC stimulated with porins. 5 X lo5 H W E C were incubated at 37°C with 10 p g / d porin for 15 min with or without addition of 4-bromodiphenacylbromide (1 FM) or 4-bromodiphenacylbromide (1 pM) plus 2-lysoPAF (10 pM). The release of ['4C]arachidonic acid was performed as described by Hirata et al. [41]. The acetyltransferase activity and the synthesis of PAF were measured as described in Material and Methods. One-way analysis of variance, with Newman-Keuls multiple-comparison test, was performed between the following : unstimulated cells and cells stimulated with porins in the absence of additions or nonstimulated cells with the addition of 4-bromodiphenacylbromide or 2- lysoPAF (probability not significant) ; porin-treated cells in the absence of additions and after addition of 4-bromodiphenacylbromide; or 4-bromodiphenacylbromide plus 2-lysoPAF; porin-treated cells in the presence of 4-bromodiphenacylbromide and after addition of 4- bromodiphenacylbromide plus 2-lysoPAF. ND, not determined.

Additions [14C]arachidonic acid Acetyltransferase PAF synthesis released activity

CPm nmol . min-' . mg protein-' n d d 0.10 2 0.1 None 9 8 8 k 44 0.51 ? 0.26

Porins 4020 ? 102l 4.21 ? 0.61' 4.40 k 0.6' Porins/4-bromodiphenacylbromide 1174 ? 116' 3.78 ? 0.72 0.70 ? 0.2l 4-Bromodiphenacylbromide 1102 -C 215 0.38 ? 0.23 0.16 ? 0.1 Porins/4-bromodiphenacylbromide/2-lysoPAF 1207 ? 178 ' ND 3.80 ? 0.8 2-LysoPAF 854 -C 166 0.71 +- 0.19 0.21 ? 0.16

'P<O.OS.

1

= 4 E

5 3

. CD

n 2 L

- 1 2 0

0.0025 0.0035 0.013 0.1 1 1.4

[Ca2+] (mM) Fig. 8. Effect of extracellular Caz+ concentrations on PAF synthesis by 5x 105 HUVEC incubated in Ca%-free balanced-salt solution (see Materials and Methods) and stimulated for 15 min with 10 pg/ml porin (dotted columns) or nonstimulated (blank columns). Ca", at the indicated concentrations, was added 10 min before stimulation with porin. Results are expressed as the mean ? SD of three experiments.

Table 3. Effect of chelation of extracellular divalent cations on porin-induced PAF synthesis. 5 X lo" H W E C were stimulated with 10 pg/d porin at 37°C for 15 min in Caz+-free balanced-salt solution or treated with 10 mM EDTA or 10 mM EGTA 10 min prior to stimulation. Values are expressed as the mean ?SD for three different experiments. One-way analysis of variance, with Newman- Keuls multiple-comparison test, was performed between nontreated cells stimulated with porins (None) and cells treated with the indicated compounds and also between cells stimulated with ponns in Caz+-free balanced-salt solution with or without the addition of Caz+ (final concentration 1.4 mM).

Treatment of cells PAF

n g / d None 4.4 ? 0.6 Caz+-free balanced-salt solution 0.5 ? 0.2l Caz+ -free balanced- salt solution/Ca* + 4.2 ? 0.4' EDTA 0.2 t 0.1' EGTA 0.3 ? 0.2l

'P<0.05.

0 10 20 30

Time (min) Fig. 9. Time course of "Ta2+ uptake by 5x105 HUVEC nonstim- dated (m) or stimulated with lOpg/ml porin (A) at 37°C in balanced-salt solution supplemented with 2 mM CaCl, and 0.5 pCi 45Ca. Results are expressed as the mean +SD of the% increase in cpm compared to time 0 from a single typical experiment performed in triplicate. Three experiments were performed with similar results.

cellular Ca2+ influx. Indeed, experiments performed with 45Ca2+ demonstrated that porins induce a rapid and persistent increase in Caz+ influx from the extracellular to the intracellular compartment (Fig. 9).

DISCUSSION The results of the present study indicate that porins, de-

rived from the cell-wall outer-membrane of Gram-negative bacteria, stimulate the synthesis of PAF by HUVEC. Vascular endothelium is highly involved in the physiopathological alterations occurring during septic shock and inflammation [47]. Release of endothelial-derived vasodilators, increased adhesiveness of the endothelial surface to leukocytes and platelets, expression of thrombogenic properties and increased permeability of vascular endothelium underlie the dynamic changes occurring in septic shock and inflammation. These functional alterations were related to the biological action of cell-wall components of Gram-negative bacteria and of mediators produced by the host such as IL-1 and TNF

692

[lo, 111. These cytokines possess a number of biological effects that are relevant to the evolution of the endothelial cell response following inflammatory injury. We recently found that the effect of TNF on the reorganization of the endothelial-cell cytoskeleton, leading to an increase in vascular permeability, is mainly dependent on an autocrine action of PAF when synthesized by TNF-stimulated endothelium [48]. The relevance of PAF to septic shock and inflammation is based on evidence obtained from several studies. The experiments indicating that PAF shares many biological effects with TNF [9]. have suggested that it may act as an important secondary mediator of inflammation and shock induced by this cytokine. The evidence that PAF is produced during endotoxic shock and experimental sepsis by Gram-negative bacteria [5, 6, 49, 501 and that PAF-receptor antagonists inhibit or reverse endotoxin-induced hypotension [5l], indicate that this mediator plays a relevant role in the physiopathological alteration occurring during Gram-negative-bacteria-induced sepsis. Indeed, PAF, when infused into experimental animals, produces hypotension, a decrease in cardiac output and hypo- volemic shock [3]. Moreover, PAF shows a wide range of biological activities that may be relevant in the recruitment of inflammatory cells. In addition to inducing the activation of platelets, PAF promotes the chemotaxis of PMN, it in- creases vascular permeability and alters the vascular tone (for a review see [3, 521). Vascular endothelial cells were shown to be both a target for PAF action and a source of this mediator after challenging with a number of stimuli including angiotensin 11, vasopressin, thrombin, leukotriene C4, leukotriene D4, histamine, bradykinin, IL-1 and TNF [13, 161. We recently demonstrated that porins directly stimulate the synthesis of PAF by PMN [21] and mesangial cells [22], suggesting that components of the outer membrane of the Gram- negative bacterial cell wall may elicit the biological actions dependent onf PAF synthesis. Porins that account for over 50% of the total outer-membrane proteins [19, 201, form transmembrane channels allowing passive diffusion across the outer membrane [23, 241. In the membrane, porins are in trimeric form and a portion of each porin molecule is ex- posed on the cell surface [53]. They are released either during cell growth or during bacteriolysis [54]. Given their resistance to proteolysis [55, 561, they are not degraded and may interact with the cells of the host organism. The released porins are incorporated into the cell membrane with marked changes in lipid and protein phase relations [26]. Depending on the dose, porins may be cytotoxic for target cells or may interfere with cell functions [25, 261. The results of the present study indicate that porins, at non-toxic concentrations, may directly stimulate PAF synthesis by HUVEC. This effect of porins does not depend on LPS contamination, since polymyxin-B-treated porins are still active. The experiments with radiolabeled precursors show two species of [3H]acetate-labeled PAF-related molecules, the 1-acyl derivative of PAF, with low biological activity, and the 1-alkyl PAF which ac- counts for platelet aggregation 141. The presence of the 1- alkyl derivative of PAF in the acetylated phospholipids, produced after porin stimulation, was confirmed by MS and by physicochemical characterization of PAF-like bioactivity. In addition, the incorporation of L3H]acetate but not [3H]choline into the newly synthesized PAF molecules suggests that transient synthesis of PAF by HUVEC, in response to porins, occurs mainly via the remodeling pathway. This involves the activation of two enzymic steps [8]. Firstly, hydrolysis of 2- lysoPAF by phospholipase A2 and secondly, acetylation of 2-lysoPAF at position 2 by a specific acetyltransferase.

The activation of phospholipase A2 was studied measur- ing the release of radiolabel from HUVEc previously incubated with [14C]arachidonic acid [41].

The evidence that the porin-induced release of [‘“Clar- achidonic acid is inhibited by 4-bromodiphenacylbromide, suggests the activation of phospholipase A2 by porins rather than a mobilization of arachidonic acid induced by the phospholipase-C-diacylglyerol-lipase pathway. The inhibition of phospholipase A2 by 4-bromodiphenacylbromide [46] also blocks the synthesis of PAF by preventing the mobilization of 2-lysoPAF, the substrate for PAF-specific CoASAc: 2-ly- soPAF acetyltransferase. Indeed, the addition of exogenous 2-lysoPAF restores the synthesis of PAF in porin-stimulated HUVEC. In addition, porins cause an increase of approximately eightfold in the activity of CoASAc: 2-lysoPAF acetyltransferase. Consistent with the activation of the remodeling pathway, which is known to require a prominent influx of extracellular Ca2+ [32, 571 rather than the mobilization of intracellular stores, the synthesis of PAF by porin-stimulated HUVEC is totally dependent upon the availability of extracellular Ca”. Indeed, the experiment performed with 45Ca2+ demonstrated that porins increased the influx of extracellular Ca”. Since the concentration of free Ca2+ in the cytosol is extremely low, compared to the extracellular fluid, a large gradient tends to drive Ca’+ into the cytosol using porins which form transmembrane channels. Both phospholipase A2 and the acetyltransferase involved in PAF synthesis are Ca2+-dependent enzymes [57, 581. The de novo pathway is not stimulated by porin probably as a consequence of the porin-induced increase in the intracellular concentration of Ca2+, which is known to inhibit the enzymes involved in this pathway [59, 601.

It was recently reported, that pore-forming toxins from Gram-positive bacteria can also induce PAF synthesis by endothelial cells, possibly by a mechanism similar to that of porins [61]. In conclusion, the synthesis of PAF, a potent mediator of inflammation and shock, by HUVEC may be relevant to pathological conditions related to Gram-negative bacterial infections. Since the porin concentration is approximately 2 X lo5 moleculeskell, the lysis of approximately 1 X lo7 Gram-negative bacteria may release an amount of porins sufficient to promote PAF synthesis. Further experiments are needed to determine whether this is the case and to determine to what extent the in vivo inflammation induced by porins [62] is dependent on the synthesis of this mediator.

This work was supported by the National Research Council (CNR); targeted project: ‘prevention and control of disease factors’, subproject: ‘causes of infective diseases’ (CT 9300607. PF41 to G. Camussi).

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outer-membrane porins from gram-negative bacteria stimulate platelet-activating-factor biosynthesis...

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