Immunology 1979 35 953
Activation of human platelets by platelet activating factor (PAF) derived from sensitized rabbit basophils* MARTHA C. O'DONNELLt, P. M. HENSON & B. A. FIEDEL Departments of Immunology, Rush Medical College, and Microbiology, University of Illinois at the Medical Center, Chicago, Illinois 60612, U.S.A. and the Department of Pediatrics, National Jewish Hospital and Research Center, Denver, Colorado 80206, U.S.A.
Received 5 January 1978; acceptedfor publication 23 March 197 8
tion reactions induced by this agent are separable and that this cross-species activation may be incomplete. Whether this is a result of the differential ability of rabbit PAF to bind to and activate rabbit as compared to human platelets or to the existence of a family of PAF molecules is not yet known. The capacity of PAF to participate in a secretory event involving human platelets lends support to the belief that PAF may play an important ubiquitous role in the cooperative, leucocyte-dependent, release of vasoactive amines which results in increased vascular permeability.
Summary. Rabbit basophil-derived platelet activating factor (PAF), a mediator of anaphylaxis, induces the aggregation and release of serotonin froin rabbit platelets. In the present study, we report that PAF obtained by challenge of specifically sensitized rabbit basophils induced the noncytotoxic release of serotonin from human platelets; maximal extent of release ranged between 34-46%. This release was unaltered in the presence of indomethacin, indicating that such secretion was not a consequence of contaminating arachidonic acid; further, as previously demonstrated with platelets of rabbit origin, it was markedly independent of a requirement for an intact prostaglandin biosynthetic pathway. In contrast to its effect upon rabbit platelets, rabbit PAF did not induce aggregation of human platelets, suggesting that the aggregation and secre-
INTRODUCTION Platelet activating factor (PAF) is a low molecular weight phospholipid (Benveniste, Le Couedic, Polonsky & Tence, 1977b; Benveniste, Kamoun and Polonsky, 1975; Kater, Austen & Goetzl, 1975) released from sensitized basophils challenged with specific antigen (Benveniste, Henson & Cochrane, 1972) or from basophils incubated under alkaline conditions (Benveniste, 1974). PAF has been shown to induce platelet aggregation and noncytotoxic release of platelet vasoactive amines (Henson, 1970). This cooperative reaction between leucocytes and platelets, originally described in the rabbit (Barbaro & Zvaifler, 1966) where PAF has been strongly
*Supported by grant AI-12870-03 from the National Institutes of Health, a grant from The Chicago Heart Association (BAF), and a Biomedical Research Support grant (No. 5-SO7RR05-14) from the National Institutes of Health
(BAF). tPresented in partial fulfillment of the requirements for the Doctor of Philosophy degree in the Graduate College, University of Illinois, at the Medical Center. Correspondence: Dr B. A. Fiedel, Department of Immunology, Rush Medical College, 1725 West Harrison Street, Chicago, Illinois 60612 U.S.A. 0019-2805/78/1200-0953$02.00 © 1978 Blackwell Scientific Publications
953
954
Mat/tha C. O'Doninell, P. M. Henson & B. A. Fiedel
implicated as a mediator of immune complex disease (Henson & Cochrane, 1971), has been reported also to exist in man (Benveniste, 1974), rat (Kater et al., 1975) and hog (Benveniste et al., 1975), suggesting that PAF may play an important role in leucocyte-dependent vasoactive amine release in higher species generally. Rabbit PAF shows a strong binding affinity for albumin (Benveniste et al., 1972) and weaker affinities for neutrophils, erythrocytes and lymphocytes (Shaw & Henson, 1976). Original reports indicated that PAF induces activation of both rabbit and human platelets. More recently, however, rabbit PAF has been reported not to activate human platelets (Shaw & Henson, 1976) and further, it has been suggested that earlier reports concerning cross-species activation may have been a result of a contamination with the prostaglandin endoperoxide precursor arachidonic acid (Benveniste, Camussi & Mencia-Haerta, 1977a). We report herein that rabbit PAF derived from antigen-stimulated basophils and prepared by simple ethanol extraction can induce the noncytotoxic release of ["4C]-serotonin(5HT) from isolated human platelets; that such activation is not due to contamination with arachidonic acid; and that this reaction can occur in the absence of an intact prostaglandin biosynthetic pathway. In addition, we report that antigen-derived rabbit PAF does not lead to the aggregation of human platelets. MATERIALS AND METHODS
Solutions The following solutions used for the preparation of PAF were prepared as previously described (Henson, 1976): isotonic phosphate-buffered saline (PBS), Tris-Tyrode's solution without Ca++ or Mg++ both in the presence and absence of ethylene glycol tetraacetate (EGTA). Tyrode's bovine serum albumin (BSA), and Tyrode's gelatin. All solutions were adjusted to pH 7-2. Diluents A, B, and C used in the isolation of platelets were plepared as described previously (Fiedel & Gewurz, 1 976a).
Immunization
Preparation ofPAF Blood was drawn from both normal and immune rabbits into 1/9 volume of 3-8 % sodium citrate with potassium soibate. The procedure thereafter was similar to that previously described (Henson, 1976). The blood was sedimented at 500 g (1600 rev/miii, 20 min, 12°) in polycarbonate roundbottomed tube3 (DuPont/Sorvall Co., Newton, Conn.), the platelet-rich plasma discarded, the buffy coat transferred to 50 ml plastic centrifuge tubes (Corning Glass Works, Corning, New York) with a siliconized Pasteur pipette, mixed with an equal volume of 2-5 % gelatin in PBS and incubated (30 min, 370) to sediment most of the contaminating erythrocytes. The supernatant cell suspension was removed and centrifuged at 500 g (1500 rev/min, 15 min, 120). The cell pellet was resuspended in a small volume of Tris-Tyrode's -EDTA solution in the absence of Ca++ or Mg++ and the remaining erythrocytes were removed by hypotonic lysis; residual platelets were removed by four consecutive washes (180 g, 8 min, 120) in Tris-Tyrode's solution lacking Ca++ or Mg++. The resultant cell pellet was readjusted to yield 106 basophils/ml with Tyrode's BSA and, on the average, contained 6 % basophils as detei mined by staining with toluidine blue. Immune cell suspensions were challenged with HRP (50 jig peroxidase/106 basophils) and incubated for 20 min at 37°. The cells were removed by centrifugation at 500 g (1500 rev/min, 10 min, 120) and supematant fluids containing PAF-albumin were removed for extraction of PAF by addition of ethanol to 80% (a procedure which also ensured the removal of any contaminating thrombin); after 15 min at room temperature the precipitated protein was removed by centrifugation at 2500 g (3000 rev/min, 15 min, 40). The PAF was stored in ethanol at -70° until the day of use, when aliquots were blown to dryness and redissolved in Tyrode's gelatin at a final concentration 0-4 - 4 x the original PAF concentration in the Tyrode's-BSA cell suspension. The secretion of platelet serotonin induced by these preparations was inhibited by theophylline and enhanced in the presence of cytochalasin B as reported (Henson &
Oades, 1976).
New Zealand white rabbits were immunized sub-
cutaneously with horseradish peroxidase (1 mg/ml in saline; HRP type 1I, Sigma Chemical Co., St. Louis, Mo.) (Henson, 1976), reimmunized with 5 mg/ml HRP at intervals of 1-3 months and bled ten days post secondary immunization via cardiac puncture.
Platelet preparation Normal rabbit or human blood was drawn into 1/9 volume acid citrate-dextrose or 1/7 volume EDTA and centrifuged at 180 g (800 rev/min, 20 min, 12'). Waslhed platelets were prepared from the platelet-
955
Activation of human platelets by rabbit PAF
rich-plasma (PRP) using previously described methods (Fiedel & Gewurz, 1976a). PRP was centrifuged at 2500 g (3000 rev/min, 15 min, 120) into 1 ml of 40% BSA (Sigma Chemical Co.), the platelets were removed with a siliconized Pasteur pipette, washed once in Diluents A and B (re-centrifuging into the BSA solution), and resuspended to 06 x 109 platelets/ml in Tyrode's gelatin. The platelet suspension was then incubated (45 min, 180) in a 50-ml plastic centrifuge tube with [14C]-5-hydroxytryptamine (serotonin) ([14C]-5HT; Amersham/Searle Corp., Arlington Heights, Illinois) at a final concentration of 0-025 pCi/ml. Release ofplatelet [14C]-5HT The standard reaction mixture in siliconized glass tubes (Chrono Log Corp, Havertown, Pa.) contained 0 4 ml of 0X6 x 109 platelets/ml (final concentration, 5 x 108 platelets/ml) and varying amounts of Tyrode's gelatin and PAF to give a final volume of 0-5 ml. The reaction tubes were incubated (15 min, 370, with stirring), cooled on ice, and centrifuged at 2500 g (3000 rev/min, 15 min, 40) (Henson, 1976). Several reaction tubes containing labelled human platelets were preincubated (10 min, 370, with stirring) with theophylline or cytochalasin B in Tyrode's gelatin or with indomethacin in saline (adjusted to pH 8 with NaOH) (Sigma Chemical Co.) prior to the addition of PAF. Aliquots (200 il) of the supernatants were assayed for radioactivity in a Beckman LS-250 Liquid Scintillation Counter (Beckman Inst. Co., Fullerton, Calif.) and released [14C]-5HT was determined. Generally, 0 4 ml of platelets contained 12-20 x 103 c.p.m.
Viability studies Human platelets were isolated using 40% BSA, labelled in the first wash by sequential addition of [14C]-5HT and sodium 5'chromate (51Cr; New England Nuclear, Boston, Mass.) at final concentrations of 0-025 and 10 ptCi/ml, respectively, and incubated for 30 min at 370 (Fiedel & Gewurz, 1976b). Platelets were washed in diluents A and B (recentrifuging into 40% BSA) and resuspended to 0-6 x 109 platelets/ml in Tyrode's gelatin. Following incubation with PAF (15 min, 370, with stirring), the cells were centrifuged at 2500 g (3000 rev/min, 15 min, 4°). The supematants were assayed for released [14C]-5HT, and both supematantaliquots (200jl) and cell pellets were assayed for 51Cr in a Nuclear-Chicago well-type gamma counter.
RESULTS The effect of antigen-stimulated rabbit basophilderived platelet activating factor (PAF) upon the secretion of serotonin from human platelets To determine whether human platelets undergo a release reaction in the presence of rabbit PAF, increasing amounts of PAF were incubated with isolated human platelets (5 x 108/ml) for 15 min and the release of [14C]-5HT was determined (Fig. 1). The amount of [14C]-5HT released increased to a maximum value of between 34 and 46% with the addition of 50 jl PAF and declined slightly as he PAF concentration was increased; incubation of PAF with labelled rabbit platelets resulted in a similar release of serotonin. To assess whether the observed release was due to a cytotoxic effect, human platelets were double-labelledwith [14C]-5HT and r'Cr, incubated with 90 pl of PAF and the differential release of each label was determined. A 34-39%releaseof ['4C]-SHTwasnotedintheabsence of any substantial leakage of the "Cr label ( < 6%), thus indicating that the PAF-induced release of serotonin was noncytotoxic in nature.
Activation of human platelets by rabbit PAF, and absence of contamination with arachidonic acid or requirement for prostaglandin intermediary metab-
olism The existence of a molecular species nominally designated as PAF that induces secretion from both rabbit and human platelets is in contrast to a report suggesting that PAF exhibits type and species 50z 40-
30°5 20to
10 10
20
30
40
50 60 F4F (1)
70
80
90
100
Figure 1. Effects of increasing amounts of rabbit basophilderived PAF upon the secretion of [14C]-5HT from human platelets. Each curve represents the mean value of duplicate samples (internal variation 20
_im (0%)
w 20cr 10-
4/ 1
3 2 TIME (min)
4
Figure 2. Effects of 20 FiM indomethacin (IND) upon arachidonic acid (0-6 mM; AA)-induced platelet aggregation and secretion of [14C]-5HT (% release shown in parenthesis) from human platelets. Insert represents the effect of IND upon the secretion of [14C]-5HT from human platelets in which PAF is used in limiting concentration.
Activation of human platelets by rabbit PAF made to elicit a similar aggregation response from human platelets. However, whether dilute or concentrated PAF preparations were utilized (or whether a secretory event was occurring), no aggregation was observed up to 20 min post challenge with PAF. It would thus appear that the effects of rabbit PAF upon human platelets are, in part, functionally distinct from its observed effects upon platelets of rabbit origin and, further, that platelet aggregation and secretion induced by this mediator are separable events. DISCUSSION
PAF appears to be an important mediator of anaphylaxis in a number of higher species. In the present investigation, we report that PAF prepared by ethanol extraction of supernatants obtained from specifically challenged suspensions of rabbit basophils can induce the secretion of serotonin from human platelets. The dose-response curves (Fig. 1) are remarkably similar in shape to curves reported for the rabbit platelet system (Henson, 1976) although the maximum release values were generally 12-24% lower in the human system. PAF-induced secretion from human platelets also demonstrated the characteristic responses to pharmacological agents reported for rabbit platelet secretion (Henson & Oades, 1976). Theophylline, an agent which increases cAMP levels in platelets (Becker & Henson, 1973), effectively inhibited secretion of [14C]5HT while cytochalasin B, a drug thought to disrupt microfilaments (Wessells, Spooner, Ash, Bradley, Luduena, Taylor & Yamada, 1971), enhanced ielease. PAF induces platelet shape change, aggregation and release of serotonin from rabbit platelets independent of endogenous ADP and products formed from arachidonate (Cazenave et al., 1977; Shaw et al., 1977). In the studies reported here, PAF-induced secretion of serotonin from human platelets was not altered in the presence of indomethacin, (Fig. 2), which inhibits arachidonateinduced secretion of serotonin, indicating that such secretion was not caused by contaminating AA and that the reactivity, as shown with rabbit platelets, is markedly independent of an intact prostaglandin biosynthetic pathway. The induction of [14C]-5HT release from human platelets by rabbit PAF contradicts a previous report that rabbit PAF does not induce human
957
platelets to secrete serotonin (Shaw & Henson, 1976). Whereas the rabbit PAF preparations utilized herein fit many of the reported criteria, they appear capable only of activating human platelets to secretion (of 5HT) without an ability to induce their aggregation in contrast to their effects upon platelets of rabbit origin. The basis for this disparity is not yet clear. Although the release from platelets of serotonin and ADP are thought often to occur in tandem, it is possible that the absence of platelet aggregation is related to an altered secretion and/or utilization of endogenous ADP by the platelet; however, in the rabbit PAF has been reported to induce platelet aggregation in the absence of a requirement for ADP (Cazenave et al., 1977). Whether this is also true for human platelets remains to be elucidated; thus, the possibility that endogenous ADP is not participating in these reactions awaits clarification. Human platelets have been reported to be 20-fold less effective in binding rabbit PAF than rabbit platelets, and the binding of PAF to the membrane has been implicated as the primary process involved in PAF-induced activation (Shaw & Henson, 1976). It is clear that appropriate internal activation events must also occur as rabbit PAF absorbs to rabbit neutrophils but does not stimulate the neutrophil to secretion, hexose monophosphate shunt activity, chemotaxis or significantly alter neutrophil response to stimuli such as zymosan-complement or C5a (Shaw & Henson, 1976). Thus, it is possible that the incomplete stimulation of human platelets by rabbit PAF might have as its basis the differential ability of PAF to bind to the respective blood elements and/or the absence of appropriate activation events. The existence, however, of a family of related but functionally distinct PAF molecules cannot be excluded; and is supported by the finding that PAF liberated by rabbit lung tissues has properties similar to basophil-derived PAF, but is not identical since platelets desensitized to basophil PAF still respond to lung-derived PAF (Kravis & Henson, 1975). Attempts are presently underway to define and compare, both biologically and chemically, PAF obtained from different species in hopes of determining the basis of this diversity. REFERENCES BARBARO J.F. & ZVAIFLER N.J. (1966) Antigen-induced histamine release from platelets of rabbits producing homologous PCA antibody. Proc. Soc. exp. Biol. Med. 122, 1245.
958
Martha C. O'Donnell, P. M. Henson & B. A. Fiedel
BECKER E.L. & HENSON P.M. (1973) In vitro studies of immunologically induced secretion of mediators from cells and related phenomena. Adv. Immunol. 17,93. BENVENISTE J., HENSON P.M. & COCHRANE C.G. (1972) Leukocyte-dependent histamine release from rabbit platelets. J. exp. Med. 136, 1356. BENVENISTE J. (1974) Platelet-activating factor, a new mediator of anaphylaxis and immune complex deposition from rabbit and human basophils. Nature (Lond.), 249, 581. BENVENISTE J., KAMOUN P. & POLONSKY J. (1975) Aggregation of human platelets by purified platelet activating factor from human and rabbit basophils. Fed. Proc. 34, 985. BENVENISTE J. & POLONSKY J. (1976) Isolation and partial characterization of platelet activating factor (PAF). Fed. Proc. 35, 516. BENVENISTE J., CAMussi G. & MENCIA-HUERTA J.M. (1977a) Cellular origin of platelet-activating factor (PAF). Fed. Proc. 36,1329. BENVENISTE J., LE COUEDIC J.P., POLONSKY J. & TENCE M. (1977b) Structural analysis of purified platelet-activating factor by lipases. Nature (Lond.), 269, 170. CAZENAVE J-P., BENVENISTE J. & MUSTARD J.F. (1977) Platelet activating factor (PAF) aggregates platelets independently of the release reaction and the arachidonate pathway. Fed. Proc. 36,454. FIEDEL B.A. & GEWURZ H. (1976a) Effects of C-reactive protein on platelet function. I. Inhibition of platelet aggregation and release reactions. J. Immunol. 116, 1289. FIEDEL B.A. & GEWURZ H. (1976b) Effects of C-reactive protein on platelet function. II. Inhibition by CRP of platelet reactivities stimulated by poly-L-lysine, ADP, epinephrine, and collagen. J. Immunol. 117,1073. FIEDEL B.A., SIMPSON, R.M. & GEWURZ, H. (1977) Effects of C-reactive protein on platelet function. III. The role of cAMP, contractile elements, and prostaglandin metabolism in CRP-induced inhibition of platelet aggregation and secretion. J. Immunol. 119, 877. HAMBERG M., SVENSSON J. & SAMUELSSON B. (1974) Prostaglandin endoperoxides: A new concept concerning the
mode of action and release of prostaglandins. Proc. nato. Acad. Sci. U.S.A. 71, 3824. HENSON P.M. (1970) Release of vasoactive amines from rabbit platelets induced by sensitized mononuclear leukocytes and antigen. J. exp. Med. 131, 287. HENSON P.M. & COCHRANE C.G. (1971) Immune complex disease in rabbits. The role of complement and of a leukocyte-dependent release of vasoactive amines from platelets. J. exp. Med. 133, 554. HENSON P.M. (1976) Activation and desensitization of platelets by platelet-activating factor (PAF) derived from IgEsensitized basophils. I. Characteristics of the secretory response. J. exp. Med. 143,937. HENSON P.M. & OADES Z.G. (1976) Activation of platelets by platelet-activating factor (PAF) derived from IgEsensitized basophils. II. The role of serine proteases, cyclic nucleotides, and contractile elements in PAF-induced secretion. J. exp. Med. 143,953. KATER L.A., AUSTEN K.F. & GOETZL E.J. (1975) Identification and partial purification of a platelet activating factor (PAF) from rat. Fed. Proc. 34,985. KRAVIs T.C. & HENSON P.M. (1975) IgE-induced release of a platelet-activating factor from rabbit lung. J. Immunol. 115, 1677. NEEDLEMAN P., MINKES M. & RAZ A. (1976) Thromboxanes: Selective biosynthesis and distinct biological properties. Science, 193,163. SHAW J.Q. & HENSON P.M. (1976) Platelet activating factor (PAF) binding to blood cellular components: type and species specificity. Fed. Proc. 35, 516. SHAW J.O., PRINTZ M., STEWART R. & HENSON P.M. (1977) Relationship of prostaglandin production to secretion in platelets stimulated with platelet activating factor. Fed. Proc. 36, 1329. WESSELLS N.K., SPOONER B.S., ASH J.F., BRADLEY M.O., LUDUENA M.A., TAYLOR E.L. & YAMADA K.M. (1971) Microfilaments in cellular and developmental processes. Contractile microfilament machinery of many cell types is reversibly inhibited by cytochalasin B. Science, 171, 135.
Activation of human platelets by platelet activating factor (PAF) derived from sensitized rabbit basophils* MARTHA C. O'DONNELLt, P. M. HENSON & B. A. FIEDEL Departments of Immunology, Rush Medical College, and Microbiology, University of Illinois at the Medical Center, Chicago, Illinois 60612, U.S.A. and the Department of Pediatrics, National Jewish Hospital and Research Center, Denver, Colorado 80206, U.S.A.
Received 5 January 1978; acceptedfor publication 23 March 197 8
tion reactions induced by this agent are separable and that this cross-species activation may be incomplete. Whether this is a result of the differential ability of rabbit PAF to bind to and activate rabbit as compared to human platelets or to the existence of a family of PAF molecules is not yet known. The capacity of PAF to participate in a secretory event involving human platelets lends support to the belief that PAF may play an important ubiquitous role in the cooperative, leucocyte-dependent, release of vasoactive amines which results in increased vascular permeability.
Summary. Rabbit basophil-derived platelet activating factor (PAF), a mediator of anaphylaxis, induces the aggregation and release of serotonin froin rabbit platelets. In the present study, we report that PAF obtained by challenge of specifically sensitized rabbit basophils induced the noncytotoxic release of serotonin from human platelets; maximal extent of release ranged between 34-46%. This release was unaltered in the presence of indomethacin, indicating that such secretion was not a consequence of contaminating arachidonic acid; further, as previously demonstrated with platelets of rabbit origin, it was markedly independent of a requirement for an intact prostaglandin biosynthetic pathway. In contrast to its effect upon rabbit platelets, rabbit PAF did not induce aggregation of human platelets, suggesting that the aggregation and secre-
INTRODUCTION Platelet activating factor (PAF) is a low molecular weight phospholipid (Benveniste, Le Couedic, Polonsky & Tence, 1977b; Benveniste, Kamoun and Polonsky, 1975; Kater, Austen & Goetzl, 1975) released from sensitized basophils challenged with specific antigen (Benveniste, Henson & Cochrane, 1972) or from basophils incubated under alkaline conditions (Benveniste, 1974). PAF has been shown to induce platelet aggregation and noncytotoxic release of platelet vasoactive amines (Henson, 1970). This cooperative reaction between leucocytes and platelets, originally described in the rabbit (Barbaro & Zvaifler, 1966) where PAF has been strongly
*Supported by grant AI-12870-03 from the National Institutes of Health, a grant from The Chicago Heart Association (BAF), and a Biomedical Research Support grant (No. 5-SO7RR05-14) from the National Institutes of Health
(BAF). tPresented in partial fulfillment of the requirements for the Doctor of Philosophy degree in the Graduate College, University of Illinois, at the Medical Center. Correspondence: Dr B. A. Fiedel, Department of Immunology, Rush Medical College, 1725 West Harrison Street, Chicago, Illinois 60612 U.S.A. 0019-2805/78/1200-0953$02.00 © 1978 Blackwell Scientific Publications
953
954
Mat/tha C. O'Doninell, P. M. Henson & B. A. Fiedel
implicated as a mediator of immune complex disease (Henson & Cochrane, 1971), has been reported also to exist in man (Benveniste, 1974), rat (Kater et al., 1975) and hog (Benveniste et al., 1975), suggesting that PAF may play an important role in leucocyte-dependent vasoactive amine release in higher species generally. Rabbit PAF shows a strong binding affinity for albumin (Benveniste et al., 1972) and weaker affinities for neutrophils, erythrocytes and lymphocytes (Shaw & Henson, 1976). Original reports indicated that PAF induces activation of both rabbit and human platelets. More recently, however, rabbit PAF has been reported not to activate human platelets (Shaw & Henson, 1976) and further, it has been suggested that earlier reports concerning cross-species activation may have been a result of a contamination with the prostaglandin endoperoxide precursor arachidonic acid (Benveniste, Camussi & Mencia-Haerta, 1977a). We report herein that rabbit PAF derived from antigen-stimulated basophils and prepared by simple ethanol extraction can induce the noncytotoxic release of ["4C]-serotonin(5HT) from isolated human platelets; that such activation is not due to contamination with arachidonic acid; and that this reaction can occur in the absence of an intact prostaglandin biosynthetic pathway. In addition, we report that antigen-derived rabbit PAF does not lead to the aggregation of human platelets. MATERIALS AND METHODS
Solutions The following solutions used for the preparation of PAF were prepared as previously described (Henson, 1976): isotonic phosphate-buffered saline (PBS), Tris-Tyrode's solution without Ca++ or Mg++ both in the presence and absence of ethylene glycol tetraacetate (EGTA). Tyrode's bovine serum albumin (BSA), and Tyrode's gelatin. All solutions were adjusted to pH 7-2. Diluents A, B, and C used in the isolation of platelets were plepared as described previously (Fiedel & Gewurz, 1 976a).
Immunization
Preparation ofPAF Blood was drawn from both normal and immune rabbits into 1/9 volume of 3-8 % sodium citrate with potassium soibate. The procedure thereafter was similar to that previously described (Henson, 1976). The blood was sedimented at 500 g (1600 rev/miii, 20 min, 12°) in polycarbonate roundbottomed tube3 (DuPont/Sorvall Co., Newton, Conn.), the platelet-rich plasma discarded, the buffy coat transferred to 50 ml plastic centrifuge tubes (Corning Glass Works, Corning, New York) with a siliconized Pasteur pipette, mixed with an equal volume of 2-5 % gelatin in PBS and incubated (30 min, 370) to sediment most of the contaminating erythrocytes. The supernatant cell suspension was removed and centrifuged at 500 g (1500 rev/min, 15 min, 120). The cell pellet was resuspended in a small volume of Tris-Tyrode's -EDTA solution in the absence of Ca++ or Mg++ and the remaining erythrocytes were removed by hypotonic lysis; residual platelets were removed by four consecutive washes (180 g, 8 min, 120) in Tris-Tyrode's solution lacking Ca++ or Mg++. The resultant cell pellet was readjusted to yield 106 basophils/ml with Tyrode's BSA and, on the average, contained 6 % basophils as detei mined by staining with toluidine blue. Immune cell suspensions were challenged with HRP (50 jig peroxidase/106 basophils) and incubated for 20 min at 37°. The cells were removed by centrifugation at 500 g (1500 rev/min, 10 min, 120) and supematant fluids containing PAF-albumin were removed for extraction of PAF by addition of ethanol to 80% (a procedure which also ensured the removal of any contaminating thrombin); after 15 min at room temperature the precipitated protein was removed by centrifugation at 2500 g (3000 rev/min, 15 min, 40). The PAF was stored in ethanol at -70° until the day of use, when aliquots were blown to dryness and redissolved in Tyrode's gelatin at a final concentration 0-4 - 4 x the original PAF concentration in the Tyrode's-BSA cell suspension. The secretion of platelet serotonin induced by these preparations was inhibited by theophylline and enhanced in the presence of cytochalasin B as reported (Henson &
Oades, 1976).
New Zealand white rabbits were immunized sub-
cutaneously with horseradish peroxidase (1 mg/ml in saline; HRP type 1I, Sigma Chemical Co., St. Louis, Mo.) (Henson, 1976), reimmunized with 5 mg/ml HRP at intervals of 1-3 months and bled ten days post secondary immunization via cardiac puncture.
Platelet preparation Normal rabbit or human blood was drawn into 1/9 volume acid citrate-dextrose or 1/7 volume EDTA and centrifuged at 180 g (800 rev/min, 20 min, 12'). Waslhed platelets were prepared from the platelet-
955
Activation of human platelets by rabbit PAF
rich-plasma (PRP) using previously described methods (Fiedel & Gewurz, 1976a). PRP was centrifuged at 2500 g (3000 rev/min, 15 min, 120) into 1 ml of 40% BSA (Sigma Chemical Co.), the platelets were removed with a siliconized Pasteur pipette, washed once in Diluents A and B (re-centrifuging into the BSA solution), and resuspended to 06 x 109 platelets/ml in Tyrode's gelatin. The platelet suspension was then incubated (45 min, 180) in a 50-ml plastic centrifuge tube with [14C]-5-hydroxytryptamine (serotonin) ([14C]-5HT; Amersham/Searle Corp., Arlington Heights, Illinois) at a final concentration of 0-025 pCi/ml. Release ofplatelet [14C]-5HT The standard reaction mixture in siliconized glass tubes (Chrono Log Corp, Havertown, Pa.) contained 0 4 ml of 0X6 x 109 platelets/ml (final concentration, 5 x 108 platelets/ml) and varying amounts of Tyrode's gelatin and PAF to give a final volume of 0-5 ml. The reaction tubes were incubated (15 min, 370, with stirring), cooled on ice, and centrifuged at 2500 g (3000 rev/min, 15 min, 40) (Henson, 1976). Several reaction tubes containing labelled human platelets were preincubated (10 min, 370, with stirring) with theophylline or cytochalasin B in Tyrode's gelatin or with indomethacin in saline (adjusted to pH 8 with NaOH) (Sigma Chemical Co.) prior to the addition of PAF. Aliquots (200 il) of the supernatants were assayed for radioactivity in a Beckman LS-250 Liquid Scintillation Counter (Beckman Inst. Co., Fullerton, Calif.) and released [14C]-5HT was determined. Generally, 0 4 ml of platelets contained 12-20 x 103 c.p.m.
Viability studies Human platelets were isolated using 40% BSA, labelled in the first wash by sequential addition of [14C]-5HT and sodium 5'chromate (51Cr; New England Nuclear, Boston, Mass.) at final concentrations of 0-025 and 10 ptCi/ml, respectively, and incubated for 30 min at 370 (Fiedel & Gewurz, 1976b). Platelets were washed in diluents A and B (recentrifuging into 40% BSA) and resuspended to 0-6 x 109 platelets/ml in Tyrode's gelatin. Following incubation with PAF (15 min, 370, with stirring), the cells were centrifuged at 2500 g (3000 rev/min, 15 min, 4°). The supematants were assayed for released [14C]-5HT, and both supematantaliquots (200jl) and cell pellets were assayed for 51Cr in a Nuclear-Chicago well-type gamma counter.
RESULTS The effect of antigen-stimulated rabbit basophilderived platelet activating factor (PAF) upon the secretion of serotonin from human platelets To determine whether human platelets undergo a release reaction in the presence of rabbit PAF, increasing amounts of PAF were incubated with isolated human platelets (5 x 108/ml) for 15 min and the release of [14C]-5HT was determined (Fig. 1). The amount of [14C]-5HT released increased to a maximum value of between 34 and 46% with the addition of 50 jl PAF and declined slightly as he PAF concentration was increased; incubation of PAF with labelled rabbit platelets resulted in a similar release of serotonin. To assess whether the observed release was due to a cytotoxic effect, human platelets were double-labelledwith [14C]-5HT and r'Cr, incubated with 90 pl of PAF and the differential release of each label was determined. A 34-39%releaseof ['4C]-SHTwasnotedintheabsence of any substantial leakage of the "Cr label ( < 6%), thus indicating that the PAF-induced release of serotonin was noncytotoxic in nature.
Activation of human platelets by rabbit PAF, and absence of contamination with arachidonic acid or requirement for prostaglandin intermediary metab-
olism The existence of a molecular species nominally designated as PAF that induces secretion from both rabbit and human platelets is in contrast to a report suggesting that PAF exhibits type and species 50z 40-
30°5 20to
10 10
20
30
40
50 60 F4F (1)
70
80
90
100
Figure 1. Effects of increasing amounts of rabbit basophilderived PAF upon the secretion of [14C]-5HT from human platelets. Each curve represents the mean value of duplicate samples (internal variation 20
_im (0%)
w 20cr 10-
4/ 1
3 2 TIME (min)
4
Figure 2. Effects of 20 FiM indomethacin (IND) upon arachidonic acid (0-6 mM; AA)-induced platelet aggregation and secretion of [14C]-5HT (% release shown in parenthesis) from human platelets. Insert represents the effect of IND upon the secretion of [14C]-5HT from human platelets in which PAF is used in limiting concentration.
Activation of human platelets by rabbit PAF made to elicit a similar aggregation response from human platelets. However, whether dilute or concentrated PAF preparations were utilized (or whether a secretory event was occurring), no aggregation was observed up to 20 min post challenge with PAF. It would thus appear that the effects of rabbit PAF upon human platelets are, in part, functionally distinct from its observed effects upon platelets of rabbit origin and, further, that platelet aggregation and secretion induced by this mediator are separable events. DISCUSSION
PAF appears to be an important mediator of anaphylaxis in a number of higher species. In the present investigation, we report that PAF prepared by ethanol extraction of supernatants obtained from specifically challenged suspensions of rabbit basophils can induce the secretion of serotonin from human platelets. The dose-response curves (Fig. 1) are remarkably similar in shape to curves reported for the rabbit platelet system (Henson, 1976) although the maximum release values were generally 12-24% lower in the human system. PAF-induced secretion from human platelets also demonstrated the characteristic responses to pharmacological agents reported for rabbit platelet secretion (Henson & Oades, 1976). Theophylline, an agent which increases cAMP levels in platelets (Becker & Henson, 1973), effectively inhibited secretion of [14C]5HT while cytochalasin B, a drug thought to disrupt microfilaments (Wessells, Spooner, Ash, Bradley, Luduena, Taylor & Yamada, 1971), enhanced ielease. PAF induces platelet shape change, aggregation and release of serotonin from rabbit platelets independent of endogenous ADP and products formed from arachidonate (Cazenave et al., 1977; Shaw et al., 1977). In the studies reported here, PAF-induced secretion of serotonin from human platelets was not altered in the presence of indomethacin, (Fig. 2), which inhibits arachidonateinduced secretion of serotonin, indicating that such secretion was not caused by contaminating AA and that the reactivity, as shown with rabbit platelets, is markedly independent of an intact prostaglandin biosynthetic pathway. The induction of [14C]-5HT release from human platelets by rabbit PAF contradicts a previous report that rabbit PAF does not induce human
957
platelets to secrete serotonin (Shaw & Henson, 1976). Whereas the rabbit PAF preparations utilized herein fit many of the reported criteria, they appear capable only of activating human platelets to secretion (of 5HT) without an ability to induce their aggregation in contrast to their effects upon platelets of rabbit origin. The basis for this disparity is not yet clear. Although the release from platelets of serotonin and ADP are thought often to occur in tandem, it is possible that the absence of platelet aggregation is related to an altered secretion and/or utilization of endogenous ADP by the platelet; however, in the rabbit PAF has been reported to induce platelet aggregation in the absence of a requirement for ADP (Cazenave et al., 1977). Whether this is also true for human platelets remains to be elucidated; thus, the possibility that endogenous ADP is not participating in these reactions awaits clarification. Human platelets have been reported to be 20-fold less effective in binding rabbit PAF than rabbit platelets, and the binding of PAF to the membrane has been implicated as the primary process involved in PAF-induced activation (Shaw & Henson, 1976). It is clear that appropriate internal activation events must also occur as rabbit PAF absorbs to rabbit neutrophils but does not stimulate the neutrophil to secretion, hexose monophosphate shunt activity, chemotaxis or significantly alter neutrophil response to stimuli such as zymosan-complement or C5a (Shaw & Henson, 1976). Thus, it is possible that the incomplete stimulation of human platelets by rabbit PAF might have as its basis the differential ability of PAF to bind to the respective blood elements and/or the absence of appropriate activation events. The existence, however, of a family of related but functionally distinct PAF molecules cannot be excluded; and is supported by the finding that PAF liberated by rabbit lung tissues has properties similar to basophil-derived PAF, but is not identical since platelets desensitized to basophil PAF still respond to lung-derived PAF (Kravis & Henson, 1975). Attempts are presently underway to define and compare, both biologically and chemically, PAF obtained from different species in hopes of determining the basis of this diversity. REFERENCES BARBARO J.F. & ZVAIFLER N.J. (1966) Antigen-induced histamine release from platelets of rabbits producing homologous PCA antibody. Proc. Soc. exp. Biol. Med. 122, 1245.
958
Martha C. O'Donnell, P. M. Henson & B. A. Fiedel
BECKER E.L. & HENSON P.M. (1973) In vitro studies of immunologically induced secretion of mediators from cells and related phenomena. Adv. Immunol. 17,93. BENVENISTE J., HENSON P.M. & COCHRANE C.G. (1972) Leukocyte-dependent histamine release from rabbit platelets. J. exp. Med. 136, 1356. BENVENISTE J. (1974) Platelet-activating factor, a new mediator of anaphylaxis and immune complex deposition from rabbit and human basophils. Nature (Lond.), 249, 581. BENVENISTE J., KAMOUN P. & POLONSKY J. (1975) Aggregation of human platelets by purified platelet activating factor from human and rabbit basophils. Fed. Proc. 34, 985. BENVENISTE J. & POLONSKY J. (1976) Isolation and partial characterization of platelet activating factor (PAF). Fed. Proc. 35, 516. BENVENISTE J., CAMussi G. & MENCIA-HUERTA J.M. (1977a) Cellular origin of platelet-activating factor (PAF). Fed. Proc. 36,1329. BENVENISTE J., LE COUEDIC J.P., POLONSKY J. & TENCE M. (1977b) Structural analysis of purified platelet-activating factor by lipases. Nature (Lond.), 269, 170. CAZENAVE J-P., BENVENISTE J. & MUSTARD J.F. (1977) Platelet activating factor (PAF) aggregates platelets independently of the release reaction and the arachidonate pathway. Fed. Proc. 36,454. FIEDEL B.A. & GEWURZ H. (1976a) Effects of C-reactive protein on platelet function. I. Inhibition of platelet aggregation and release reactions. J. Immunol. 116, 1289. FIEDEL B.A. & GEWURZ H. (1976b) Effects of C-reactive protein on platelet function. II. Inhibition by CRP of platelet reactivities stimulated by poly-L-lysine, ADP, epinephrine, and collagen. J. Immunol. 117,1073. FIEDEL B.A., SIMPSON, R.M. & GEWURZ, H. (1977) Effects of C-reactive protein on platelet function. III. The role of cAMP, contractile elements, and prostaglandin metabolism in CRP-induced inhibition of platelet aggregation and secretion. J. Immunol. 119, 877. HAMBERG M., SVENSSON J. & SAMUELSSON B. (1974) Prostaglandin endoperoxides: A new concept concerning the
mode of action and release of prostaglandins. Proc. nato. Acad. Sci. U.S.A. 71, 3824. HENSON P.M. (1970) Release of vasoactive amines from rabbit platelets induced by sensitized mononuclear leukocytes and antigen. J. exp. Med. 131, 287. HENSON P.M. & COCHRANE C.G. (1971) Immune complex disease in rabbits. The role of complement and of a leukocyte-dependent release of vasoactive amines from platelets. J. exp. Med. 133, 554. HENSON P.M. (1976) Activation and desensitization of platelets by platelet-activating factor (PAF) derived from IgEsensitized basophils. I. Characteristics of the secretory response. J. exp. Med. 143,937. HENSON P.M. & OADES Z.G. (1976) Activation of platelets by platelet-activating factor (PAF) derived from IgEsensitized basophils. II. The role of serine proteases, cyclic nucleotides, and contractile elements in PAF-induced secretion. J. exp. Med. 143,953. KATER L.A., AUSTEN K.F. & GOETZL E.J. (1975) Identification and partial purification of a platelet activating factor (PAF) from rat. Fed. Proc. 34,985. KRAVIs T.C. & HENSON P.M. (1975) IgE-induced release of a platelet-activating factor from rabbit lung. J. Immunol. 115, 1677. NEEDLEMAN P., MINKES M. & RAZ A. (1976) Thromboxanes: Selective biosynthesis and distinct biological properties. Science, 193,163. SHAW J.Q. & HENSON P.M. (1976) Platelet activating factor (PAF) binding to blood cellular components: type and species specificity. Fed. Proc. 35, 516. SHAW J.O., PRINTZ M., STEWART R. & HENSON P.M. (1977) Relationship of prostaglandin production to secretion in platelets stimulated with platelet activating factor. Fed. Proc. 36, 1329. WESSELLS N.K., SPOONER B.S., ASH J.F., BRADLEY M.O., LUDUENA M.A., TAYLOR E.L. & YAMADA K.M. (1971) Microfilaments in cellular and developmental processes. Contractile microfilament machinery of many cell types is reversibly inhibited by cytochalasin B. Science, 171, 135.
