INFECTION AND IMMUNITY, June 1990, p.

1591-1599

Vol. 58, No. 6

0019-9567/90/061591-09$02.00/0 Copyright 1990, American Society for Microbiology

Signal Transduction in Human Platelets and Inflammatory Mediator Release Induced by Genetically Cloned Hemolysin-Positive and -Negative Escherichia coli Strains
B. KONIG, W. SCHONFELD, J. SCHEFFER, AND W. KONIG* Lehrstuhl fur Medizinische Mikrobiologie und Immunologie, Arbeitsgruppe fur Infektabwehrmechanismen, Ruhr-Universitat Bochum, 4630 Bochum, Federal Republic of Germany
Received 31 August 1989/Accepted 22 February 1990

Incubation of human platelets with the hemolysin-producing Escherichia coli strain K-12 (pANNS211) induced the activation of protein kinase C, aggregation of platelets, calcium influx, low amounts of 12-hydroxyeicosatetraenoic acid (12-HETE), and release of serotonin from dense granules. Nonhemolytic isogenic strains of E. coil 536/21 which differed only in their types of adhesins (MSH+ MS-Fim+; S-MRH+ S-Fim+; P-MRH+ P-Fim+) released neither serotonin nor 12-HETE from human platelets nor induced platelet aggregation. All hemolysin-negative bacteria except E. coli 536/21, without any adhesins, were able to activate protein kinase C reversibly but did not induce calcium influx. Activation of platelets with fluoride, an activator of the GTP-binding protein, was associated with protein kinase C activation, calcium influx, platelet aggregation, serotonin release, and 12-HETE formation. The simultaneous stimulation of platelets with NaF and the nonhemolytic E. coli strains suppressed several of the NaF-induced platelet responses. Membrane preparations isolated from stimulated platelets with hemolysin-negative and hemolysin-positive E. coli showed increased binding of guanylylimidodiphosphate, a nonhydrolyzable GTP analog, and enhanced GTPase activity.

In previous studies with human polymorphonuclear granulocytes (19), it was shown that bacteria differ in their ability to trigger release of inflammatory mediator from cells depending on the receptor structure of their fimbriae as well as the distribution of receptors on the target cells. Pathogenic Escherichia coli strains generally exhibit several characteristics which contribute to their virulence, including the ability to adhere to epithelial cells (14, 22, 36a, 39), to become serum resistant, and to express hemolysin. Two general classes of bacterial adhesins have been described. Type 1 fimbriae demonstrate mannose-sensitive adherence and mannose-resistant pili, with cell attachment unaffected by mannose. The mannose-resistant adhesins defined by their receptor specificities include P-fimbriae, which recognize a glycolipid receptor containing a-galactosyl-1,4-galactose (29, 38); S-fimbriae, which bind to a sialic acidcontaining receptor; and M-fimbriae, which recognize a glycoprotein common to M blood group antigens. Hemolysin has been shown to be a potent virulence factor and a stimulus for mediator release from a variety of cells (3, 8, 34). Little is known about the role of adhesins and the E. coli a-hemolysin in human platelet activation. Platelets exposed to collagen, thrombin, or the calcium ionophore A23187 aggregate and discharge their granular contents in a secretory process (4, 23). In addition to dense granule release, the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) may have a significant role in mediating or modulating inflammatory processes. Platelets are the major source of 12-HETE, which is a potent chemokinetic compound for neutrophils and may mediate leukocyte diapedesis through the vascular endothelium. There is also increasing evidence that HETEs are involved in pathological processes, including a number of inflammatory diseases. In platelets the
*

protein kinase C is supposed to be involved in the cellular activation process. Protein kinase C is a protein-phosphorylating enzyme that is activated by endogenous diacylglycerol or exogenous phorbol esters (15, 19). Upon activation, the cytoplasmic enzyme activity is translocated to the plasma membrane (5, 21). In platelets the translocation of protein kinase C to the particulate fraction is accompanied by the additional appearance of a phospholipid-independent protein kinase C, which has been suggested to be a proteolytic product of protein kinase C (25, 36). However, the physiological role of membrane-associated phospholipid-dependent and -independent protein kinase activity is unknown (5). The disappearance of protein kinase C from the cytosol may provide indirect evidence for C-kinase activation. It is still not clear how protein kinase activity is involved in the platelet activation process. Recently, the role of G-proteins in the activation of cells has been shown (28, 35). The guanine nucleotide-binding proteins serve as membranebound transducers of chemically and physically encoded information. Up to now, G-protein involvement in the cellular responses induced by bacteria has not been studied. Recently we showed that NaF, a direct G-protein activator, is a potent inducer of mediator release in human platelets. It was the purpose of our present study to analyze the signal transduction process for platelet activation induced by adhesin-carrying E. coli strains (mannose-resistant hemagglutination [MRH] and mannose-sensitive hemagglutination [MSH]) and by an isogenic E. coli strain expressing ahemolysin.
MATERIALS AND METHODS Materials. Brain-heart infusion was obtained from Oxoid,

Corresponding author.
1591

and [3H]serotonin (hydroxytryptamine, creatine sulfate, 30.4 Ci/mmol) were purchased from New England Nuclear Corp. DE-52 cellulose was from Whatman. Synthetic leukotrienes

Basingstoke, Hampshire, England). [_y-32P]ATP (5 Ci/mmol)

1592

KONIG ET AL.
TABLE 1. Strains used Relevant characteristics

INFECT. IMMUN.

E. coli strain

536/21 536/21(pGB30int) 536/21(pANN801-4) 536/21(pANN921)

K-12

Hly- MSH- MS-Fim- MRHHly- MSH+ MS-Fim+ Hly- MSH- MS-FimHly- MSH- MS-FimHly- MSH+ MS-Fim+ Hly+ MSH- MS-FimMRH-

K-12(pANN5211)

MR-FimMR-FimS-MRH+ S-Fim+ P-MRH+ P-Fim+ MRH- MR-FimMRH- MR-Fim-

gift from J. Rokach (Merck-Frosst, PointClaire, Quebec, Canada). All other chemicals were purchased from Sigma Chemical Co., Deisenhofen, FRG. Buffer solutions. The medium used for washing the peripheral leukocytes and for mediator release was phosphatebuffered saline (PBS; 0.2 M phosphate, 0.1% NaCl, pH 7.4). CaCl2 (0.6 mM) and MgCl2 (1 mM) were added shortly before the cells were stimulated. Buffer A consists of 20 mM Tris hydrochloride (Tris-HCl, pH 7.5), 0.5 mM EGTA (ethylene glycol tetraacetic acid), 0.5 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Bacterial strains. E. coli 536, isolated from a patient with a urinary tract infection, was the parent strain for the E. coli 536 variants. Strain 536/21 is a spontaneous mutant of strain 536 that has lost the ability to produce hemolysin, the Mrh phenotype, and MSH. E. coli 536/21 was transformed with recombinant plasmids to form E. coli 536/21 variants expressing different types of adhesins (Table 1). Cloning and functional characterization of the plasmid-encoded determinants were performed at the Institut fur Genetik und Mikrobiologie, Universitat Wurzburg, Wurzburg, FRG. Bacterial growth. Brain-heart infusion broth (10 ml) was inoculated with 100 ,ul of an overnight culture; bacterial growth proceeded for 3.5 h at 37C on a shaker (150 rpm) (34). Hemolysin assay. The production of hemolysin was tested on sheep blood agar plates. A quantitative hemolysin assay was performed as described previously (18). Preparation of cells. Platelets were prepared from freshly drawn blood. Briefly, EDTA-blood was centrifuged at 200 x g for 25 min to obtain platelet-rich plasma. Platelets were obtained by centrifugation at 2,200 rpm at 4C. The platelets were washed in PBS and resuspended at about 2 x 108/ml. The purity of the platelets was 98%. Viability of the cells was assayed by light microscopy with trypan blue exclusion as well as by the analysis of lactate dehydrogenase (LDH) release from stimulated and nonstimulated cells. Analysis of LDH was carried out as described previously (17). Platelet aggregometry. A platelet suspension (108 platelets per ml) was stirred at 37C in an aggregometer (Chrono-log Corporation). Aggregation was followed by turbidimetric measurement. The baseline for minimal light transmission was adjusted to the optical density of platelets suspended in buffer. Maximal (100%) light transmission was defined as the optical density of the platelet-free medium (buffer). Platelet aggregation was detected as an increase in light transmission. The lag phase of aggregation was defined as the time interval between the addition of bacteria to the platelet suspension and the onset of aggregation. Ca ionophore A23187, arachidonic acid, and thrombin served as the control agonists for platelet aggregation. Assay for the translocation of protein kinase C activity. Human platelets (2 x 108/ml) were incubated for 10 min at 37C in PBS containing calcium and magnesium. The cells were subsequently activated by the addition of NaF and
were a generous

washed E. coli bacteria, as indicated in the figure legends. Activation of the cells was stopped by centrifugation at 2,200 rpm for 10 min; the cells were then resuspended in 1 ml of buffer A and subsequently sonicated (amplitude, 50 p.m, twice for 10 s each; Branson Sonifier 250). The crude homogenate was centrifuged at 13,000 rpm for 10 min in an Eppendorf centrifuge. The cytosolic fractions were chromatographed on DEAE- cellulose columns (0.5 by 3 cm) equilibrated with buffer A and washed with 3 ml of buffer A. Protein kinase C was eluted with 1.5 ml of buffer A containing 100 mM NaCl. Protein kinase C assay. The reaction mixture (190 ,ud) contained 20 mM Tris-HCl (pH 7.5), 10 mM magnesium acetate, 500 ,ug of histone type IIIS per ml, 10 ,uM [-y32P)ATP (105 cpm/nmol), 2.5 ,ug of diolein, 25 ,ug of phosphatidylserine, 1 mM CaCl2, and the enzyme preparation under study. All reagents were dissolved in distilled water. The quantitation of background phosphotransferase activity was performed by analyzing histone phosphorylation in the absence of calcium and phospholipid. After 10 min at 30C, the reaction was stopped by the addition of 0.75 ml of 25% trichloroacetic acid. Acid-precipitable materials were collected on Millipore membrane filters (pore size, 0.45 ,um; Millititer HA filtration plates; Millipore Corp., Eschborn, FRG). The filters were washed twice with 1 ml of 10% trichloroacetic acid. The dried filters were transferred into scintillation vials, distilled water was added, and the radioactivity was measured by Cerenkov radiation (Rack beta 1209; LKB, Turku, Finland). Protein kinase C activity was calculated by subtracting background activities, and values are reported as percent cytosolic protein kinase C activity in untreated cells. Loading cells with fura-2. The acetoxymethylester of fura2 fura-2AM, was added to 2 x 108 platelets in HEPES buffer (145 mM NaCl, 5 mM KCI, 1 mM Na2HPO4, 1 mM CaCl2, 0.5 mM MgSO4, 5 mM glucose, and 10 mM HEPES [N-

2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7.4]) at a final concentration of 3 ,uM. The cells were incubated at 37C with intermittent mixing to allow complete uptake of the dye. After 30 min, the cells were washed with HEPES buffer. Fluorescence measurements. Freshly loaded platelets were spun down and resuspended at a concentration of 2 x 107 cells per ml in HEPES buffer supplemented with CaCl2 (1 mM) and MgCl2 (0.8 mM). A Perkin-Elmer spectrofluorometer was used with an excitation wavelength set at 340 nm and emission measured at 510 nm. Stimuli were added as indicated in the Results. Maximum fluorescence values (Fmax) were obtained by adding digitonine up to a final concentration of S ,ug/ml, thus permeabilizing the cells for calcium and ensuring that essentially all fura-2 was complexed with calcium. Minimum fluorescence values (Fmin) were subsequently obtained by adding EGTA up to a final concentration of 6.6 mM, to chelate free calcium and to ensure that essentially no fura-2 was complexed with calcium. Intracellular free calcium concentrations were calculated by methods described previously by Tsien et al. 12-HETE release from human platelets. Human platelets (108) were suspended in 0.5 ml of PBS buffer (pH 7.4) with 0.8 mM MgCl2 and 1 mM CaCl2. For stimulation, 100 ,ul of bacterial cell suspension (5 x 108 bacteria) was added to the cells and incubated as described in Results. The supernatant of stimulated cells was analyzed for 12-HETE formation by high-pressure liquid chromatography (17). The mobile phase was a mixture of a K2HPO4 solution (17 mM) containing

VOL. 58, 1990

HEMOLYSIN-POSITIVE AND -NEGATIVE E. COLI

1593

0.05% EDTA, acetonitrile, and methanol (1:0.75:0.75, by

volume) adjusted to pH 5.0 with phosphoric acid. Assay for serotonin release. Washed human platelets were resuspended at a concentration of 109 platelets per ml in PBS containing [3H]serotonin (5-hydroxy[3H]tryptamine; 0.2 piCi/108 cells). The suspension was incubated for 30 min at 37C, during this time, 75% of the 5-hydroxy[3H]tryptamine was incorporated. The labeled platelets were washed twice with PBS and finally suspended at a concentration of 2 x 108 platelets per ml. The release of [3H]serotonin was determined after mixing the platelet suspension with 0.5 ml of 3% paraformaldehyde in PBS. The formaldehyde-treated samples were centrifuged at 1,200 x g for 10 min, and the supernatant was analyzed for 3H radioactivity by liquid scintillation spectrometry. Released [3H]serotonin is expressed as a percentage of the total platelet-bound
LI)

E-zl

zi

C-CZ-

-Z

O_-

10 s

time (mlinutes)

10000
28. mlM5
l-c
Al
C;i

[3H]serotonin.
Binding of Gpp(NH)p. Cells were incubated in the presence of NaF (25 mM), E. coli K-12 (109 cells), and E. coli K-12(pANN5211) (109 cells) or in the absence of any stimulus (buffer control) for 40 min at 37C. The cell suspensions were washed, suspended in 0.5 M Tris-0.34 M sucrose buffer (pH 7.5), and EGTA (1 mM), dithiothreitol (1 mM), and leupeptin (100 ,ug/ml) were added. Cell disruption was carried out by sonication (Branson Sonifier 250). The sonicates were centrifuged at 300 x g for 10 min, and the resulting postnuclear supernatant was further differentiated by centrifugation at 10,000 x g for 20 min (J2-21 centrifuge with a JA-20 rotor; Beckman Instruments, Palo Alto, Calif.) and a 100,000 x g for 60 min (L8-70 Ultracentrifuge, SW 6OTi rotor; Beckman). The 100,000 x g pellet was suspended in Tris-sucrose buffer, assayed for protein content by the method of Bradford (1), and stored at -70C. The binding of 3H-labeled guanylylimidodiphosphate ([3H]-Gpp (NH) p) was determined by a modification of the procedure of Matsumoto et al. (23a). The membrane fraction (10 ,ug of protein) was incubated in 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 1.14 mM ATP, 0.5 mM App(NH)p, 0.25 mM ouabain, and 0.5 ,um[3H]Gpp (NH)p. Nonspecific binding was defined as the amount of [3H]Gpp (NH)p bound in the presence of a 1,000-fold molar excess of nonlabeled Gpp(NH)p. Incubations were terminated after 120 min at room temperature by rapid filtration through cellulose ester membranes (pore size, 0.45 ,um; Millititer HA filtration plates; Millipore Corp. Eschborn, FRG). The filters were washed four times with 20 mM Tris buffer containing 0.25 mM MgCl2 and 50 puM EGTA. The dried filters were measured for radioactivity by liquid scintillation counting. GTPase assay. Membrane protein (10 jig) was added to 170 ,ul of 20 mM Tris-HCl buffer (pH 7.6) containing 150 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 1.14 mM ATP, 0.5 mM App(NH)p, 5 mM phosphocreatinine, 50 U of creatine phosphokinase per ,ul, and 0.25 ,uM [y-32P]GTP (30 Ci/mmol; Amersham Corp.). The assay was run for 120 min and was terminated by addition of 1 ml of 20 mM potassium phosphate buffer (pH 7.5) containing 5% charcoal, 0.5% bovine serum albumin, and 0.1% dextran. The samples were centrifuged for 8 min at 13,000 x g, and the radioactivity in 0.2 ml of the supernatant was counted. GTPase activity is reported as 32Pi released from [-y-32P]GTP. Determination of LDH. LDH (EC 1.1.1.27) was determined as described previously (18). Enzyme release was quantified as percentage of total activities from sonicated cells. Statistics. If not stated otherwise, all data were calculated

-4

0 0/0

"

B
10

10 15 t irre (minutes)

'/
C-I
o,

-I

,F ai

-c
)

15 0~~~~~~~~~~~~~~~~~~~~~~~~~~~

NaF-

31

time (rrlnutes ) FIG. 1. Effect of hemolysin, NaF, and NaF in the presence of E. coli 536/21 on platelet aggregation. Suspensions of platelets were stimulated with (A) 109 E. coli K-12(pANN5211) cells expressing different hemolysin activities, (B) NaF at the concentrations indicated, and (C) either (12.5 mM) NaF or 12.5 mM NaF in the presence of 109 washed bacteria. Aggregation was recorded continuously. Values represent the results from six different platelet preparations.

as means standard deviation

(SD) of three independent

experiments.
RESULTS Platelet aggregation by E. coli strains. Aggregation studies were carried out as a parameter of platelet activation. To 800 ,ul of washed platelets (108/ml) in PBS with calcium and magnesium (0.8 mM), washed bacteria (100 ,ul, 109) or culture supernatant (100 ,ul) was added. The hemolysinnegative bacteria did not induce platelet aggregation. Subsequently, washed E. coli K-12(pANN5211), which expresses hemolysin (from 10 to 100%), in contrast to E. coli K-12, was studied. As shown in Fig. 1, the hemolysin-expressing strain was able to induce platelet aggregation in a dose-dependent manner. Depending on the hemolysin concentration, platelet aggregation started after a lag period of 30 s (100% hemolysin activity) to 3 min (10% hemolysin activity) and reached maximal values within 5 to 30 min from the onset. To verify that platelet aggregation was induced by hemolysin itself,

1594

KONIG ET AL.
6050
4030
20-

INFECT. IMMUN.

hemolysin-containing and hemolysin-free culture supernatants were applied as stimuli. The hemolysin-positive culture supernatant induced platelet aggregation in a dose-dependent manner, while the hemolysin-negative supernatant failed to do so (data not shown). Hemolysin did not act through platelet cytotoxicity, as was examined by light microscopy and LDH release. Since G-proteins have been described to exert a regulatory role in signal transduction, NaF was added as a costimulus for platelet aggregation. Figure 1B shows the aggregation pattern of human platelets induced by NaF. Aggregation started after a lag period of 1 min and reached a maximum within 5 min from the onset at a NaF concentration of 12.5 mM. At lower NaF concentrations, the lag phase increased and maximal values were reached at later incubation times. In the presence of NaF, the onset of aggregation induced by the hemolysin-positive E. coli K-12(pANN5211) was slightly faster than with the bacteria alone. When platelets were incubated with NaF in the presence of the nonhemolytic E. coli strains, which by themselves did not induce platelet aggregation, NaF-induced aggregation was completely inhibited, as is shown for E. coli 536/21 (Fig. 1C). Adhesinbearing and adhesin-deficient strains were similar in their inhibitory effects. Addition of the bacterial strains at various time points (1 to 10 min) after activation of the platelets with NaF did not inhibit platelet aggregation, suggesting that the simultaneous presence of NaF and bacteria is required for suppression. Serotonin release from human platelets. Platelets (108) labeled with [3H]serotonin were incubated with the various E. coli strains. Serotonin release was monitored from 30 s up to 40 min of incubation. The hemolysin-negative strains, independent of their type of adhesin, did not induce serotonin release even at concentrations up to 1010 bacteria. Platelets stimulated with E. coli K-12(pANN5211) revealed a serotonin release which ranged from 10% (hemolysin activity, 55%) to 50% (hemolysin activity, 100%) (Fig. 2A). The release of serotonin obviously occurred 30 s after the stimulus was added and reached a plateau after 20 to 40 min, depending on the hemolysin activity. In addition to bacteriabound hemolysin, the hemolysin-positive culture supernatants ("free" hemolysin) were studied as stimuli for serotonin release. The release of serotonin also occurred 30 s after the stimulus was added and reached a plateau after 20 to 40 min, dependent on the hemolysin activity (data not shown). The release occurred under noncytotoxic conditions, as assessed by trypan blue exclusion and failure of LDH release. NaF is also a potent stimulus for serotonin release. Human platelets respond with the release of serotonin in a time- and dose-dependent manner (Fig. 2B). When nonhemolytic E. coli bacteria and NaF were added simultaneously to platelets, the pattern of the NaF-induced serotonin release was changed slightly compared with the pattern in experiments with NaF alone. E. coli 536/21 and mannose-sensitive E. coli bacteria had no or an inhibitory effect (10 to 20%), depending on the NaF concentration (Fig. 3). The E. coli strains bearing mannose-resistant adhesins showed at low NaF concentrations a stimulatory effect (30 to 50%) as was shown by repeated experiments (Fig. 3). With higher NaF concentrations (>10 mM), the effects were more pronounced. Preincubation of platelets with adherent bacteria for 1 h had no effect on subsequent NaF-induced serotonin release (data not shown). When platelets were incubated with NaF and E. coli K-12(pANN5211) (hemolysin activity, 80%), the results showed that the hemolysin-induced sero-

serotonin release 1% of controll

-*
-A,

E. coli K12 55% hemolyt. activ./

75%

-.-*+-

hemolyt. activ.
-

82% hemolyt. activ.

100% hemolyt. activ.

10
5

10

20

time [mini

serotonin release [% of totall

601 -*- 2.5mM

- A-

12.5mM

40

*- 25mM

II
A~~~~

10

20 time [min]

30

40

FIG. 2. Dose-dependent effect of hemolysin and NaF on serotonin release. Platelets were stimulated with E. coli bacteria of various hemolytic activities (hemolyt. activ.) (A) and with NaF at the indicated concentrations (B) at 37C. Serotonin release was monitored for up to 20 min. Each value represents the mean + SD of at least three independent experiments.

tonin release was not influenced by NaF. There was no additive effect with regard to the amount of serotonin released (data not shown).
Induction of 12-HETE formation from human platelets by E. coli strains. We then studied the effect of the various washed E. coli strains on 12-HETE formation from human platelets. E. coli 536/21 failed to induce the release of 12-HETE. E. coli strains with adhesins induced minimal amounts of 12-HETE (12 5 ng), which were slightly higher than baseline levels, i.e., platelets in the presence of buffer. The differences among the various adhesins did not affect the formation of 12-HETE. With E. coli K-12(pANN5211) as the stimulus, a slight increase in 12-HETE formation compared with the nonhemolytic E. coli strains was observed. The values ranged from 19 6 ng at a hemolysin activity of 13% up to 25 6 ng at a hemolysin activity of 80%. NaF was a very effective stimulus for 12-HETE release from intact platelets. The response was dependent on the concentration of sodium fluoride. The amounts of 12-HETE ranged from 90 29 ng at a NaF concentration of 2.5 mM up to 1,200 300 ng at a concentration of 12.5 mM and declined at an NaF concentration of 25 mM to 900 150 ng. The ability of NaF to induce the release of 12-HETE was markedly reduced (by 85%) in the presence of the nonhemolytic E. coli strains and also by E. coli K-12(pANN5211). Among the nonhemolytic E. coli strains, adhesin-expressing bacteria were better able to inhibit NaF-induced 12-HETE formation (P < 0.05) than E. coli 536/21 (data not shown). As examples are shown the kinetics of NaF-induced 12-HETE

VOL. 58, 1990

HEMOLYSIN-POSITIVE AND -NEGATIVE E. COLI

TABLE 2. Protein kinase C activationa
E. coli strain

1595

Mean hemolysin SD (%) activity

Mean cytosolic protein kinase C activity SD (% of control')

536/21 K-12 K-12(pANN5211)

100 10 82 9 75 6 55 6 133

90 4 58 6 12 2 28 4 43 6 65 12 984

-------

S-MRH P-MRH S-MRH S-Fim P-Fim

PBS MSH MS-Fim

5040-

serotonin release [% of

total]

a Results are means of three independent experiments performed in triplicate. b Control activity was determined with buffer instead of bacteria.

3020-

TI

10v
-------

S-MRH P-MRH S-MRH S-Fim P-Fim

PBS MSH MS-Fim

FIG. 3. Interaction of washed E. coli bacteria with NaF-induced serotonin release from platelets. Platelets were incubated with NaF in the presence of no bacteria or of E. coli bacteria expressing different adhesins. Upper panel, 2.5 mM NaF; lower panel, 25 mM NaF. The incubation time was 15 min at 37C. Each value represents the mean + SD of at least three independent experiments. *, Significant compared with the control.

formation in the presence of E. coli 536/21 (Fig. 4). Maximal inhibition was observed after 20 to 30 min of incubation. Biochenistry of platelet activation. Adhesins do not seem to play an important role in direct platelet activation, which differs from the results obtained with human granulocytes. For this purpose, the biochemistry of platelet activation was studied with only three E. coli strains, E. coli 536/21 (Hly-), E. coli K-12 (Hly-), and E. coli K-12(pANN5211) (Hly+). Translocation of protein kinase C activity. Human platelets (2 x 108) in 1 ml of PBS with calcium (1 mM) and magnesium (0.8 mM) were incubated with the different E. coli bacteria (5 x 108) for various time intervals. The remaining cytosolic
12-HETE release [% of controll
100'
8060 4020' 0 10
20 30

\i
40

time

[min]

FIG. 4. Time course of 12-HETE release from fluoride-stimulated platelets in the presence of E. coli 536/21. Each value represents the mean + SD of at least three independent experiments.

protein kinase C activities were quantitated. As a control, platelets were incubated with phorbol myristic acid (10-8 M) for 10 min. With the loss of protein kinase C from the cytosolic fraction (up to 40% of the buffer control), a concomitant increase in membrane-associated protein kinase C activity (120 versus 100% of the control value, i.e., unstimulated cells) was observed. All E. coli strains except E. coli 536/21 induced protein kinase C activation. The decrease in cytosolic protein kinase C activity was detectable within 5 to 10 min after the addition of bacteria. Cytosolic protein kinase activities in the range of 60 to 80% of the control levels were obtained. The protein kinase C levels of the cytosol returned to basal levels within 20 to 40 min of incubation (data not shown). Platelets incubated with E. coli K-12(pANN5211) (Hly+) showed a dose-dependent activation of protein kinase by hemolysin (Table 2). The translocation proceeded with a lag phase of 2 to 10 min and was most pronounced in cells incubated with higher hemolysin activities (Fig. SA). A return to basal levels was noted after 20 (Hly+ = 20%) to 60 (Hly+ = 80%) min of incubation. To rule out protein kinase C inactivation by hemolysin itself, partially purified protein kinase C from unstimulated platelets was incubated with hemolysin-positive E. coli bacteria and with hemolysin-containing culture supernatants. Inactivation of protein kinase activity was not obtained (data not shown). With fluoride as the stimulus, the translocation of protein kinase was detectable 3 to 5 min after the addition of NaF. Control levels were reached after 20 to 30 min of incubation (Fig. SB). In subsequent experiments, the simultaneous addition of NaF and E. coli bacteria was studied. With E. coli K-12, it became apparent that combined stimulation with NaF did not differ from the bacterially induced protein kinase C activation. Similarly, the pattern of activation by E. coli K-12(pANN5211) was not modulated in the presence of NaF (data not shown). Elevation of intracellular calcium levels. Platelets were loaded with the calcium-sensitive fluorescent dye fura-2, and the intracellular Ca2" concentration (Ca2"i) was measured in subsequent experiments. Calcium ionophore A23187 (10-6 M) as a control resulted in an extremely rapid increase in fluorescence up to values obtained with digitonin for Fmax. E. coli K-12(pANN5211) induced rapid substantial increases in platelet calcium over a concentration range of 10 to 100% of hemolysin activity (Table 3). The increase started after 30 s and reached the maximal value of 1,000 nM within 3 min with bacteria expressing 100% hemolysin (Fig. 6). Bacteria with 10% hemolysin reached a value of 200 nM after 3 min, while for those with 60% hemolysin a value of 500 to 700 nM was obtained. No increase in Ca2"i was observed when platelets were stimulated with the nonhemolytic strain E.

1596

KONIG ET AL.

INFECT. IMMUN.

A cytosolic PKC activity [% of controli 100I

80 -I 6040 -

--------

0) C) 0) C) 0 0)
0

200

10 time [mini

20

30

80% hemolytic activ. * 60% hemolytic activ. 10% hemolytic activity

a

B PKC activity [%
100

of controll
(a

0)

8060-*-

cz

C)

0)

1\.

0,

6 mM 12.5 mM 28.5 mM 5

401
200

-,-

i
0 2 4 m

-0-

0
10 time [mini
20

[min]

30

FIG. 5. Time- and dose-dependent activation of cytosolic protein kinase C (PKC) by hemolysin and NaF in human platelets. Platelets were treated with hemolysin of the indicated hemolytic activity (activ.) (A) or with NaF at the indicated concentrations (B) for various times. The control was untreated. Results are means SD from three independent experiments.

FIG. 6. Calcium influx caused by E. coli K-12(pANN5211) and NaF. Typical fluorescence tracing after addition of (upper panel) E. coli K-12(pANN5211) bacteria (50% hemolytic activity) and (lower panel) (25 mM) NaF to human platelets. Experiments were carried out as described in Materials and Methods.

coli K-12 (data not shown). When NaF was used as a stimulus for platelet activation at concentrations from 2.5 to 12.5 mM, the rise was less (500 nM at 5 mM and at higher concentrations) compared with the increase obtained with the calcium ionophore A23187 and E. coli K-12(pANN5211). Figure 6 presents a typical fluorescence tracing after stimulation of human platelets with NaF (25 mM) at the optimal concentration. The simultaneous stimulation of platelets with NaF (12.5 mM) and nonhemolytic E. coli strains (K-12
TABLE 3. Effect of Ca ionophore A23187, NaF, and bacteria on intracellular calcium levels in human plateletsa
Stimulus
Mean increase in Ca2.i + SD (nM)

Lag phase (min)

Time of maximal Ca2+i (min postaddition)

Ca ionophore A23187 NaF 2.5 mM 12.5 mM 25 mM E. coli K-12(pANN5211) hemolysin (% activity) 100 60

720 120

<1

<1

300 40 310 60 300 60

1 <1 <1

6 <1 <1

10 a Results are means for experiments performed in triplicate with three different donor cells.

920 180 730 120 300 40

<1 <1 >1

1 1-3 2-4

and 536/21) did not change the elevation of Ca2 i induced by NaF alone. Binding of Gpp(NH) to cell membranes. The binding of nonhydrolyzable Gpp(NH)p to isolated membranes of platelets was determined. Intact platelets (2 x 108/ml) were incubated in the absence of any stimulus (PBS control) or in the presence of NaF (25 mM), E. coli K-12, or E. coli K-12(pANNS211) expressing hemolysin with an activity ranging from 10 to 40%. After an incubation time of 40 min at 37C, the reaction was stopped. The membrane fraction was prepared as described in Materials and Methods, and the specific binding of [3H]Gpp(NH)p (0.5 puM) was examined. It is apparent (Fig. 7) that platelet membranes revealed an increased binding capacity for Gpp(NH)p after NaF stimulation compared with unstimulated cells. An increased binding capacity for Gpp(NH)p was also seen after stimulation with E. coli K-12 and E. coli K-12(pANN5211) compared with unstimulated cells. In comparison to E. coli K-12-stimulated platelets, an increased binding capacity for Gpp(NH)p after K-12(pANN5211) stimulation was obtained; the increase was dependent on the hemolysin activity. The results reveal that hemolysin is a stimulus which augments the specific binding of Gpp(NH)p to platelet membranes. GTPase activity in stimulated platelet membranes. GTPase activity in membranes from platelets incubated with NaF, E coli K-12, and E. coli K-12(pANN5211) (Hly') was monitored. Intact platelets (2 x 108/ml) were incubated either with buffer (PBS) or in the presence of NaF (25 mM), of E. coli K-12, or of E. coli K-12(pANN5211) expressing hemolysin with an activity of 80%. After an incubation time of 15 min at 37C, the reaction was stopped. The membrane fraction was prepared as described in Materials and Meth-

VOL. 58, 1990

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30002500200015001000-

Binding of Gpp(NH)p [cpml

500 NaF K12 hemolytic activity 0%

PBS

FIG. 7. Binding of Gpp(NH)p to

were

K12pANN 5211 10% 40% 20% platelet membranes. Platelets

stimulated with buffer, 25 mM NaF, E. coli K-12, or E. coli K-12(pANN5211) expressing different hemolysin activities. The incubation proceeded for 40 min. The experiment shown is representative of at least three experiments.

ods, and the hydrolysis of [32P]GTP

was determined. A slight stimulation of GTPase activity was detected upon addition of E. coli K-12 (Acpm, 3,044 84) compared with the PBS control (Acpm, 10 30). NaF and E. coli K12(pANN5211) (Hly+) increased GTPase activity 6-fold (Acpm, 20,870 580) and 10-fold (Acpm, 28,224 245), respectively.

DISCUSSION Our data clearly demonstrate that hemolysin-positive E. coli bacteria and hemolysin-negative E. coli strains activate the signal transduction cascase of platelets in a different
way, as

shown by platelet activation (serotonin release,

aggregation, 12-HETE formation) and early stimulus-transducing events (G-protein involvement, protein kinase C, calcium mobilization, and G-protein-mediated activation by NaF). Isogenic E. coli strains with defined types of adhesins and hemolysin expression were studied. The hemolysin-positive E. coli strain K-12(pANN5211) (Hly+) as well as the hemolysin-positive culture supernatants induced platelet aggregation and serotonin release in a dose-dependent manner. E. coli K-12(pANN5211) also induced 12-HETE formation. All nonhemolytic bacteria failed to induce serotonin release from platelet dense granules. Consistent with these results, the nonhemolytic bacteria, unlike the hemolysin-positive E. coli strains, did not induce irreversible platelet aggregation. In earlier investigations, it could be demonstrated that the various adhesins and the hemolysin of E. coli are potent
pathogenicity factors (19). In human granulocytes and rat mast cells, E. coli with S-type mannose-resistant adhesins was the most potent inducer for leukotriene formation and histamine release (17); E. coli bacteria with P-type mannoseresistant adhesins were less active. Mannose-sensitive adhesins seemed to play no role in leukotriene formation and histamine release. With human lymphocytes, monocytes,

and basophils, the various kinds of adhesins differed in their ability to inhibit cellular responses by a subsequent stimulus. The activation of the inflammatory cells was significantly enhanced in the presence of hemolysin. It is apparent from our data that E. coli a-hemolysin is a potent component in platelet activation. In contrast to human polymorphonuclear

leukocytes, lymphocytes, monocytes, and basophils, human platelets did not show adhesin-specific mediator release. A failure of receptor specificity on human platelets may account for the different results obtained. No data exist as to the signal-transducing events in platelet activation by whole bacteria or defined pathogenicity factors of E. coli, e.g., the a-hemolysin. The role of protein kinase C in platelet activation and regulation has been the subject of numerous investigations (26, 32, 33). It has been suggested that some human platelet responses are mediated at least in part through the activation of the protein kinase C system. Stimulation of platelets by neurotransmitters, hormones, and biologically active substances results in the activation of two enzymes, myosin light-chain kinase and protein kinase C (13, 32). Myosin light-chain kinase, a calmodulin-dependent enzyme, requires an increase in free cytoplasmic calcium concentration for its activation (9), while protein kinase C stimulation also occurs at resting calcium levels (30). The hemolysin-positive E. coli K-12(pANN5211) induced protein kinase C activation in a dose-dependent manner as well as a rise in intracellular calcium levels. Hemolysin activity greater than 50% was required to induce serotonin release. These activities exceeded those required for protein kinase activation in platelets. These results suggest that serotonin release by hemolysin might not be due to the activation of protein kinase alone. All nonhemolytic bacteria except E. coli 536/21, which has no adhesins, activated protein kinase C but did not induce calcium influx. These results suggest that stimulation of platelet aggregation and serotonin release might not be due to the activation of protein kinase C alone. The data are consistent with earlier observations in which the independent induction of protein kinase C activation and calcium mobilization was shown by using the exogenous addition of synthetic diacylglycerol or 12-O-tetradecanoylphorbol-13-acetate for the former and the calcium ionophore A23187 for the latter activation process (2, 9, 40). Under appropriate conditions, protein kinase C activation and calcium mobilization also act synergistically to elicit a physiological response of the platelets, e.g., the release of serotonin. Human platelets respond to agonists by generating free arachidonic acid, the precursor of thromboxane A2 and 12-HETE. E. coli K-12(pANN5211) induced only low amounts of 12-HETE. All hemolysin-negative E. coli strains, independent of their adhesins, induced the release of only negligible amounts of 12-HETE. Although the regulatory mechanisms which underlie the arachidonic acid release process are not clearly understood, a crucial role for calcium is indicated by the ability of the Ca ionophore A23187 to generate arachidonic acid and 12-HETE from platelets. The existence of a calcium- and phospholipid-independent form of protein kinase C as well as genetically different subsets of protein kinase C (7, 11, 12, 16, 20, 24) suggest that the preferential activation of these kinases may modulate different aspects of the cellular responses. To transduce the information across the cell membrane, most tissues seem to have at least two major receptor classes. One class depends on the generation of cyclic AMP as a second messenger, while the other class of receptors induce a rapid turnover of inositol phospholipids as well as mobilizing Ca2". Although the role of the two guanine regulatory proteins G. and Gi in the adenylate cyclase system is well established, their possible involvement in platelet responses induced after stimulation with E. coli hemolysin or adherent E. coli bacteria has not yet been examined.

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KONIG ET AL.

INFECT. IMMUN.

Fluoride by itself induces serotonin release, platelet aggregation, and 12-HETE formation in a time- and dosedependent manner. Its activation is due to activation of G-protein by mimicking the -y-phosphate of GTP. Since hemolysin-positive E. coli induce similar activities in platelets even though 12-HETE formation is rather low, it appears that the G-protein system is closely involved in signal transduction. Our results show that G-proteins are involved in platelet activation induced by hemolysin-positive and hemolysinnegative E. coli bacteria. An increased binding of the nonhydrolyzable GTP analog Gpp(NH)p to stimulated platelet membranes as well as an increase in GTPase activity was obtained. The role of G-proteins as regulatory units was shown by the simultaneous stimulation of platelets with NaF and E. coli bacteria. NaF does not alter the response of platelets towards E. coli K-12(pANN5211) with regard to platelet aggregation and serotonin release. In comparison to nonhemolytic E. coli strains, E. coli K-12(pANN5211) does not inhibit 12-HETE release induced by NaF. Thus, NaF and hemolysin do not counteract each other in the cellular transduction system. One may suggest that the state of activation of G-proteins (Gi, Gj) is an important parameter for cellular responsiveness. In fact we recently demonstrated that the ligand receptor-induced leukotriene formation by formyl-methionyl-leucyl-phenylalanine (fMLP) is modulated in the presence of NaF and further that previous activation of granulocytes by NaF also modulates the fMLP-induced cellular responses. These results emphasize the important role of G-protein prestimulation for the subsequent response. Several conclusions can be drawn from the results observed in platelets: (i) activation of protein kinase C alone is not sufficient for the complete signal transduction leading to inflamnmatory mediator release; (ii) aggregation and serotonin release are regulated differently from 12-HETE formation; (iii) the G-protein system seems to be involved in cell activation by hemolysin and nonhemolytic bacteria; and (iv) nonhemolytic E. coli strains counteract the NaF-induced platelet activation while no effect is obtained for the ahemolysin. Experiments will be carried out to study the role of protein kinase C and guanine nucleotide-binding proteins in subsequent platelet activation by hemolysin-positive and hemolysin-negative E. coli strains.
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